PHYTOCHROME C Is an Essential Light Receptor for Photoperiodic Flowering in the Temperate Grass, Brachypodium distachyon.
Genetics. 2014 Jul 14;
Authors: Woods DP, Ream TS, Minevich G, Hobert O, Amasino RM
We show that in the temperate grass, Brachypodium distachyon, PHYTOCHROME C (PHYC), is necessary for photoperiodic flowering. In loss-of-function phyC mutants, flowering is extremely delayed in inductive photoperiods. PHYC was identified as the causative locus by utilizing a mapping by sequencing pipeline (Cloudmap) optimized for identification of induced mutations in Brachypodium. In phyC mutants the expression of Brachypodium homologs of key flowering time genes in the photoperiod pathway such as GIGANTEA (GI), PHOTOPERIOD 1 (PPD1/PRR37), CONSTANS (CO), and florigen/FT are greatly attenuated. PHYC also controls the day-length dependence of leaf size as the effect of day-length on leaf size is abolished in phyC mutants. The control of genes upstream of florigen production by PHYC was likely to have been a key feature of the evolution of a long-day flowering response in temperate pooid grasses.
PMID: 25023399 [PubMed - as supplied by publisher]
Starvation-Induced Transgenerational Inheritance of Small RNAs in C. elegans.
Cell. 2014 Jul 9;
Authors: Rechavi O, Houri-Ze'evi L, Anava S, Goh WS, Kerk SY, Hannon GJ, Hobert O
Evidence from animal studies and human famines suggests that starvation may affect the health of the progeny of famished individuals. However, it is not clear whether starvation affects only immediate offspring or has lasting effects; it is also unclear how such epigenetic information is inherited. Small RNA-induced gene silencing can persist over several generations via transgenerationally inherited small RNA molecules in C. elegans, but all known transgenerational silencing responses are directed against foreign DNA introduced into the organism. We found that starvation-induced developmental arrest, a natural and drastic environmental change, leads to the generation of small RNAs that are inherited through at least three consecutive generations. These small, endogenous, transgenerationally transmitted RNAs target genes with roles in nutrition. We defined genes that are essential for this multigenerational effect. Moreover, we show that the F3 offspring of starved animals show an increased lifespan, corroborating the notion of a transgenerational memory of past conditions.
PMID: 25018105 [PubMed - as supplied by publisher]
Left-right asymmetry: Advances and enigmas.
Genesis. 2014 Jun;52(6):451-4
Authors: Halpern ME, Hobert O, Wright CV
PMID: 24943067 [PubMed - in process]
Maintenance of postmitotic neuronal cell identity.
Nat Neurosci. 2014 Jul;17(7):899-907
Authors: Deneris ES, Hobert O
The identity of specific cell types in the nervous system is defined by the expression of neuron type-specific gene batteries. How the expression of such batteries is initiated during nervous system development has been under intensive study over the past few decades. However, comparatively little is known about how gene batteries that define the terminally differentiated state of a neuron type are maintained throughout the life of an animal. Here we provide an overview of studies in invertebrate and vertebrate model systems that have carved out the general and not commonly appreciated principle that neuronal identity is maintained in postmitotic neurons by the sustained, and often autoregulated, expression of the same transcription factors that initiate terminal differentiation in a developing organism. Disruption of postmitotic maintenance mechanisms may result in neuropsychiatric and neurodegenerative conditions.
PMID: 24929660 [PubMed - in process]
Progressive degeneration of dopaminergic neurons through TRP channel-induced cell death.
J Neurosci. 2014 Apr 23;34(17):5738-46
Authors: Nagarajan A, Ning Y, Reisner K, Buraei Z, Larsen JP, Hobert O, Doitsidou M
Progressive neurodegenerative diseases are among the most frequently occurring aging-associated human pathologies. In a screen for Caenorhabditis elegans mutant animals that lack their normal complement of dopaminergic neurons, we identified two strains with progressive loss of dopaminergic neurons during postembryonic life. Through whole-genome sequencing we show that both strains harbor dominant (d), gain-of-function mutations in the Transient Receptor Potential (TRP) mechanosensory channel trp-4, a member of the invertebrate and vertebrate TRPN-type of the TRP family channels. Gain-of-function mutations in TRP channels have not been previously implicated in dopaminergic neuronal degeneration. We show that trp-4(d) induces cell death in dopamine neurons through a defined, calcium-related downstream pathway.
PMID: 24760834 [PubMed - indexed for MEDLINE]
TargetOrtho: a phylogenetic footprinting tool to identify transcription factor targets.
Genetics. 2014 May;197(1):61-76
Authors: Glenwinkel L, Wu D, Minevich G, Hobert O
The identification of the regulatory targets of transcription factors is central to our understanding of how transcription factors fulfill their many key roles in development and homeostasis. DNA-binding sites have been uncovered for many transcription factors through a number of experimental approaches, but it has proven difficult to use this binding site information to reliably predict transcription factor target genes in genomic sequence space. Using the nematode Caenorhabditis elegans and other related nematode species as a starting point, we describe here a bioinformatic pipeline that identifies potential transcription factor target genes from genomic sequences. Among the key features of this pipeline is the use of sequence conservation of transcription-factor-binding sites in related species. Rather than using aligned genomic DNA sequences from the genomes of multiple species as a starting point, TargetOrtho scans related genome sequences independently for matches to user-provided transcription-factor-binding motifs, assigns motif matches to adjacent genes, and then determines whether orthologous genes in different species also contain motif matches. We validate TargetOrtho by identifying previously characterized targets of three different types of transcription factors in C. elegans, and we use TargetOrtho to identify novel target genes of the Collier/Olf/EBF transcription factor UNC-3 in C. elegans ventral nerve cord motor neurons. We have also implemented the use of TargetOrtho in Drosophila melanogaster using conservation among five species in the D. melanogaster species subgroup for target gene discovery.
PMID: 24558259 [PubMed - in process]
Development of left/right asymmetry in the Caenorhabditis elegans nervous system: From zygote to postmitotic neuron.
Genesis. 2014 Jun;52(6):528-43
Authors: Hobert O
Despite their gross morphological symmetry, animal nervous systems can perceive and process information in a left/right asymmetric manner. How left/right asymmetric functional features develop in the context of a bilaterally symmetric structure is a very poorly understood problem, in part because very few morphological or molecular correlates of functional asymmetries have been identified so far in vertebrate or invertebrate nervous systems. One of the very few systems in which a molecular correlate for functional lateralization has been uncovered is the taste sensory system of the nematode Caenorhabditis elegans, which is composed of a pair of bilaterally symmetric neurons, ASE left (ASEL) and ASE right (ASER). ASEL and ASER are similar in morphology, connectivity, and molecular composition, but they express distinct members of a putative chemoreceptor gene family and respond in a fundamentally distinct manner to taste cues. Extensive forward and reverse genetic analysis has uncovered a complex gene regulatory network, composed of transcription factors, miRNAs, chromatin regulators, and intercellular signals, that instruct the asymmetric features of these two neurons. In this review, this system is described in detail, drawing a relatively complete picture of asymmetry control in a nervous system. genesis 52:528-543, 2014. © 2014 Wiley Periodicals, Inc.
PMID: 24510690 [PubMed - in process]
Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1.
Genes Dev. 2014 Jan 1;28(1):34-43
Authors: Cochella L, Tursun B, Hsieh YW, Galindo S, Johnston RJ, Chuang CF, Hobert O
Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ("antisymmetries") or found stereotypically on one particular side of an animal ("directional asymmetries"). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. We describe here an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs.
PMID: 24361693 [PubMed - indexed for MEDLINE]
The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types.
Development. 2014 Jan;141(2):422-35
Authors: Zhang F, Bhattacharya A, Nelson JC, Abe N, Gordon P, Lloret-Fernandez C, Maicas M, Flames N, Mann RS, Colón-Ramos DA, Hobert O
Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.
PMID: 24353061 [PubMed - indexed for MEDLINE]
Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins.
Cell. 2013 Oct 24;155(3):659-73
Authors: Serrano-Saiz E, Poole RJ, Felton T, Zhang F, De La Cruz ED, Hobert O
The choice of using one of many possible neurotransmitter systems is a critical step in defining the identity of an individual neuron type. We show here that the key defining feature of glutamatergic neurons, the vesicular glutamate transporter EAT-4/VGLUT, is expressed in 38 of the 118 anatomically defined neuron classes of the C. elegans nervous system. We show that distinct cis-regulatory modules drive expression of eat-4/VGLUT in distinct glutamatergic neuron classes. We identify 13 different transcription factors, 11 of them homeodomain proteins, that act in distinct combinations in 25 different glutamatergic neuron classes to initiate and maintain eat-4/VGLUT expression. We show that the adoption of a glutamatergic phenotype is linked to the adoption of other terminal identity features of a neuron, including cotransmitter phenotypes. Examination of mouse orthologs of these homeodomain proteins resulted in the identification of mouse LHX1 as a regulator of glutamatergic neurons in the brainstem.
PMID: 24243022 [PubMed - indexed for MEDLINE]
The neuronal genome of Caenorhabditis elegans.
The ~100 MB genome of C. elegans codes for ~20,000 protein-coding genes many of which are required for the function of the nervous system, composed of 302 neurons in the adult hermaphrodite and of 383 neurons in the adult male. In addition to housekeeping genes, a differentiated neuron is thought to express many hundreds if not thousands of genes that define its functional properties. These genes code for ion channels, G-protein-coupled receptors, neurotransmitter-synthesizing enzymes, transporters and receptors, neuropeptides and their receptors, cell adhesion molecules, motor proteins, signaling molecules and many others. Collectively such genes have been referred to as "terminal differentiation genes" or "effector genes". The differential expression of distinct combinations of terminal differentiation genes define different neuron types. This paper provides a compendium of more than 2,800 putative terminal differentiation genes. One pervasive theme revealed by the analysis of many gene families is the nematode-specific expansions of many neuron function-related gene families, including, for example, many types of ion channel families, sensory receptors and neurotransmitter receptors. The gene lists provided here can serve multiple purposes. They can serve as quick reference guides for individual gene families or they can be used to mine large datasets (e.g., expression datasets) for genes with likely functions in the nervous system. They also serve as a starting point for future projects. For example, a comprehensive understanding of the regulation of the often complex expression patterns of these genes in the nervous system will eventually explain how nervous systems are built.
PMID: 24081909 [PubMed - in process]
Microbeam irradiation of C. elegans nematode in microfluidic channels.
Radiat Environ Biophys. 2013 Nov;52(4):531-7
Authors: Buonanno M, Garty G, Grad M, Gendrel M, Hobert O, Brenner DJ
To perform high-throughput studies on the biological effects of ionizing radiation in vivo, we have implemented a microfluidic tool for microbeam irradiation of Caenorhabditis elegans. The device allows the immobilization of worms with minimal stress for a rapid and controlled microbeam irradiation of multiple samples in parallel. Adapted from an established design, our microfluidic clamp consists of 16 tapered channels with 10-μm-thin bottoms to ensure charged particle traversal. Worms are introduced into the microfluidic device through liquid flow between an inlet and an outlet, and the size of each microchannel guarantees that young adult worms are immobilized within minutes without the use of anesthesia. After site-specific irradiation with the microbeam, the worms can be released by reversing the flow direction in the clamp and collected for analysis of biological endpoints such as repair of radiation-induced DNA damage. For such studies, minimal sample manipulation and reduced use of drugs such as anesthetics that might interfere with normal physiological processes are preferable. By using our microfluidic device that allows simultaneous immobilization and imaging for irradiation of several whole living samples on a single clamp, here we show that 4.5-MeV proton microbeam irradiation induced DNA damage in wild-type C. elegans, as assessed by the formation of Rad51 foci that are essential for homologous repair of radiation-induced DNA damage.
PMID: 23942865 [PubMed - indexed for MEDLINE]
A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans.
Genes Dev. 2013 Jun 15;27(12):1391-405
Authors: Doitsidou M, Flames N, Topalidou I, Abe N, Felton T, Remesal L, Popovitchenko T, Mann R, Chalfie M, Hobert O
Terminal differentiation programs in the nervous system are encoded by cis-regulatory elements that control the expression of terminal features of individual neuron types. We decoded the regulatory information that controls the expression of five enzymes and transporters that define the terminal identity of all eight dopaminergic neurons in the nervous system of the Caenorhabditis elegans hermaphrodite. We show that the tightly coordinated, robust expression of these dopaminergic enzymes and transporters ("dopamine pathway") is ensured through a combinatorial cis-regulatory signature that is shared by all dopamine pathway genes. This signature is composed of an Ets domain-binding site, recognized by the previously described AST-1 Ets domain factor, and two distinct types of homeodomain-binding sites that act in a partially redundant manner. Through genetic screens, we identified the sole C. elegans Distalless/Dlx ortholog, ceh-43, as a factor that acts through one of the homeodomain sites to control both induction and maintenance of terminal dopaminergic fate. The second type of homeodomain site is a Pbx-type site, which is recognized in a partially redundant and neuron subtype-specific manner by two Pbx factors, ceh-20 and ceh-40, revealing novel roles of Pbx factors in the context of terminal neuron differentiation. Taken together, we revealed a specific regulatory signature and cognate, terminal selector-type transcription factors that define the entire dopaminergic nervous system of an animal. Dopaminergic neurons in the mouse olfactory bulb express a similar combinatorial transcription factor collective of Ets/Dlx/Pbx factors, suggesting deep phylogenetic conservation of dopaminergic regulatory programs.
PMID: 23788625 [PubMed - indexed for MEDLINE]
Defining specificity determinants of cGMP mediated gustatory sensory transduction in Caenorhabditis elegans.
Genetics. 2013 Aug;194(4):885-901
Authors: Smith HK, Luo L, O'Halloran D, Guo D, Huang XY, Samuel AD, Hobert O
Cyclic guanosine monophosphate (cGMP) is a key secondary messenger used in signal transduction in various types of sensory neurons. The importance of cGMP in the ASE gustatory receptor neurons of the nematode Caenorhabditis elegans was deduced by the observation that multiple receptor-type guanylyl cyclases (rGCs), encoded by the gcy genes, and two presently known cyclic nucleotide-gated ion channel subunits, encoded by the tax-2 and tax-4 genes, are essential for ASE-mediated gustatory behavior. We describe here specific mechanistic features of cGMP-mediated signal transduction in the ASE neurons. First, we assess the specificity of the sensory functions of individual rGC proteins. We have previously shown that multiple rGC proteins are expressed in a left/right asymmetric manner in the functionally lateralized ASE neurons and are required to sense distinct salt cues. Through domain swap experiments among three different rGC proteins, we show here that the specificity of individual rGC proteins lies in their extracellular domains and not in their intracellular, signal-transducing domains. Furthermore, we find that rGC proteins are also sufficient to confer salt sensory responses to other neurons. Both findings support the hypothesis that rGC proteins are salt receptor proteins. Second, we identify a novel, likely downstream effector of the rGC proteins in gustatory signal transduction, a previously uncharacterized cyclic nucleotide-gated (CNG) ion channel, encoded by the che-6 locus. che-6 mutants show defects in gustatory sensory transduction that are similar to defects observed in animals lacking the tax-2 and tax-4 CNG channels. In contrast, thermosensory signal transduction, which also requires tax-2 and tax-4, does not require che-6, but requires another CNG, cng-3. We propose that CHE-6 may form together with two other CNG subunits, TAX-2 and TAX-4, a gustatory neuron-specific heteromeric CNG channel complex.
PMID: 23695300 [PubMed - indexed for MEDLINE]
The SWI/SNF chromatin remodeling complex selectively affects multiple aspects of serotonergic neuron differentiation.
Genetics. 2013 May;194(1):189-98
Authors: Weinberg P, Flames N, Sawa H, Garriga G, Hobert O
Regulatory programs that control the specification of serotonergic neurons have been investigated by genetic mutant screens in the nematode Caenorhabditis elegans. Loss of a previously uncloned gene, ham-3, affects migration and serotonin antibody staining of the hermaphrodite-specific neuron (HSN) pair. We characterize these defects here in more detail, showing that the defects in serotonin antibody staining are paralleled by a loss of the transcription of all genes involved in serotonin synthesis and transport. This loss is specific to the HSN class as other serotonergic neurons appear to differentiate normally in ham-3 null mutants. Besides failing to migrate appropriately, the HSNs also display axon pathfinding defects in ham-3 mutants. However, the HSNs are still generated and express a subset of their terminal differentiation features in ham-3 null mutants, demonstrating that ham-3 is a specific regulator of select features of the HSNs. We show that ham-3 codes for the C. elegans ortholog of human BAF60, Drosophila Bap60, and yeast Swp73/Rsc6, which are subunits of the yeast SWI/SNF and vertebrate BAF chromatin remodeling complex. We show that the effect of ham-3 on serotonergic fate can be explained by ham-3 regulating the expression of the Spalt/SALL-type Zn finger transcription factor sem-4, a previously identified regulator of serotonin expression in HSNs and of the ham-2 Zn transcription factor, a previously identified regulator of HSN migration and axon outgrowth. Our findings provide the first evidence for the involvement of the BAF complex in the acquisition of terminal neuronal identity and constitute genetic proof by germline knockout that a BAF complex component can have cell-type-specific roles during development.
PMID: 23457234 [PubMed - indexed for MEDLINE]
Embryonic priming of a miRNA locus predetermines postmitotic neuronal left/right asymmetry in C. elegans.
Cell. 2012 Dec 7;151(6):1229-42
Authors: Cochella L, Hobert O
The mechanisms by which functional left/right asymmetry arises in morphologically symmetric nervous systems are poorly understood. Here, we provide a mechanistic framework for how functional asymmetry in a postmitotic neuron pair is specified in C. elegans. A key feature of this mechanism is a temporally separated, two-step activation of the lsy-6 miRNA locus. The lsy-6 locus is first "primed" by chromatin decompaction in the precursor for the left neuron, but not the right neuron, several divisions before the neurons are born. lsy-6 expression is then "boosted" to functionally relevant levels several divisions later in the mother of the left neuron, through the activity of a bilaterally expressed transcription factor that can only activate lsy-6 in the primed neuron. This study shows how cells can become committed during early developmental stages to execute a specific fate much later in development and provides a conceptual framework for understanding the generation of neuronal diversity.
PMID: 23201143 [PubMed - indexed for MEDLINE]
Removal of Polycomb repressive complex 2 makes C. elegans germ cells susceptible to direct conversion into specific somatic cell types.
Cell Rep. 2012 Nov 29;2(5):1178-86
Authors: Patel T, Tursun B, Rahe DP, Hobert O
How specific cell types can be directly converted into other distinct cell types is a matter of intense investigation with wide-ranging basic and biomedical implications. Here, we show that removal of the histone 3 lysine 27 (H3K27) methyltransferase Polycomb repressor complex 2 (PRC2) permits ectopically expressed, neuron-type-specific transcription factors ("terminal selectors") to convert Caenorhabditis elegans germ cells directly into specific neuron types. Terminal-selector-induced germ-cell-to-neuron conversion can be observed not only upon genome-wide loss of H3K27 methylation in PRC2(-) animals but also upon genome-wide redistribution of H3K27 methylation patterns in animals that lack the H3K36 methyltransferase MES-4. Manipulation of the H3K27 methylation status not only permits conversion of germ cells into neurons but also permits hlh-1/MyoD-dependent conversion of germ cells into muscle cells, indicating that PRC2 protects the germline from the aberrant execution of multiple distinct somatic differentiation programs. Taken together, our findings demonstrate that the normally multistep process of development from a germ cell via a zygote to a terminally differentiated somatic cell type can be short-cut by providing an appropriate terminal selector transcription factor and manipulating histone methylation patterns.
PMID: 23103163 [PubMed - indexed for MEDLINE]
CloudMap: a cloud-based pipeline for analysis of mutant genome sequences.
Genetics. 2012 Dec;192(4):1249-69
Authors: Minevich G, Park DS, Blankenberg D, Poole RJ, Hobert O
Whole genome sequencing (WGS) allows researchers to pinpoint genetic differences between individuals and significantly shortcuts the costly and time-consuming part of forward genetic analysis in model organism systems. Currently, the most effort-intensive part of WGS is the bioinformatic analysis of the relatively short reads generated by second generation sequencing platforms. We describe here a novel, easily accessible and cloud-based pipeline, called CloudMap, which greatly simplifies the analysis of mutant genome sequences. Available on the Galaxy web platform, CloudMap requires no software installation when run on the cloud, but it can also be run locally or via Amazon's Elastic Compute Cloud (EC2) service. CloudMap uses a series of predefined workflows to pinpoint sequence variations in animal genomes, such as those of premutagenized and mutagenized Caenorhabditis elegans strains. In combination with a variant-based mapping procedure, CloudMap allows users to sharply define genetic map intervals graphically and to retrieve very short lists of candidate variants with a few simple clicks. Automated workflows and extensive video user guides are available to detail the individual analysis steps performed (http://usegalaxy.org/cloudmap). We demonstrate the utility of CloudMap for WGS analysis of C. elegans and Arabidopsis genomes and describe how other organisms (e.g., Zebrafish and Drosophila) can easily be accommodated by this software platform. To accommodate rapid analysis of many mutants from large-scale genetic screens, CloudMap contains an in silico complementation testing tool that allows users to rapidly identify instances where multiple alleles of the same gene are present in the mutant collection. Lastly, we describe the application of a novel mapping/WGS method ("Variant Discovery Mapping") that does not rely on a defined polymorphic mapping strain, and we integrate the application of this method into CloudMap. CloudMap tools and documentation are continually updated at http://usegalaxy.org/cloudmap.
PMID: 23051646 [PubMed - indexed for MEDLINE]
Extending our experimental reach: Toolbox reviews in Genetics.
Genetics. 2012 Sep;192(1):1
Authors: Johnston M, Hobert O
PMID: 22964833 [PubMed - indexed for MEDLINE]
The secreted immunoglobulin domain proteins ZIG-5 and ZIG-8 cooperate with L1CAM/SAX-7 to maintain nervous system integrity.
PLoS Genet. 2012;8(7):e1002819
Authors: Bénard CY, Blanchette C, Recio J, Hobert O
During nervous system development, neuronal cell bodies and their axodendritic projections are precisely positioned through transiently expressed patterning cues. We show here that two neuronally expressed, secreted immunoglobulin (Ig) domain-containing proteins, ZIG-5 and ZIG-8, have no detectable role during embryonic nervous system development of the nematode Caenorhabditis elegans but are jointly required for neuronal soma and ventral cord axons to maintain their correct position throughout postembryonic life of the animal. The maintenance defects observed upon removal of zig-5 and zig-8 are similar to those observed upon complete loss of the SAX-7 protein, the C. elegans ortholog of the L1CAM family of adhesion proteins, which have been implicated in several neurological diseases. SAX-7 exists in two isoforms: a canonical, long isoform (SAX-7L) and a more adhesive shorter isoform lacking the first two Ig domains (SAX-7S). Unexpectedly, the normally essential function of ZIG-5 and ZIG-8 in maintaining neuronal soma and axon position is completely suppressed by genetic removal of the long SAX-7L isoform. Overexpression of the short isoform SAX-7S also abrogates the need for ZIG-5 and ZIG-8. Conversely, overexpression of the long isoform disrupts adhesion, irrespective of the presence of the ZIG proteins. These findings suggest an unexpected interdependency of distinct Ig domain proteins, with one isoform of SAX-7, SAX-7L, inhibiting the function of the most adhesive isoform, SAX-7S, and this inhibition being relieved by ZIG-5 and ZIG-8. Apart from extending our understanding of dedicated neuronal maintenance mechanisms, these findings provide novel insights into adhesive and anti-adhesive functions of IgCAM proteins.
PMID: 22829780 [PubMed - indexed for MEDLINE]
Diverse functions of microRNAs in nervous system development.
Curr Top Dev Biol. 2012;99:115-43
MicroRNAs (miRNAs) are integral parts of the gene regulatory networks that control most developmental processes. Through their regulatory action, miRNAs introduce an additional layer of genetic complexity that can translate into increased cellular diversity, something that is extremely relevant to nervous system structure. In addition, miRNAs sharpen the spatial and temporal boundaries between different cellular states during development. Here, we illustrate these roles with a number of specific miRNAs that act during distinct steps of neural development. We further discuss specific aspects of miRNA function that make these regulators particularly suited to provide the robustness and complexity that are essential for the dynamic nature of both the development and activity of the nervous system.
PMID: 22365737 [PubMed - indexed for MEDLINE]
From genes to function: the C. elegans genetic toolbox.
Wiley Interdiscip Rev Dev Biol. 2012 Jan-Feb;1(1):114-37
Authors: Boulin T, Hobert O
This review aims to provide an overview of the technologies which make the nematode Caenorhabditis elegans an attractive genetic model system. We describe transgenesis techniques and forward and reverse genetic approaches to isolate mutants and clone genes. In addition, we discuss the new possibilities offered by genome engineering strategies and next-generation genome analysis tools.
PMID: 23801671 [PubMed - indexed for MEDLINE]
Coordinated regulation of cholinergic motor neuron traits through a conserved terminal selector gene.
Nat Neurosci. 2012 Feb;15(2):205-14
Authors: Kratsios P, Stolfi A, Levine M, Hobert O
Cholinergic motor neurons are defined by the coexpression of a battery of genes encoding proteins that act sequentially to synthesize, package and degrade acetylcholine and reuptake its breakdown product, choline. How expression of these critical motor neuron identity determinants is controlled and coordinated is not understood. We show here that, in the nematode Caenorhabditis elegans, all members of the cholinergic gene battery, as well as many other markers of terminal motor neuron fate, are co-regulated by a shared cis-regulatory signature and a common trans-acting factor, the phylogenetically conserved COE (Collier, Olf, EBF)-type transcription factor UNC-3. UNC-3 initiated and maintained expression of cholinergic fate markers and was sufficient to induce cholinergic fate in other neuron types. UNC-3 furthermore operated in negative feedforward loops to induce the expression of transcription factors that repress individual UNC-3-induced terminal fate markers, resulting in diversification of motor neuron differentiation programs in specific motor neuron subtypes. A chordate ortholog of UNC-3, Ciona intestinalis COE, was also both required and sufficient for inducing a cholinergic fate. Thus, UNC-3 is a terminal selector for cholinergic motor neuron differentiation whose function is conserved across phylogeny.
PMID: 22119902 [PubMed - indexed for MEDLINE]
Transgenerational inheritance of an acquired small RNA-based antiviral response in C. elegans.
Cell. 2011 Dec 9;147(6):1248-56
Authors: Rechavi O, Minevich G, Hobert O
Induced expression of the Flock House virus in the soma of C. elegans results in the RNAi-dependent production of virus-derived, small-interfering RNAs (viRNAs), which in turn silence the viral genome. We show here that the viRNA-mediated viral silencing effect is transmitted in a non-Mendelian manner to many ensuing generations. We show that the viral silencing agents, viRNAs, are transgenerationally transmitted in a template-independent manner and work in trans to silence viral genomes present in animals that are deficient in producing their own viRNAs. These results provide evidence for the transgenerational inheritance of an acquired trait, induced by the exposure of animals to a specific, biologically relevant physiological challenge. The ability to inherit such extragenic information may provide adaptive benefits to an animal.
PMID: 22119442 [PubMed - indexed for MEDLINE]
Regulation of terminal differentiation programs in the nervous system.
Annu Rev Cell Dev Biol. 2011;27:681-96
The generation of individual neuron types in the nervous system is a multistep process whose endpoint is the expression of neuron type-specific batteries of terminal differentiation genes that determine the functional properties of a neuron. This review focuses on the regulatory mechanisms that are involved in controlling the terminally differentiated state of a neuron. I review several case studies from invertebrate and vertebrate nervous systems that reveal that many terminal differentiation features of a neuron are coregulated via terminal selector transcription factors that initiate and maintain terminal differentiation programs.
PMID: 21985672 [PubMed - indexed for MEDLINE]
Temporal and spatial regulation of microRNA activity with photoactivatable cantimirs.
ACS Chem Biol. 2011 Dec 16;6(12):1332-8
Authors: Zheng G, Cochella L, Liu J, Hobert O, Li WH
MicroRNAs (miRNAs) are small non-coding RNAs that play numerous important roles in physiology and human diseases. During animal development, many miRNAs are expressed continuously from early embryos throughout adults, yet it is unclear whether these miRNAs are actually required at all the stages of development. Current techniques of manipulating microRNA function lack the required spatial and temporal resolution to adequately address the functionality of a given microRNA at a specific time or at single-cell resolution. To examine stage- or cell-specific function of miRNA during development and to achieve precise control of miRNA activity, we have developed photoactivatable antisense oligonucleotides against miRNAs. These caged oligonucleotides can be activated with 365 nm light with extraordinarily high efficiency to release potent antisense reagents to inhibit miRNAs. Initial application of these caged antimirs in a model organism (C. elegans) revealed that the activity of a miRNA (lsy-6) is required specifically around the comma stage during embryonic development to control a left/right asymmetric differentiation program in the C. elegans nervous system. This suggests that a transient input of lsy-6 during development is sufficient to specify the neuronal cell fate.
PMID: 21977972 [PubMed - indexed for MEDLINE]
Notch-dependent induction of left/right asymmetry in C. elegans interneurons and motoneurons.
Curr Biol. 2011 Jul 26;21(14):1225-31
Authors: Bertrand V, Bisso P, Poole RJ, Hobert O
Although nervous systems are largely bilaterally symmetric on a structural level, they display striking degrees of functional left/right (L/R) asymmetry. In Caenorhabditis elegans, two structurally symmetric pairs of sensory neurons, ASE and AWC, display two distinctly controlled types of functional L/R asymmetries (stereotyped versus stochastic asymmetry). Beyond these two cases, the extent of neuronal asymmetry in the C. elegans nervous system was unclear. Here, we report that the Beta3/Olig-type bHLH transcription factor hlh-16 is L/R asymmetrically expressed in several distinct, otherwise bilaterally symmetric interneuron and motoneuron pairs that are part of a known navigation circuit. We find that hlh-16 asymmetry is generated during gastrulation by an asymmetric LAG-2/Delta signal originating from the mesoderm that promotes hlh-16 expression in neurons on the left side through direct binding of the Notch effector LAG-1/Su(H)/CBF to a cis-regulatory element in the hlh-16 locus. Removal of hlh-16 reveals an unanticipated asymmetry in the ability of the axons of the AIY interneurons to extend into the nerve ring, with the left AIY axon requiring elevated hlh-16 expression for correct extension. Our study suggests that the extent of molecular L/R asymmetry in the C. elegans nervous system is broader than previously anticipated, establishes a novel signaling mechanism that crosses germ layers to diversify bilaterally symmetric neuronal lineages, and reveals L/R asymmetric control of axonal outgrowth of bilaterally symmetric neurons.
PMID: 21737278 [PubMed - indexed for MEDLINE]
A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans.
PLoS Genet. 2011 Jun;7(6):e1002109
Authors: Poole RJ, Bashllari E, Cochella L, Flowers EB, Hobert O
One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.
PMID: 21698137 [PubMed - indexed for MEDLINE]
Transcriptional control of the terminal fate of monoaminergic neurons.
Annu Rev Neurosci. 2011;34:153-84
Authors: Flames N, Hobert O
Monoaminergic neurons are critical functional components of all nervous systems across phylogeny. The terminally differentiated state of individual types of monoaminergic neurons is defined by the coordinated expression of a battery of genes that instructs the synthesis and transport of specific monoamines, such as serotonin or dopamine. Dysfunction or deregulation of several of these enzymes and transporter system has been proposed to be the underlying basis of several pathological conditions. We review here the state of knowledge of the nature of the transcriptional regulatory programs that control the expression of what we term monoaminergic gene batteries (enzymes and transporters for specific monoamines) and thereby define the terminally differentiated state of monoaminergic neurons. We review several case studies in vertebrate and invertebrate model systems and propose that the coordinated expression of the genes that define individual monoaminergic cell types may be brought about by transcriptional coregulatory strategies.
PMID: 21692658 [PubMed - indexed for MEDLINE]
A left/right asymmetric neuronal differentiation program is controlled by the Caenorhabditis elegans lsy-27 zinc-finger transcription factor.
Genetics. 2011 Jul;188(3):753-9
Authors: Zhang F, O'Meara MM, Hobert O
Functional diversification across the left/right axis is a common feature of many nervous systems. The genetic programs that control left/right asymmetric neuron function and gene expression in the nervous system are, however, poorly understood. We describe here the molecular characterization of two phenotypically similar mutant Caenorhabditis elegans strains in which left/right asymmetric gene expression programs of two gustatory neurons, called ASEL and ASER, are disrupted such that the differentiation program of the ASER neuron is derepressed in the ASEL neuron. We show that in one mutant strain the LIM homeobox gene lim-6 is defective whereas in another strain a novel member of a nematode-specific, fast-evolving family of C2H2 zinc-finger transcription factors, lsy-27, is mutated, as revealed by whole-genome sequencing. lsy-27 is broadly and exclusively expressed in the embryo and acts during the initiation, but not during the maintenance phase of ASE asymmetry control to assist in the initiation of lim-6 expression.
PMID: 21555395 [PubMed - indexed for MEDLINE]
Maintaining a memory by transcriptional autoregulation.
Curr Biol. 2011 Feb 22;21(4):R146-7
One of the key features of cellular differentiation programs is stability. Although differentiation is reversible in principle, many components of the gene batteries induced upon terminal differentiation are maintained throughout a cell's life. For example, muscle cells continuously express the myosin gene, and GABAergic neurons continuously express genes for GABA synthesis and transport. Maintaining gene expression patterns in the nervous system is a particular challenge given the non-renewing nature and therefore extensive life span of many neuronal cell types.
PMID: 21334290 [PubMed - indexed for MEDLINE]
Direct conversion of C. elegans germ cells into specific neuron types.
Science. 2011 Jan 21;331(6015):304-8
Authors: Tursun B, Patel T, Kratsios P, Hobert O
The ability of transcription factors to directly reprogram the identity of cell types is usually restricted and is defined by cellular context. Through the ectopic expression of single Caenorhabditis elegans transcription factors, we found that the identity of mitotic germ cells can be directly converted into that of specific neuron types: glutamatergic, cholinergic, or GABAergic. This reprogramming event requires the removal of the histone chaperone LIN-53 (RbAp46/48 in humans), a component of several histone remodeling and modifying complexes, and this removal can be mimicked by chemical inhibition of histone deacetylases. Our findings illustrate the ability of germ cells to be directly converted into individual, terminally differentiated neuron types and demonstrate that a specific chromatin factor provides a barrier for cellular reprogramming.
PMID: 21148348 [PubMed - indexed for MEDLINE]
Developmental control of lateralized neuron size in the nematode Caenorhabditis elegans.
Neural Dev. 2010;5:33
Authors: Goldsmith AD, Sarin S, Lockery S, Hobert O
BACKGROUND: Nervous systems are generally bilaterally symmetric on a gross structural and organizational level but are strongly lateralized (left/right asymmetric) on a functional level. It has been previously noted that in vertebrate nervous systems, symmetrically positioned, bilateral groups of neurons in functionally lateralized brain regions differ in the size of their soma. The genetic mechanisms that control these left/right asymmetric soma size differences are unknown. The nematode Caenorhabditis elegans offers the opportunity to study this question with single neuron resolution. A pair of chemosensory neurons (ASEL and ASER), which are bilaterally symmetric on several levels (projections, synaptic connectivity, gene expression patterns), are functionally lateralized in that they express distinct chemoreceptors and sense distinct chemosensory cues.
RESULTS: We describe here that ASEL and ASER also differ substantially in size (soma volume, axonal and dendritic diameter), a feature that is predicted to change the voltage conduction properties of the two sensory neurons. This difference in size is not dependent on sensory input or neuronal activity but developmentally programmed by a pathway of gene regulatory factors that also control left/right asymmetric chemoreceptor expression of the two ASE neurons. This regulatory pathway funnels via the DIE-1 Zn finger transcription factor into the left/right asymmetric distribution of nucleoli that contain the rRNA regulator Fibrillarin/FIB-1, a RNA methyltransferase implicated in the non-hereditary immune disease scleroderma, which we find to be essential to establish the size differences between ASEL and ASER.
CONCLUSIONS: Taken together, our findings reveal a remarkable conservation of the linkage of functional lateralization with size differences across phylogeny and provide the first insights into the developmentally programmed regulatory mechanisms that control neuron size lateralities.
PMID: 21122110 [PubMed - indexed for MEDLINE]
C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy.
PLoS One. 2010;5(11):e15435
Authors: Doitsidou M, Poole RJ, Sarin S, Bigelow H, Hobert O
Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.
PMID: 21079745 [PubMed - indexed for MEDLINE]
The neurexin superfamily of Caenorhabditis elegans.
Gene Expr Patterns. 2011 Jan-Feb;11(1-2):144-50
Authors: Haklai-Topper L, Soutschek J, Sabanay H, Scheel J, Hobert O, Peles E
The neurexin superfamily is a group of transmembrane molecules mediating cell-cell contacts and generating specialized membranous domains in polarized epithelial and nerves cells. We describe here the domain organization and expression of the entire, core neurexin superfamily in the nematode Caenorhabditis elegans, which is composed of three family members. One of the superfamily members, nrx-1, is an ortholog of vertebrate neurexin, the other two, itx-1 and nlr-1, are orthologs of the Caspr subfamily of neurexin-like genes. Based on reporter gene analysis, we find that nrx-1 is exclusively expressed in most if not all cells of the nervous system and localizes to presynaptic specializations. itx-1 and nrx-1 reporter genes are expressed in non-overlapping patterns within and outside the nervous system. ITX-1 protein co-localizes with β-G-spectrin to a subapical domain within intestinal cells. These studies provide a starting point for further functional analysis of this family of proteins.
PMID: 21055481 [PubMed - indexed for MEDLINE]
Maintenance of neuronal laterality in Caenorhabditis elegans through MYST histone acetyltransferase complex components LSY-12, LSY-13 and LIN-49.
Genetics. 2010 Dec;186(4):1497-502
Authors: O'Meara MM, Zhang F, Hobert O
Left/right asymmetrically expressed genes permit an animal to perform distinct tasks with the right vs. left side of its brain. Once established during development, lateralized gene expression patterns need to be maintained during the life of the animal. We show here that a histone modifying complex, composed of the LSY-12 MYST-type histone acetyltransferase, the ING-family PHD domain protein LSY-13, and PHD/bromodomain protein LIN-49, is required to first initiate and then actively maintain lateralized gene expression in the gustatory system of the nematode Caenorhabditis elegans. Similar defects are observed upon postembryonic removal of two C2H2 zinc finger transcription factors, die-1 and che-1, demonstrating that a combination of transcription factors, which recognize DNA in a sequence-specific manner, and a histone modifying enzyme complex are responsible for inducing and maintaining neuronal laterality.
PMID: 20923973 [PubMed - indexed for MEDLINE]
Neurogenesis in the nematode Caenorhabditis elegans.
The nervous system represents the most complex tissue of C. elegans both in terms of numbers (302 neurons and 56 glial cells = 37% of the somatic cells in a hermaphrodite) and diversity (118 morphologically distinct neuron classes). The lineage and morphology of each neuron type has been described in detail and neuronal fate markers exists for virtually all neurons in the form of fluorescent reporter genes. The ability to "phenotype" neurons at high resolution combined with the amenability of C. elegans to genetic mutant analysis make the C. elegans nervous system a prime model system to elucidate the nature of the gene regulatory programs that build a nervous system-a central question of developmental neurobiology. Discussing a number of regulatory genes involved in neuronal lineage determination and neuronal differentiation, I will try to carve out in this review a few general principles of neuronal development in C. elegans. These principles may be conserved across phylogeny.
PMID: 20891032 [PubMed - indexed for MEDLINE]
Gene regulation: enhancers stepping out of the shadow.
Curr Biol. 2010 Sep 14;20(17):R697-9
The expression of many animal genes has been shown to be controlled by two--rather than one--enhancers with similar regulatory content. Such enhancer redundancy ensures robustness of gene expression under adverse environmental or genetic conditions.
PMID: 20833307 [PubMed - indexed for MEDLINE]
The molecular and gene regulatory signature of a neuron.
Trends Neurosci. 2010 Oct;33(10):435-45
Authors: Hobert O, Carrera I, Stefanakis N
Neuron-type specific gene batteries define the morphological and functional diversity of cell types in the nervous system. Here, we discuss the composition of neuron-type specific gene batteries and illustrate gene regulatory strategies which determine the unique gene expression profiles and molecular composition of individual neuronal cell types from C. elegans to higher vertebrates. Based on principles learned from prokaryotic gene regulation, we argue that neuronal terminal gene batteries are functionally grouped into parallel-acting 'regulons'. The theoretical concepts discussed here provide testable hypotheses for future experimental analysis of the exact gene-regulatory mechanisms employed in the generation of neuronal diversity and identity.
PMID: 20663572 [PubMed - indexed for MEDLINE]
Lineage programming: navigating through transient regulatory states via binary decisions.
Curr Opin Genet Dev. 2010 Aug;20(4):362-8
Authors: Bertrand V, Hobert O
Lineage-based mechanisms are widely used to generate cell type diversity in both vertebrates and invertebrates. For the past few decades, the nematode Caenorhabditis elegans has served as a primary model system to study this process because of its fixed and well-characterized cell lineage. Recent studies conducted at the level of single cells and individual cis-regulatory elements suggest a general model by which cellular diversity is generated in this organism. During its developmental history a cell passes through multiple transient regulatory states characterized by the expression of specific sets of transcription factors. The transition from one state to another is driven by a general binary decision mechanism acting at each successive division in a reiterative manner and ending up with the activation of the terminal differentiation program upon terminal division. A similar cell fate specification system seems to play a role in generating cellular diversity in the nervous system of more complex organisms such as Drosophila and vertebrates.
PMID: 20537527 [PubMed - indexed for MEDLINE]
Analysis of multiple ethyl methanesulfonate-mutagenized Caenorhabditis elegans strains by whole-genome sequencing.
Genetics. 2010 Jun;185(2):417-30
Authors: Sarin S, Bertrand V, Bigelow H, Boyanov A, Doitsidou M, Poole RJ, Narula S, Hobert O
Whole-genome sequencing (WGS) of organisms displaying a specific mutant phenotype is a powerful approach to identify the genetic determinants of a plethora of biological processes. We have previously validated the feasibility of this approach by identifying a point-mutated locus responsible for a specific phenotype, observed in an ethyl methanesulfonate (EMS)-mutagenized Caenorhabditis elegans strain. Here we describe the genome-wide mutational profile of 17 EMS-mutagenized genomes as assessed with a bioinformatic pipeline, called MAQGene. Surprisingly, we find that while outcrossing mutagenized strains does reduce the total number of mutations, a striking mutational load is still observed even in outcrossed strains. Such genetic complexity has to be taken into account when establishing a causative relationship between genotype and phenotype. Even though unintentional, the 17 sequenced strains described here provide a resource of allelic variants in almost 1000 genes, including 62 premature stop codons, which represent candidate knockout alleles that will be of further use for the C. elegans community to study gene function.
PMID: 20439776 [PubMed - indexed for MEDLINE]
The Groucho ortholog UNC-37 interacts with the short Groucho-like protein LSY-22 to control developmental decisions in C. elegans.
Development. 2010 Jun;137(11):1799-805
Authors: Flowers EB, Poole RJ, Tursun B, Bashllari E, Pe'er I, Hobert O
Transcriptional co-repressors of the Groucho/TLE family are important regulators of development in many species. A subset of Groucho/TLE family members that lack the C-terminal WD40 domains have been proposed to act as dominant-negative regulators of Groucho/TLE proteins, yet such a role has not been conclusively proven. Through a mutant screen for genes controlling a left/right asymmetric cell fate decision in the nervous system of the nematode C. elegans, we have retrieved loss-of-function alleles in two distinct loci that display identical phenotypes in neuronal fate specification and in other developmental contexts. Using the novel technology of whole-genome sequencing, we find that these loci encode the C. elegans ortholog of Groucho, UNC-37, and, surprisingly, a short Groucho-like protein, LSY-22, that is similar to truncated Groucho proteins in other species. Besides their phenotypic similarities, unc-37 and lsy-22 show genetic interactions and UNC-37 and LSY-22 proteins also physically bind to each other in vivo. Our findings suggest that rather than acting as negative regulators of Groucho, small Groucho-like proteins may promote Groucho function. We propose that Groucho-mediated gene regulatory events involve heteromeric complexes of distinct Groucho-like proteins.
PMID: 20431118 [PubMed - indexed for MEDLINE]
Hypoxia activates a latent circuit for processing gustatory information in C. elegans.
Nat Neurosci. 2010 May;13(5):610-4
Authors: Pocock R, Hobert O
Dedicated neuronal circuits enable animals to engage in specific behavioral responses to environmental stimuli. We found that hypoxic stress enhanced gustatory sensory perception via previously unknown circuitry in Caenorhabditis elegans. The hypoxia-inducible transcription factor HIF-1 upregulated serotonin (5-HT) expression in specific sensory neurons that are not normally required for chemosensation. 5-HT subsequently promoted hypoxia-enhanced sensory perception by signaling through the metabotropic G protein-coupled receptor SER-7 in an unusual peripheral neuron, the M4 motor neuron. M4 relayed this information back into the CNS via the FMRFamide-related neuropeptide FLP-21 and its cognate receptor, NPR-1. Thus, physiological detection of hypoxia results in the activation of an additional, previously unrecognized circuit for processing sensory information that is not required for sensory processing under normoxic conditions.
PMID: 20400959 [PubMed - indexed for MEDLINE]
Questions over the scientific basis of epigenome project.
Nature. 2010 Mar 25;464(7288):487
Authors: Ptashne M, Hobert O, Davidson E
PMID: 20336114 [PubMed - indexed for MEDLINE]
Curr Opin Neurobiol. 2010 Feb;20(1):2-5
Authors: Guillemot F, Hobert O
PMID: 20149634 [PubMed - indexed for MEDLINE]
The impact of whole genome sequencing on model system genetics: get ready for the ride.
Genetics. 2010 Feb;184(2):317-9
Much of our understanding of how organisms develop and function is derived from the extraordinarily powerful, classic approach of screening for mutant organisms in which a specific biological process is disrupted. Reaping the fruits of such forward genetic screens in metazoan model systems like Drosophila, Caenorhabditis elegans, or zebrafish traditionally involves time-consuming positional cloning strategies that result in the identification of the mutant locus. Whole genome sequencing (WGS) has begun to provide an effective alternative to this approach through direct pinpointing of the molecular lesion in a mutated strain isolated from a genetic screen. Apart from significantly altering the pace and costs of genetic analysis, WGS also provides new perspectives on solving genetic problems that are difficult to tackle with conventional approaches, such as identifying the molecular basis of multigenic and complex traits.
PMID: 20103786 [PubMed - indexed for MEDLINE]
Neuron-type specific regulation of a 3'UTR through redundant and combinatorially acting cis-regulatory elements.
RNA. 2010 Feb;16(2):349-63
Authors: Didiano D, Cochella L, Tursun B, Hobert O
3' Untranslated region (UTR)-dependent post-transcriptional regulation has emerged as a critical mechanism of controlling gene expression in various physiological contexts, including cellular differentiation events. Here, we examine the regulation of the 3'UTR of the die-1 transcription factor in a single neuron of the nematode C. elegans. This 3'UTR shows the intriguing feature of being differentially regulated across the animal's left/right axis. In the left gustatory neuron, ASEL, in which DIE-1 protein is normally expressed in adult animals, the 3'UTR confers no regulatory information, while in the right gustatory neuron, ASER, where DIE-1 is normally not expressed, this 3'UTR confers negative regulatory information. Here, we systematically analyze the cis-regulatory architecture of the die-1 3'UTR using a transgenic, in vivo assay system. Through extensive mutagenesis and sequence insertions into heterologous 3'UTR contexts, we describe three 25-base-pair (bp) sequence elements that are both required and sufficient to mediate the ASER-specific down-regulation of the die-1 3'UTR. These three 25-bp sequence elements operate in both a redundant and combinatorial manner. Moreover, there are not only redundant elements within the die-1 3'UTR regulating its left/right asymmetric activity but asymmetric 3'UTR regulation is itself redundant with other regulatory mechanisms to achieve asymmetric DIE-1 protein expression and function in ASEL versus ASER. The features of 3'UTR regulation we describe here may apply to some of the vast number of genes in animal genomes whose expression is predicted to be regulated through their 3'UTR.
PMID: 20040592 [PubMed - indexed for MEDLINE]
The small, secreted immunoglobulin protein ZIG-3 maintains axon position in Caenorhabditis elegans.
Genetics. 2009 Nov;183(3):917-27
Authors: Bénard C, Tjoe N, Boulin T, Recio J, Hobert O
Vertebrate and invertebrate genomes contain scores of small secreted or transmembrane proteins with two immunoglobulin (Ig) domains. Many of them are expressed in the nervous system, yet their function is not well understood. We analyze here knockout alleles of all eight members of a family of small secreted or transmembrane Ig domain proteins, encoded by the Caenorhabditis elegans zig ("zwei Ig Domänen") genes. Most of these family members display the unusual feature of being coexpressed in a single neuron, PVT, whose axon is located along the ventral midline of C. elegans. One of these genes, zig-4, has previously been found to be required for maintaining axon position postembryonically in the ventral nerve cord of C. elegans. We show here that loss of zig-3 function results in similar postdevelopmental axon maintenance defects. The maintenance function of both zig-3 and zig-4 serves to counteract mechanical forces that push axons around, as well as various intrinsic attractive forces between axons that cause axon displacement if zig genes like zig-3 or zig-4 are deleted. Even though zig-3 is expressed only in a limited number of neurons, including PVT, transgenic rescue experiments show that zig-3 can function irrespective of which cell or tissue type it is expressed in. Double mutant analysis shows that zig-3 and zig-4 act together to affect axon maintenance, yet they are not functionally interchangeable. Both genes also act together with other, previously described axon maintenance factors, such as the Ig domain proteins DIG-1 and SAX-7, the C. elegans ortholog of the human L1 protein. Our studies shed further light on the use of dedicated factors to maintain nervous system architecture and corroborate the complexity of the mechanisms involved.
PMID: 19737747 [PubMed - indexed for MEDLINE]
The C. elegans Tailless/TLX transcription factor nhr-67 controls neuronal identity and left/right asymmetric fate diversification.
Development. 2009 Sep;136(17):2933-44
Authors: Sarin S, Antonio C, Tursun B, Hobert O
An understanding of the molecular mechanisms of cell fate determination in the nervous system requires the elucidation of transcriptional regulatory programs that ultimately control neuron-type-specific gene expression profiles. We show here that the C. elegans Tailless/TLX-type, orphan nuclear receptor NHR-67 acts at several distinct steps to determine the identity and subsequent left/right (L/R) asymmetric subtype diversification of a class of gustatory neurons, the ASE neurons. nhr-67 controls several broad aspects of sensory neuron development and, in addition, triggers the expression of a sensory neuron-type-specific selector gene, che-1, which encodes a zinc-finger transcription factor. Subsequent to its induction of overall ASE fate, nhr-67 diversifies the fate of the two ASE neurons ASEL and ASER across the L/R axis by promoting ASER and inhibiting ASEL fate. This function is achieved through direct expression activation by nhr-67 of the Nkx6-type homeobox gene cog-1, an inducer of ASER fate, that is inhibited in ASEL through the miRNA lsy-6. Besides controlling bilateral and asymmetric aspects of ASE development, nhr-67 is also required for many other neurons of diverse lineage history and function to appropriately differentiate, illustrating the broad and diverse use of this type of transcription factor in neuronal development.
PMID: 19641012 [PubMed - indexed for MEDLINE]
MAQGene: software to facilitate C. elegans mutant genome sequence analysis.
Nat Methods. 2009 Aug;6(8):549
Authors: Bigelow H, Doitsidou M, Sarin S, Hobert O
PMID: 19620971 [PubMed - indexed for MEDLINE]
Wnt asymmetry and the terminal division of neuronal progenitors.
Cell Cycle. 2009 Jul 1;8(13):1973-4
PMID: 19550137 [PubMed - indexed for MEDLINE]
Lateralized gustatory behavior of C. elegans is controlled by specific receptor-type guanylyl cyclases.
Curr Biol. 2009 Jun 23;19(12):996-1004
Authors: Ortiz CO, Faumont S, Takayama J, Ahmed HK, Goldsmith AD, Pocock R, McCormick KE, Kunimoto H, Iino Y, Lockery S, Hobert O
BACKGROUND: Even though functional lateralization is a common feature of many nervous systems, it is poorly understood how lateralized neural function is linked to lateralized gene activity. A bilaterally symmetric pair of C. elegans gustatory neurons, ASEL and ASER, senses a number of chemicals in a left/right asymmetric manner and therefore serves as a model to study the genetic basis of functional lateralization. The extent of functional lateralization of the ASE neurons and genes responsible for the left/right asymmetric activity of ASEL and ASER is unknown.
RESULTS: We show here that a substantial number of salt ions are sensed in a left/right asymmetric manner and that lateralized salt responses allow the worm to discriminate between distinct salt cues. To identify molecules that may be involved in sensing salt ions and/or transmitting such sensory information, we examined the chemotaxis behavior of animals harboring mutations in eight different receptor-type, transmembrane guanylyl cyclases (encoded by gcy genes), which are expressed in either ASEL (gcy-6, gcy-7, gcy-14), ASER (gcy-1, gcy-4, gcy-5, gcy-22), or ASEL and ASER (gcy-19). Disruption of a particular ASER-expressed gcy gene, gcy-22, results in a broad chemotaxis defect to nearly all salts sensed by ASER, as well as to a left/right asymmetrically sensed amino acid. In contrast, disruption of other gcy genes resulted in highly salt ion-specific chemosensory defects.
CONCLUSIONS: Our findings broaden our understanding of lateralities in neural function, provide insights into how this laterality is molecularly encoded, and reveal an unusual multitude of molecules involved in gustatory signal transduction.
PMID: 19523832 [PubMed - indexed for MEDLINE]
Looking beyond development: maintaining nervous system architecture.
Curr Top Dev Biol. 2009;87:175-94
Authors: Bénard C, Hobert O
Neuronal circuitries established in development must persist throughout life. This poses a serious challenge to the structural integrity of an embryonically patterned nervous system as an animal dramatically increases its size postnatally, remodels parts of its anatomy, and incorporates new neurons. In addition, body movements, injury, and ageing generate physical stress on the nervous system. Specific molecular pathways maintain intrinsic properties of neurons in the mature nervous system. Other factors ensure that the overall organization of entire neuronal ensembles into ganglia and fascicles is appropriately maintained upon external challenges. Here, we discuss different molecules underlying these neuronal maintenance mechanisms, with a focus on lessons learned from the nematode Caenorhabditis elegans.
PMID: 19427520 [PubMed - indexed for MEDLINE]
Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans.
Dev Cell. 2009 Apr;16(4):563-75
How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. We establish here a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/beta-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. Our study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/beta-catenin pathway is widely used to control the identity of neuronal lineages.
PMID: 19386265 [PubMed - indexed for MEDLINE]
Gene regulatory logic of dopamine neuron differentiation.
Nature. 2009 Apr 16;458(7240):885-9
Dopamine signalling regulates a variety of complex behaviours, and defects in dopamine neuron function or survival result in severe human pathologies, such as Parkinson's disease. The common denominator of all dopamine neurons is the expression of dopamine pathway genes, which code for a set of phylogenetically conserved proteins involved in dopamine synthesis and transport. Gene regulatory mechanisms that result in the direct activation of dopamine pathway genes and thereby ultimately determine the identity of dopamine neurons are poorly understood in all systems studied so far. Here we show that a simple cis-regulatory element, the dopamine (DA) motif, controls the expression of all dopamine pathway genes in all dopaminergic cell types in Caenorhabditis elegans. The DA motif is activated by the ETS transcription factor AST-1. Loss of ast-1 results in the failure of all distinct dopaminergic neuronal subtypes to terminally differentiate. Ectopic expression of ast-1 is sufficient to activate the dopamine pathway in some cellular contexts. Vertebrate dopamine pathway genes also contain phylogenetically conserved DA motifs that can be activated by the mouse ETS transcription factor Etv1 (also known as ER81), and a specific class of dopamine neurons fails to differentiate in mice lacking Etv1. Moreover, ectopic Etv1 expression induces dopaminergic fate marker expression in neuronal primary cultures. Mouse Etv1 can also functionally substitute for ast-1 in C. elegans. Our studies reveal a simple and apparently conserved regulatory logic of dopamine neuron terminal differentiation and may provide new entry points into the diagnosis or therapy of conditions in which dopamine neurons are defective.
PMID: 19287374 [PubMed - indexed for MEDLINE]
A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.
PLoS One. 2009;4(3):e4625
Authors: Tursun B, Cochella L, Carrera I, Hobert O
Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination ("recombineering") represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5' and 3' cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.
PMID: 19259264 [PubMed - indexed for MEDLINE]
Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro.
Authors: Tung JJ, Hobert O, Berryman M, Kitajewski J
New capillaries are formed through angiogenesis and an integral step in this process is endothelial tubulogenesis. The molecular mechanisms driving tube formation during angiogenesis are not yet delineated. Recently, the chloride intracellular channel 4 (CLIC4)-orthologue EXC-4 was found to be necessary for proper development and maintenance of the Caenorhabditis elegans excretory canal, implicating CLIC4 as a regulator of tubulogenesis. Here, we studied the role of CLIC4 in angiogenesis and endothelial tubulogenesis. We report the effects of inhibiting or inducing CLIC4 expression on distinct aspects of endothelial cell behavior in vitro. Our experiments utilized RNA interference to establish cultured human endothelial cell lines with significant reduction of CLIC4 expression, and a CLIC4-expressing lentiviral plasmid was used to establish CLIC4 overexpression in endothelial cells. We observed no effect on cell migration and a modest effect on cell survival. Reduced CLIC4 expression decreased cell proliferation, capillary network formation, capillary-like sprouting, and lumen formation. This suggests that normal endogenous CLIC4 expression is required for angiogenesis and tubulogenesis. Accordingly, increased CLIC4 expression promoted proliferation, network formation, capillary-like sprouting, and lumen formation. We conclude that CLIC4 functions to promote endothelial cell proliferation and to regulate endothelial morphogenesis, and is thus involved in multiple steps of in vitro angiogenesis.
PMID: 19247789 [PubMed - indexed for MEDLINE]
Cis-regulatory mutations in the Caenorhabditis elegans homeobox gene locus cog-1 affect neuronal development.
Genetics. 2009 Apr;181(4):1679-86
Authors: O'Meara MM, Bigelow H, Flibotte S, Etchberger JF, Moerman DG, Hobert O
We apply here comparative genome hybridization as a novel tool to identify the molecular lesion in two Caenorhabditis elegans mutant strains that affect a neuronal cell fate decision. The phenotype of the mutant strains resembles those of the loss-of-function alleles of the cog-1 homeobox gene, an inducer of the fate of the gustatory neuron ASER. We find that both lesions map to the cis-regulatory control region of cog-1 and affect a phylogenetically conserved binding site for the C2H2 zinc-finger transcription factor CHE-1, a previously known regulator of cog-1 expression in ASER. Identification of this CHE-1-binding site as a critical regulator of cog-1 expression in the ASER in vivo represents one of the rare demonstrations of the in vivo relevance of an experimentally determined or predicted transcription-factor-binding site. Aside from the mutationally defined CHE-1-binding site, cog-1 contains a second, functional CHE-1-binding site, which in isolation is sufficient to drive reporter gene expression in the ASER but in an in vivo context is apparently insufficient for promoting appropriate ASER expression. The cis-regulatory control regions of other ASE-expressed genes also contain ASE motifs that can promote ASE neuron expression when isolated from their genomic context, but appear to depend on multiple ASE motifs in their normal genomic context. The multiplicity of cis-regulatory elements may ensure the robustness of gene expression.
PMID: 19189954 [PubMed - indexed for MEDLINE]
Comparing platforms for C. elegans mutant identification using high-throughput whole-genome sequencing.
PLoS One. 2008;3(12):e4012
Authors: Shen Y, Sarin S, Liu Y, Hobert O, Pe'er I
BACKGROUND: Whole-genome sequencing represents a promising approach to pinpoint chemically induced mutations in genetic model organisms, thereby short-cutting time-consuming genetic mapping efforts.
PRINCIPAL FINDINGS: We compare here the ability of two leading high-throughput platforms for paired-end deep sequencing, SOLiD (ABI) and Genome Analyzer (Illumina; "Solexa"), to achieve the goal of mutant detection. As a test case we used a mutant C. elegans strain that harbors a mutation in the lsy-12 locus which we compare to the reference wild-type genome sequence. We analyzed the accuracy, sensitivity, and depth-coverage characteristics of the two platforms. Both platforms were able to identify the mutation that causes the phenotype of the mutant C. elegans strain, lsy-12. Based on a 4 MB genomic region in which individual variants were validated by Sanger sequencing, we observe tradeoffs between rates of false positives and false negatives when using both platforms under similar coverage and mapping criteria.
SIGNIFICANCE: In conclusion, whole-genome sequencing conducted by either platform is a viable approach for the identification of single-nucleotide variations in the C. elegans genome.
PMID: 19107202 [PubMed - indexed for MEDLINE]
Regulatory logic of neuronal diversity: terminal selector genes and selector motifs.
Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20067-71
Individual neuronal cell types are defined by the expression of unique batteries of terminal differentiation genes. The elucidation of the cis-regulatory architecture of several distinct, single neuron type-specific gene batteries in Caenorhabditis elegans has revealed a strikingly simple cis-regulatory logic, in which small cis-regulatory motifs are activated in postmitotic neurons by autoregulating transcription factors (TFs). Loss of the TFs results in the loss of the identity of the individual neuron type. I propose to term these TFs "terminal selector genes" and their cognate cis-regulatory target sites "terminal selector motifs." Terminal selector genes assign individual neuronal identities by directly controlling the expression of downstream, terminal differentiation genes and act in specific regulatory network configurations. The simplicity of the cis-regulatory logic on which the terminal selector gene concept is based may contribute to the evolvability of neuronal diversity.
PMID: 19104055 [PubMed - indexed for MEDLINE]
Extracellular sugar modifications provide instructive and cell-specific information for axon-guidance choices.
Curr Biol. 2008 Dec 23;18(24):1978-85
Authors: Bülow HE, Tjoe N, Townley RA, Didiano D, van Kuppevelt TH, Hobert O
Heparan sulfates (HSs) are extraordinarily complex extracellular sugar molecules that are critical components of multiple signaling systems controlling neuronal development. The molecular complexity of HSs arises through a series of specific modifications, including sulfations of sugar residues and epimerizations of their glucuronic acid moieties. The modifications are introduced nonuniformly along protein-attached HS polysaccharide chains by specific enzymes. Genetic analysis has demonstrated the importance of specific HS-modification patterns for correct neuronal development. However, it remains unclear whether HS modifications provide a merely permissive substrate or whether they provide instructive patterning information during development. We show here with single-cell resolution that highly stereotyped motor axon projections in C. elegans depend on specific HS-modification patterns. By manipulating extracellular HS-modification patterns, we can cell specifically reroute axons, indicating that HS modifications are instructive. This axonal rerouting is dependent on the HS core protein lon-2/glypican and both the axon guidance cue slt-1/Slit and its receptor eva-1. These observations suggest that a changed sugar environment instructs slt-1/Slit-dependent signaling via eva-1 to redirect axons. Our experiments provide genetic in vivo evidence for the "HS code" hypothesis which posits that specific combinations of HS modifications provide specific and instructive information to mediate the specificity of ligand/receptor interactions.
PMID: 19062279 [PubMed - indexed for MEDLINE]
Cis-regulatory mechanisms of left/right asymmetric neuron-subtype specification in C. elegans.
Development. 2009 Jan;136(1):147-60
Authors: Etchberger JF, Flowers EB, Poole RJ, Bashllari E, Hobert O
Anatomically and functionally defined neuron types are sometimes further classified into individual subtypes based on unique functional or molecular properties. To better understand how developmental programs controlling neuron type specification are mechanistically linked to programs controlling neuronal subtype specification, we have analyzed a neuronal subtype specification program that occurs across the left/right axis in the nervous system of the nematode C. elegans. A terminal selector transcription factor, CHE-1, is required for the specification of the ASE neuron class, and a gene regulatory feedback loop of transcription factors and miRNAs is required to diversify the two ASE neurons into an asymmetric left and right subtype (ASEL and ASER). However, the link between the CHE-1-dependent ASE neuron class specification and the ensuing left-right subtype specification program is poorly understood. We show here that CHE-1 has genetically separable functions in controlling bilaterally symmetric ASE neuron class specification and the ensuing left-right subtype specification program. Both neuron class specification and asymmetric subclass specification depend on CHE-1-binding sites (;ASE motifs') in symmetrically and asymmetrically expressed target genes, but in the case of asymmetrically expressed target genes, the activity of the ASE motif is modulated through a diverse set of additional cis-regulatory elements. Depending on the target gene, these cis-regulatory elements either promote or inhibit the activity of CHE-1. The activity of these L/R asymmetric cis-regulatory elements is indirectly controlled by che-1 itself, revealing a feed-forward loop configuration in which che-1 restricts its own activity. Relative binding affinity of CHE-1 to ASE motifs also depends on whether a gene is expressed bilaterally or in a left/right asymmetric manner. Our analysis provides insights into the molecular mechanisms of neuronal subtype specification, demonstrating that the activity of a neuron type-specific selector gene is modulated by a variety of distinct means to diversify individual neuron classes into specific subclasses. It also suggests that feed-forward loop motifs may be a prominent feature of neuronal diversification events.
PMID: 19060335 [PubMed - indexed for MEDLINE]
Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans.
Nat Methods. 2008 Oct;5(10):869-72
Authors: Doitsidou M, Flames N, Lee AC, Boyanov A, Hobert O
We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.
PMID: 18758453 [PubMed - indexed for MEDLINE]
Caenorhabditis elegans mutant allele identification by whole-genome sequencing.
Nat Methods. 2008 Oct;5(10):865-7
Authors: Sarin S, Prabhu S, O'Meara MM, Pe'er I, Hobert O
Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.
PMID: 18677319 [PubMed - indexed for MEDLINE]
Oxygen levels affect axon guidance and neuronal migration in Caenorhabditis elegans.
Nat Neurosci. 2008 Aug;11(8):894-900
Oxygen deprivation can cause severe defects in human brain development, yet the precise cellular and molecular consequences of varying oxygen levels on nervous system development are unknown. We found that hypoxia caused specific axon pathfinding and neuronal migration defects in C. elegans that result from the stabilization of the transcription factor HIF-1 (hypoxia-inducible factor 1) in neurons and muscle. Stabilization of HIF-1 through removal of the proteasomal HIF-1 degradatory pathway phenocopies the hypoxia-induced neuronal defects. Hypoxia-mediated defects in nervous system development depended on signaling through the insulin-like receptor DAF-2, which serves to control the level of reactive oxygen species that also affects axon pathfinding. Hypoxia exerted its effect on axon pathfinding, at least in part, through HIF-1-dependent regulation of the Eph receptor VAB-1. HIF-1-mediated upregulation of VAB-1 protected embryos from hypoxia-induced lethality, but increased VAB-1 levels elicited aberrant axon pathfinding. Similar genetic pathways may cause aberrant human brain development under hypoxic conditions.
PMID: 18587389 [PubMed - indexed for MEDLINE]
Molecular architecture of a miRNA-regulated 3' UTR.
RNA. 2008 Jul;14(7):1297-317
Authors: Didiano D, Hobert O
Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.
PMID: 18463285 [PubMed - indexed for MEDLINE]
Gene regulation by transcription factors and microRNAs.
Science. 2008 Mar 28;319(5871):1785-6
The properties of a cell are determined by the genetic information encoded in its genome. Understanding how such information is differentially and dynamically retrieved to define distinct cell types and cellular states is a major challenge facing molecular biology. Gene regulatory factors that control the expression of genomic information come in a variety of flavors, with transcription factors and microRNAs representing the most numerous gene regulatory factors in multicellular genomes. Here, I review common principles of transcription factor- and microRNA-mediated gene regulatory events and discuss conceptual differences in how these factors control gene expression.
PMID: 18369135 [PubMed - indexed for MEDLINE]
Vector-free DNA constructs improve transgene expression in C. elegans.
Nat Methods. 2008 Jan;5(1):3
Authors: Etchberger JF, Hobert O
PMID: 18165801 [PubMed - indexed for MEDLINE]
Functional dissection of the C. elegans cell adhesion molecule SAX-7, a homologue of human L1.
Mol Cell Neurosci. 2008 Jan;37(1):56-68
Authors: Pocock R, Bénard CY, Shapiro L, Hobert O
Cell adhesion molecules of the Immunoglobulin superfamily (IgCAMs) play important roles in neuronal development, homeostasis and disease. Here, we use an animal in vivo assay system to study the function of sax-7, the Caenorhabditis elegans homologue of the human L1 IgCAM, a homophilic adhesion molecule involved in several neurological diseases. We show that the 6 Ig/5 FnIII domain protein SAX-7 acts autonomously in the nervous system to maintain axon position in the ventral nerve cord of the nematode. As previously reported, sax-7 is also required to maintain the relative positioning of neuronal cell bodies in several head ganglia. We use the loss of cellular adhesiveness in sax-7 null mutants as an assay system to investigate the contribution of individual domains and sequence motifs to the function of SAX-7, utilizing transgenic rescue approaches. By shortening the hinge region between the Ig1+2 and Ig3+4 domains, we improve the adhesive function of SAX-7, thereby providing support for a previously proposed autoinhibitory "horseshoe" conformation of IgCAMs. However, we find that Ig3+4 are the only Ig domains required and sufficient for the adhesive function of SAX-7. Previous models of L1-type IgCAMs that invoke an important role of the first two Ig domains in controlling adhesion therefore do not appear to apply to SAX-7. Moreover, we find that neither the 5 FnIII domains, nor the protease cleavage site embedded in them, are required for the adhesive function of SAX-7. Lastly, we show that of the several protein binding motifs present in the intracellular region of SAX-7, only its ankyrin binding motif is required and also solely sufficient to confer the adhesive functions of SAX-7.
PMID: 17933550 [PubMed - indexed for MEDLINE]
miRNAs play a tune.
Cell. 2007 Oct 5;131(1):22-4
Two new studies describe functionally relevant interactions between microRNAs (miRNAs) and their targets in the immune system and the brain (Xiao et al., 2007; Karres et al., 2007). Furthermore, these studies illustrate the involvement of miRNAs in tuning the expression of target genes to physiologically relevant levels.
PMID: 17923083 [PubMed - indexed for MEDLINE]
Genetic screens for Caenorhabditis elegans mutants defective in left/right asymmetric neuronal fate specification.
Genetics. 2007 Aug;176(4):2109-30
Authors: Sarin S, O'Meara MM, Flowers EB, Antonio C, Poole RJ, Didiano D, Johnston RJ, Chang S, Narula S, Hobert O
We describe here the results of genetic screens for Caenorhabditis elegans mutants in which a single neuronal fate decision is inappropriately executed. In wild-type animals, the two morphologically bilaterally symmetric gustatory neurons ASE left (ASEL) and ASE right (ASER) undergo a left/right asymmetric diversification in cell fate, manifested by the differential expression of a class of putative chemoreceptors and neuropeptides. Using single cell-specific gfp reporters and screening through a total of almost 120,000 haploid genomes, we isolated 161 mutants that define at least six different classes of mutant phenotypes in which ASEL/R fate is disrupted. Each mutant phenotypic class encompasses one to nine different complementation groups. Besides many alleles of 10 previously described genes, we have identified at least 16 novel "lsy" genes ("laterally symmetric"). Among mutations in known genes, we retrieved four alleles of the miRNA lsy-6 and a gain-of-function mutation in the 3'-UTR of a target of lsy-6, the cog-1 homeobox gene. Using newly found temperature-sensitive alleles of cog-1, we determined that a bistable feedback loop controlling ASEL vs. ASER fate, of which cog-1 is a component, is only transiently required to initiate but not to maintain ASEL and ASER fate. Taken together, our mutant screens identified a broad catalog of genes whose molecular characterization is expected to provide more insight into the complex genetic architecture of a left/right asymmetric neuronal cell fate decision.
PMID: 17717195 [PubMed - indexed for MEDLINE]
The molecular signature and cis-regulatory architecture of a C. elegans gustatory neuron.
Genes Dev. 2007 Jul 1;21(13):1653-74
Authors: Etchberger JF, Lorch A, Sleumer MC, Zapf R, Jones SJ, Marra MA, Holt RA, Moerman DG, Hobert O
Taste receptor cells constitute a highly specialized cell type that perceives and conveys specific sensory information to the brain. The detailed molecular composition of these cells and the mechanisms that program their fate are, in general, poorly understood. We have generated serial analysis of gene expression (SAGE) libraries from two distinct populations of single, isolated sensory neuron classes, the gustatory neuron class ASE and the thermosensory neuron class AFD, from the nematode Caenorhabditis elegans. By comparing these two libraries, we have identified >1000 genes that define the ASE gustatory neuron class on a molecular level. This set of genes contains determinants of the differentiated state of the ASE neuron, such as a surprisingly complex repertoire of transcription factors (TFs), ion channels, neurotransmitters, and receptors, as well as seven-transmembrane receptor (7TMR)-type putative gustatory receptor genes. Through the in vivo dissection of the cis-regulatory regions of several ASE-expressed genes, we identified a small cis-regulatory motif, the "ASE motif," that is required for the expression of many ASE-expressed genes. We demonstrate that the ASE motif is a binding site for the C2H2 zinc finger TF CHE-1, which is essential for the correct differentiation of the ASE gustatory neuron. Taken together, our results provide a unique view of the molecular landscape of a single neuron type and reveal an important aspect of the regulatory logic for gustatory neuron specification in C. elegans.
PMID: 17606643 [PubMed - indexed for MEDLINE]
Reporter gene fusions.
Authors: Boulin T, Etchberger JF, Hobert O
PMID: 18050449 [PubMed - indexed for MEDLINE]
Specification of the nervous system.
Nervous systems are characterized by an astounding degree of cellular diversity. The nematode Caenorhabditis elegans has served as a valuable model system to define the genetic programs that serve to generate cellular diversity in the nervous system. This review discusses neuronal diversity in C. elegans and provides an overview of the molecular mechanisms that define and specify neuronal cell types in C. elegans.
PMID: 18050401 [PubMed - indexed for MEDLINE]
Architecture of a microRNA-controlled gene regulatory network that diversifies neuronal cell fates.
Cold Spring Harb Symp Quant Biol. 2006;71:181-8
Individual cell types are defined by the expression of specific gene batteries. Regulatory networks that control cell-type-specific gene expression programs in the nervous system are only beginning to be understood. This paper summarizes a complex gene regulatory network, composed of several transcription factors and microRNAs (miRNAs), that controls neuronal subclass specification in the nervous system of the nematode Caenorhabditis elegans.
PMID: 17381295 [PubMed - indexed for MEDLINE]
Early embryonic programming of neuronal left/right asymmetry in C. elegans.
Curr Biol. 2006 Dec 5;16(23):2279-92
Authors: Poole RJ, Hobert O
BACKGROUND: Nervous systems are largely bilaterally symmetric on a morphological level but often display striking degrees of functional left/right (L/R) asymmetry. How L/R asymmetric functional features are superimposed onto an essentially bilaterally symmetric structure and how nervous-system laterality relates to the L/R asymmetry of internal organs are poorly understood. We address these questions here by using the establishment of L/R asymmetry in the ASE chemosensory neurons of C. elegans as a paradigm. This bilaterally symmetric neuron pair is functionally lateralized in that it senses a distinct class of chemosensory cues and expresses a putative chemoreceptor family in a L/R asymmetric manner.
RESULTS: We show that the directionality of the asymmetry of the two postmitotic ASE neurons ASE left (ASEL) and ASE right (ASER) in adults is dependent on a L-/R-symmetry-breaking event at a very early embryonic stage, the six-cell stage, which also establishes the L/R asymmetric placement of internal organs. However, the L/R asymmetry of the ASE neurons per se is dependent on an even earlier anterior-posterior (A/P) Notch signal that specifies embryonic ABa/ABp blastomere identities at the four-cell stage. This Notch signal, which functions through two T box genes, acts genetically upstream of a miRNA-controlled bistable feedback loop that regulates the L/R asymmetric gene-expression program in the postmitotic ASE cells.
CONCLUSIONS: Our results link adult neuronal laterality to the generation of the A/P axis at the two-cell stage and raise the possibility that neural asymmetries observed across the animal kingdom are similarly established by very early embryonic interactions.
PMID: 17141609 [PubMed - indexed for MEDLINE]
A novel Eph receptor-interacting IgSF protein provides C. elegans motoneurons with midline guidepost function.
Curr Biol. 2006 Oct 10;16(19):1871-83
Authors: Boulin T, Pocock R, Hobert O
BACKGROUND: The ventral midline is a prominent structure in vertebrate and invertebrate nervous systems that provides crucial topological information for guiding axons to their appropriate target destinations. Rather than being composed of specialized midline glia cells as in many other species, the embryonic midline of the nematode Caenorhabditis elegans is physically defined by motoneuron cell bodies that separate the left from the right ventral cord fascicles. Their function during development, if any, is not known.
RESULTS: We show here that besides being components of the postembryonic locomotory circuit, these embryonic motoneurons (eMNs) actively provide midline guidance information for a specific subset of ventral midline axons. This information is provided in the form of a novel, cell-surface-anchored immunoglobulin superfamily (IgSF) member, WRK-1. WRK-1 acts in eMNs to prevent follower axons from inappropriately crossing the ventral midline. We describe the function of the Eph receptor vab-1 and multiple ephrin ligands at the midline, and we show by double mutant analysis and physical interaction tests that WRK-1 functionally interacts with the Eph receptor system. This interaction appears to occur exclusively in the context of axon guidance at the ventral midline but not in other cellular contexts, thereby suggesting that Eph receptor signaling is mechanistically distinct in different tissue types.
CONCLUSIONS: Our studies reveal cellular and molecular components of axon midline patterning and suggest that Ephrin signaling relies on previously unknown accessory components.
PMID: 17027485 [PubMed - indexed for MEDLINE]
Curr Biol. 2006 Apr 4;16(7):R233-4
PMID: 16927459 [PubMed - indexed for MEDLINE]
Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions.
Nat Struct Mol Biol. 2006 Sep;13(9):849-51
We use Caenorhabditis elegans to test proposed general rules for microRNA (miRNA)-target interactions. We show that G.U base pairing is tolerated in the 'seed' region of the lsy-6 miRNA interaction with its in vivo target cog-1, and that 6- to 8-base-pair perfect seed pairing is not a generally reliable predictor for an interaction of lsy-6 with a 3' untranslated region (UTR). Rather, lsy-6 can functionally interact with its target site only in specific 3' UTR contexts. Our findings illustrate the difficulty of establishing generalizable rules of miRNA-target interactions.
PMID: 16921378 [PubMed - indexed for MEDLINE]
An unusual Zn-finger/FH2 domain protein controls a left/right asymmetric neuronal fate decision in C. elegans.
Development. 2006 Sep;133(17):3317-28
Authors: Johnston RJ, Copeland JW, Fasnacht M, Etchberger JF, Liu J, Honig B, Hobert O
Gene regulatory networks that control the terminally differentiated state of a cell are, by and large, only superficially understood. In a mutant screen aimed at identifying regulators of gene batteries that define the differentiated state of two left/right asymmetric C. elegans gustatory neurons, ASEL and ASER, we have isolated a mutant, fozi-1, with a novel mixed-fate phenotype, characterized by de-repression of ASEL fate in ASER. fozi-1 codes for a protein that functions in the nucleus of ASER to inhibit the expression of the LIM homeobox gene lim-6, neuropeptide-encoding genes and putative chemoreceptors of the GCY gene family. The FOZI-1 protein displays a highly unusual domain architecture, that combines two functionally essential C2H2 zinc-finger domains, which are probably involved in transcriptional regulation, with a formin homology 2 (FH2) domain, normally found only in cytosolic regulators of the actin cytoskeleton. We demonstrate that the FH2 domain of FOZI-1 has lost its actin polymerization function but maintains its phylogenetically ancient ability to homodimerize. fozi-1 genetically interacts with several transcription factors and micro RNAs in the context of specific regulatory network motifs. These network motifs endow the system with properties that provide insights into how cells adopt their stable terminally differentiated states.
PMID: 16887832 [PubMed - indexed for MEDLINE]
DIG-1, a novel giant protein, non-autonomously mediates maintenance of nervous system architecture.
Development. 2006 Sep;133(17):3329-40
Authors: Bénard CY, Boyanov A, Hall DH, Hobert O
Dedicated mechanisms exist to maintain the architecture of an animal's nervous system after development is completed. To date, three immunoglobulin superfamily members have been implicated in this process in the nematode Caenorhabditis elegans: the secreted two-Ig domain protein ZIG-4, the FGF receptor EGL-15 and the L1-like SAX-7 protein. These proteins provide crucial information for neuronal structures, such as axons, that allows them to maintain the precise position they acquired during development. Yet, how widespread this mechanism is throughout the nervous system, and what other types of factors underlie such a maintenance mechanism, remains poorly understood. Here, we describe a new maintenance gene, dig-1, that encodes a predicted giant secreted protein containing a large number of protein interaction domains. With 13,100 amino acids, the DIG-1 protein is the largest secreted protein identifiable in any genome database. dig-1 functions post-developmentally to maintain axons and cell bodies in place within axonal fascicles and ganglia. The failure to maintain axon and cell body position is accompanied by defects in basement membrane structure, as evidenced by electron microscopy analysis of dig-1 mutants. Expression pattern and mosaic analysis reveals that dig-1 is produced by muscles to maintain nervous system architecture, demonstrating that dig-1 functions non-autonomously to preserve the proper layout of neural structures. We propose that DIG-1 is a component of the basement membrane that mediates specific contacts between cellular surfaces and their environment through the interaction with a cell-type specific set of other maintenance factors.
PMID: 16887823 [PubMed - indexed for MEDLINE]
The molecular diversity of glycosaminoglycans shapes animal development.
Annu Rev Cell Dev Biol. 2006;22:375-407
Authors: Bülow HE, Hobert O
Proteoglycans (PGs), molecules in which glycosaminoglycans (GAGs) are covalently linked to a protein core, are components of the extracellular matrix of all multicellular organisms. Sugar moieties in GAGs are often extensively modified, which make these molecules enormously complex. We discuss here the role of PGs during animal development, emphasizing the in vivo significance of sugar modifications. We explore a model in which the modification patterns of GAG chains may provide a specific code that contributes to the correct development of a multicellular organism.
PMID: 16805665 [PubMed - indexed for MEDLINE]
Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo.
J Mol Biol. 2006 Jun 23;359(5):1316-33
Authors: Berry KL, Hobert O
Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ''PTM'' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.
PMID: 16737711 [PubMed - indexed for MEDLINE]
Developmental regulation of whole cell capacitance and membrane current in identified interneurons in C. elegans.
J Neurophysiol. 2006 Jun;95(6):3665-73
Authors: Faumont S, Boulin T, Hobert O, Lockery SR
Postembryonic developmental changes in electrophysiological properties of the AIY interneuron class were investigated using whole cell voltage clamp. AIY interneurons displayed an increase in cell capacitance during larval development, whereas steady-state current amplitude did not increase. The time course of the outward membrane current, carried at least in part by K+ ions, matured, from a slowly activating, sustained current to a rapidly activating, decaying current. We also investigated how the development of capacitance and outward current was altered by loss-of-function mutations in genes expressed in AIY. One such gene, the LIM homeobox gene ttx-3, is known to be involved in the specification of the AIY neuronal subtype. In ttx-3 mutants, capacitance and outward current matured precociously. In mutants of the gene wrk-1, an immunoglobulin superfamily (IgSF) member whose expression is regulated by ttx-3, capacitance matured normally, whereas outward current matured precociously. We conclude that AIY interneurons contain distinct pathways for regulating capacitance and membrane current.
PMID: 16554520 [PubMed - indexed for MEDLINE]
Searching for neuronal left/right asymmetry: genomewide analysis of nematode receptor-type guanylyl cyclases.
Genetics. 2006 May;173(1):131-49
Authors: Ortiz CO, Etchberger JF, Posy SL, Frøkjaer-Jensen C, Lockery S, Honig B, Hobert O
Functional left/right asymmetry ("laterality") is a fundamental feature of many nervous systems, but only very few molecular correlates to functional laterality are known. At least two classes of chemosensory neurons in the nematode Caenorhabditis elegans are functionally lateralized. The gustatory neurons ASE left (ASEL) and ASE right (ASER) are two bilaterally symmetric neurons that sense distinct chemosensory cues and express a distinct set of four known chemoreceptors of the guanylyl cyclase (gcy) gene family. To examine the extent of lateralization of gcy gene expression patterns in the ASE neurons, we have undertaken a genomewide analysis of all gcy genes. We report the existence of a total of 27 gcy genes encoding receptor-type guanylyl cyclases and of 7 gcy genes encoding soluble guanylyl cyclases in the complete genome sequence of C. elegans. We describe the expression pattern of all previously uncharacterized receptor-type guanylyl cyclases and find them to be highly biased but not exclusively restricted to the nervous system. We find that >41% (11/27) of all receptor-type guanylyl cyclases are expressed in the ASE gustatory neurons and that one-third of all gcy genes (9/27) are expressed in a lateral, left/right asymmetric manner in the ASE neurons. The expression of all laterally expressed gcy genes is under the control of a gene regulatory network composed of several transcription factors and miRNAs. The complement of gcy genes in the related nematode C. briggsae differs from C. elegans as evidenced by differences in chromosomal localization, number of gcy genes, and expression patterns. Differences in gcy expression patterns in the ASE neurons of C. briggsae arise from a difference in cis-regulatory elements and trans-acting factors that control ASE laterality. In sum, our results indicate the existence of a surprising multitude of putative chemoreceptors in the gustatory ASE neurons and suggest the existence of a substantial degree of laterality in gustatory signaling mechanisms in nematodes.
PMID: 16547101 [PubMed - indexed for MEDLINE]
Uses of GFP in Caenorhabditis elegans.
Methods Biochem Anal. 2006;47:203-26
Authors: Hobert O, Loria P
PMID: 16335715 [PubMed - indexed for MEDLINE]
A novel C. elegans zinc finger transcription factor, lsy-2, required for the cell type-specific expression of the lsy-6 microRNA.
Development. 2005 Dec;132(24):5451-60
Authors: Johnston RJ, Hobert O
The two Caenorhabditis elegans gustatory neurons, ASE left (ASEL) and ASE right (ASER) are morphologically bilaterally symmetric, yet left/right asymmetric in function and in the expression of specific chemosensory signaling molecules. The ASEL versus ASER cell-fate decision is controlled by a complex gene regulatory network composed of microRNAs (miRNAs) and transcription factors. Alterations in the activities of each of these regulatory factors cause a complete lateral cell-fate switch. Here, we describe lsy-2, a novel C2H2 zinc finger transcription factor that is required for the execution of the ASEL stable state. In lsy-2 null mutants, the ASEL neuron adopts the complete ASER gene expression profile, including both upstream regulatory and terminal effector genes. The normally left/right asymmetric ASE neurons are therefore ;symmetrized' in lsy-2 mutants. Cell-specific rescue experiments indicate that lsy-2 is required autonomously in ASEL for the activation of ASEL-specifying factors and the repression of ASER-specifying factors. Genetic epistasis experiments demonstrate that lsy-2 exerts its activity by regulating the transcription of the lsy-6 miRNA in the ASEL neuron, thereby making lsy-2 one of the few factors known to control the cell-type specificity of miRNA gene expression.
PMID: 16291785 [PubMed - indexed for MEDLINE]
MicroRNAs acting in a double-negative feedback loop to control a neuronal cell fate decision.
Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12449-54
Authors: Johnston RJ, Chang S, Etchberger JF, Ortiz CO, Hobert O
The elucidation of the architecture of gene regulatory networks that control cell-type specific gene expression programs represents a major challenge in developmental biology. We describe here a cell fate decision between two alternative neuronal fates and the architecture of a gene regulatory network that controls this cell fate decision. The two Caenorhabditis elegans taste receptor neurons "ASE left" (ASEL) and "ASE right" (ASER) share many bilaterally symmetric features, but each cell expresses a distinct set of chemoreceptors that endow the gustatory system with the capacity to sense and discriminate specific environmental inputs. We show that these left/right asymmetric fates develop from a precursor state in which both ASE neurons express equivalent features. This hybrid precursor state is unstable and transitions into the stable ASEL or ASER terminal end state. Although this transition is spatially stereotyped in wild-type animals, mutant analysis reveals that each cell has the potential to transition into either the ASEL or ASER stable end state. The stability and irreversibility of the terminal differentiated state is ensured by the interactions of two microRNAs (miRNAs) and their transcription factor targets in a double-negative feedback loop. Simple feedback loops are found as common motifs in many gene regulatory networks, but the loop described here is unusually complex and involves miRNAs. The interaction of miRNAs in double-negative feedback loops may not only be a means for miRNAs to regulate their own expression but may also represent a general paradigm for how terminal cell fates are selected and stabilized.
PMID: 16099833 [PubMed - indexed for MEDLINE]
An interneuronal chemoreceptor required for olfactory imprinting in C. elegans.
Science. 2005 Jul 29;309(5735):787-90
Authors: Remy JJ, Hobert O
Animals alter their behavioral patterns in an experience-dependent manner. Olfactory imprinting is a process in which the exposure of animals to olfactory cues during specific and restricted time windows leaves a permanent memory ("olfactory imprint") that shapes the animal's behavior upon encountering the olfactory cues at later times. We found that Caenorhabditis elegans displays olfactory imprinting behavior that is mediated by a single pair of interneurons. To function in olfactory imprinting, this interneuron pair must express a G protein-coupled chemoreceptor family member encoded by the sra-11 gene. Our study provides insights into the cellular and molecular basis of olfactory imprinting and reveals a function for a chemosensory receptor family member in interneurons.
PMID: 16051801 [PubMed - indexed for MEDLINE]
MicroRNAs: all gone and then what?
Curr Biol. 2005 May 24;15(10):R387-9
MicroRNAs are abundant gene regulatory factors whose function in animal development and homeostasis is poorly understood. A new study reports the genetic elimination of miRNA function on a full genomic scale and identifies a subfamily of miRNAs involved in brain morphogenesis.
PMID: 15916942 [PubMed - indexed for MEDLINE]
Common logic of transcription factor and microRNA action.
Trends Biochem Sci. 2004 Sep;29(9):462-8
Over the past few years, microRNAs (miRNAs) have emerged as abundant regulators of gene expression. Like many transcription factors (TFs), miRNAs are important determinants of cellular fate specification. Here I provide a conceptual framework for miRNA action in the context of creating cellular diversity in a developing organism, and emphasize the conceptual similarity of TF- and miRNA-mediated control of gene expression. Both TFs and miRNAs are trans-acting factors that exert their activity through composite cis-regulatory elements that are 'hard-wired' into DNA or RNA. TFs and miRNAs act in a largely combinatorial manner - that is, many different TFs or miRNAs control one gene - and they act cooperatively on their targets - that is, there are several cis-regulatory elements for a single TF or miRNA species in a target gene. Just as the set of TFs in a given cell type has been proposed to constitute a 'code' that specifies cellular differentiation, so 'miRNA codes' are likely to have conceptually similar roles in the specification of cell types.
PMID: 15337119 [PubMed - indexed for MEDLINE]
MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode.
Nature. 2004 Aug 12;430(7001):785-9
Authors: Chang S, Johnston RJ, Frøkjaer-Jensen C, Lockery S, Hobert O
Animal microRNAs (miRNAs) are gene regulatory factors that prevent the expression of specific messenger RNA targets by binding to their 3' untranslated region. The Caenorhabditis elegans lsy-6 miRNA (for lateral symmetry defective) is required for the left/right asymmetric expression of guanyl cyclase (gcy) genes in two chemosensory neurons termed ASE left (ASEL) and ASE right (ASER). The asymmetric expression of these putative chemoreceptors in turn correlates with the functional lateralization of the ASE neurons. Here we find that a mutation in the die-1 zinc-finger transcription factor disrupts both the chemosensory laterality and left/right asymmetric expression of chemoreceptor genes in the ASE neurons. die-1 controls chemosensory laterality by activating the expression of lsy-6 specifically in ASEL, but not in ASER, where die-1 expression is downregulated through two sites in its 3' untranslated region. These two sites are complementary to mir-273, a previously uncharacterized miRNA, whose expression is strongly biased towards ASER. Forced bilateral expression of mir-273 in ASEL and ASER causes a loss of asymmetric die-1 expression and ASE laterality. Thus, an inverse distribution of two sequentially acting miRNAs in two bilaterally symmetric neurons controls laterality of the nematode chemosensory system.
PMID: 15306811 [PubMed - indexed for MEDLINE]
Caenorhabditis elegans ABL-1 antagonizes p53-mediated germline apoptosis after ionizing irradiation.
Nat Genet. 2004 Aug;36(8):906-12
Authors: Deng X, Hofmann ER, Villanueva A, Hobert O, Capodieci P, Veach DR, Yin X, Campodonico L, Glekas A, Cordon-Cardo C, Clarkson B, Bornmann WG, Fuks Z, Hengartner MO, Kolesnick R
c-Abl, a conserved nonreceptor tyrosine kinase, integrates genotoxic stress responses, acting as a transducer of both pro- and antiapoptotic effector pathways. Nuclear c-Abl seems to interact with the p53 homolog p73 to elicit apoptosis. Although several observations suggest that cytoplasmic localization of c-Abl is required for antiapoptotic function, the signals that mediate its antiapoptotic effect are largely unknown. Here we show that worms carrying an abl-1 deletion allele, abl-1(ok171), are specifically hypersensitive to radiation-induced apoptosis in the Caenorhabditis elegans germ line. Our findings delineate an apoptotic pathway antagonized by ABL-1, which requires sequentially the cell cycle checkpoint genes clk-2, hus-1 and mrt-2; the C. elegans p53 homolog, cep-1; and the genes encoding the components of the conserved apoptotic machinery, ced-3, ced-9 and egl-1. ABL-1 does not antagonize germline apoptosis induced by the DNA-alkylating agent ethylnitrosourea. Furthermore, worms treated with the c-Abl inhibitor STI-571 (Gleevec; used in human cancer therapy), two newly synthesized STI-571 variants or PD166326 had a phenotype similar to that generated by abl-1(ok171). These studies indicate that ABL-1 distinguishes proapoptotic signals triggered by two different DNA-damaging agents and suggest that C. elegans might provide tissue models for development of anticancer drugs.
PMID: 15273685 [PubMed - indexed for MEDLINE]
Genomic cis-regulatory architecture and trans-acting regulators of a single interneuron-specific gene battery in C. elegans.
Dev Cell. 2004 Jun;6(6):757-70
Authors: Wenick AS, Hobert O
Gene batteries are sets of coregulated genes with common cis-regulatory elements that define the differentiated state of a cell. The nature of gene batteries for individual neuronal cellular subtypes and their linked cis-regulatory elements is poorly defined. Through molecular dissection of the highly modular cis-regulatory architecture of individual neuronally expressed genes, we have defined a conserved 16 bp cis-regulatory motif that drives gene expression in a single interneuron subtype, termed AIY, in the nematode Caenorhabditis elegans. This motif is bound and activated by the Paired- and LIM-type homeodomain proteins CEH-10 and TTX-3. Using genome-wide phylogenetic footprinting, we delineated the location, distribution, and evolution of AIY-specific cis-regulatory elements throughout the genome and thereby defined a large battery of AIY-expressed genes, all of which represent direct Paired/LIM homeodomain target genes. The identity of these homeodomain targets provides novel insights into the biology of the AIY interneuron.
PMID: 15177025 [PubMed - indexed for MEDLINE]
Differential functions of the C. elegans FGF receptor in axon outgrowth and maintenance of axon position.
Neuron. 2004 May 13;42(3):367-74
Authors: Bülow HE, Boulin T, Hobert O
Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon outgrowth. We show here that the C. elegans fibroblast growth factor receptor (FGFR), EGL-15, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-15 tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate outgrowth of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-15(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon outgrowth but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-15(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position.
PMID: 15134634 [PubMed - indexed for MEDLINE]
The immunoglobulin superfamily in Caenorhabditis elegans and Drosophila melanogaster.
Development. 2004 May;131(10):2237-8; author reply 2238-40
Authors: Hobert O, Hutter H, Hynes RO
PMID: 15128663 [PubMed - indexed for MEDLINE]
CisOrtho: a program pipeline for genome-wide identification of transcription factor target genes using phylogenetic footprinting.
BMC Bioinformatics. 2004 Mar 12;5:27
Authors: Bigelow HR, Wenick AS, Wong A, Hobert O
BACKGROUND: All known genomes code for a large number of transcription factors. It is important to develop methods that will reveal how these transcription factors act on a genome wide level, that is, through what target genes they exert their function.
RESULTS: We describe here a program pipeline aimed at identifying transcription factor target genes in whole genomes. Starting from a consensus binding site, represented as a weight matrix, potential sites in a pre-filtered genome are identified and then further filtered by assessing conservation of the putative site in the genome of a related species, a process called phylogenetic footprinting. CisOrtho has been successfully used to identify targets for two homeodomain transcription factors in the genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.
CONCLUSIONS: CisOrtho will identify targets of other nematode transcription factors whose DNA binding specificity is known and can be easily adapted to search other genomes for transcription factor targets.
PMID: 15113408 [PubMed - indexed for MEDLINE]
A genetic screen for neurite outgrowth mutants in Caenorhabditis elegans reveals a new function for the F-box ubiquitin ligase component LIN-23.
Genetics. 2004 Mar;166(3):1253-67
Authors: Mehta N, Loria PM, Hobert O
Axon pathfinding and target recognition are highly dynamic and tightly regulated cellular processes. One of the mechanisms involved in regulating protein activity levels during axonal and synaptic development is protein ubiquitination. We describe here the isolation of several Caenorhabditis elegans mutants, termed eno (ectopic/erratic neurite outgrowth) mutants, that display defects in axon outgrowth of specific neuron classes. One retrieved mutant is characterized by abnormal termination of axon outgrowth in a subset of several distinct neuron classes, including ventral nerve cord motor neurons, head motor neurons, and mechanosensory neurons. This mutant is allelic to lin-23, which codes for an F-box-containing component of an SCF E3 ubiquitin ligase complex that was previously shown to negatively regulate postembryonic cell divisions. We demonstrate that LIN-23 is a broadly expressed cytoplasmically localized protein that is required autonomously in neurons to affect axon outgrowth. Our newly isolated allele of lin-23, a point mutation in the C-terminal tail of the protein, displays axonal outgrowth defects similar to those observed in null alleles of this gene, but does not display defects in cell cycle regulation. We have thus defined separable activities of LIN-23 in two distinct processes, cell cycle control and axon patterning. We propose that LIN-23 targets distinct substrates for ubiquitination within each process.
PMID: 15082545 [PubMed - indexed for MEDLINE]
Differential sulfations and epimerization define heparan sulfate specificity in nervous system development.
Neuron. 2004 Mar 4;41(5):723-36
Heparan sulfate proteoglycans (HSPG) are components of the extracellular matrix through which axons navigate to reach their targets. The heparan sulfate (HS) side chains of HSPGs show complex and differentially regulated patterns of secondary modifications, including sulfations of distinct hydroxyl groups and epimerization of an asymmetric carbon atom. These modifications endow the HSPG-containing extracellular matrix with the potential to code for an enormous molecular diversity. Attempting to decode this diversity, we analyzed C. elegans animals lacking three HS-modifying enzymes, glucuronyl C5-epimerase, heparan 6O-sulfotransferase, and 2O-sulfotransferase. Each of the mutant animals exhibit distinct as well as overlapping axonal and cellular guidance defects in specific neuron classes. We have linked individual HS modifications to two specific guidance systems, the sax-3/Robo and kal-1/Anosmin-1 systems, whose activity is dependent on different HS modifications in different cellular contexts. Our results demonstrate that the molecular diversity in HS encodes information that is crucial for different aspects of neuronal development.
PMID: 15003172 [PubMed - indexed for MEDLINE]
A conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans.
J Neurosci. 2004 Mar 3;24(9):2191-201
Authors: Loria PM, Hodgkin J, Hobert O
For a motor unit to function, neurons and muscle cells need to adopt their correct cell fate, form appropriate cellular contacts, and assemble a specific repertoire of signaling proteins into presynaptic and postsynaptic structures. In the nematode Caenorhabditis elegans, a disruption of any of these steps causes uncoordinated locomotory behavior (unc phenotype). We report here the positional cloning of a new unc gene, unc-122, which we show by mosaic analysis and tissue-specific rescue experiments to act in muscle to affect locomotory behavior. unc-122 codes for a phylogenetically conserved type II transmembrane protein with collagen repeats and a cysteine-rich olfactomedin domain. Together with uncharacterized proteins in C. elegans, Drosophila, and vertebrates, UNC-122 defines a novel family of proteins that we term "Colmedins." UNC-122 protein is expressed exclusively in muscle and coelomocytes and localizes to the postsynaptic surface of GABAergic and cholinergic neuromuscular junctions (NMJs). Presynaptic and postsynaptic structures are present and properly aligned in unc-122 mutant animals, yet the animals display neurotransmission defects characterized by an altered sensitivity toward drugs that interfere with cholinergic signaling. Moreover, unc-122 mutant animals display anatomical defects in motor axons that are likely a secondary consequence of neurotransmission defects. Both the neuroanatomical and locomotory defects worsen progressively during the life of an animal, consistent with a role of unc-122 in acute signaling at the NMJ. On the basis of motifs in the UNC-122 protein sequence that are characteristic of extracellular matrix proteins, we propose that UNC-122 is involved in maintaining a structural microenvironment that allows efficient neuromuscular signaling.
PMID: 14999070 [PubMed - indexed for MEDLINE]
A microRNA controlling left/right neuronal asymmetry in Caenorhabditis elegans.
Nature. 2003 Dec 18;426(6968):845-9
How left/right functional asymmetry is layered on top of an anatomically symmetrical nervous system is poorly understood. In the nematode Caenorhabditis elegans, two morphologically bilateral taste receptor neurons, ASE left (ASEL) and ASE right (ASER), display a left/right asymmetrical expression pattern of putative chemoreceptor genes that correlates with a diversification of chemosensory specificities. Here we show that a previously undefined microRNA termed lsy-6 controls this neuronal left/right asymmetry of chemosensory receptor expression. lsy-6 mutants that we retrieved from a genetic screen for defects in neuronal left/right asymmetry display a loss of the ASEL-specific chemoreceptor expression profile with a concomitant gain of the ASER-specific profile. A lsy-6 reporter gene construct is expressed in less than ten neurons including ASEL, but not ASER. lsy-6 exerts its effects on ASEL through repression of cog-1, an Nkx-type homeobox gene, which contains a lsy-6 complementary site in its 3' untranslated region and that has been shown to control ASE-specific chemoreceptor expression profiles. lsy-6 is the first microRNA to our knowledge with a role in neuronal patterning, providing new insights into left/right axis formation.
PMID: 14685240 [PubMed - indexed for MEDLINE]
A C. elegans CLIC-like protein required for intracellular tube formation and maintenance.
Science. 2003 Dec 19;302(5653):2134-7
Authors: Berry KL, Bülow HE, Hall DH, Hobert O
The Caenorhabditis elegans excretory canal is composed of a single elongated and branched cell that is tunneled by an inner lumen of apical character. Loss of the exc-4 gene causes a cystic enlargement of this intracellular tube. exc-4 encodes a member of the chloride intracellular channel (CLIC) family of proteins. EXC-4 protein localizes to various tubular membranes in distinct cell types, including the lumenal membrane of the excretory tubes. A conserved 55-amino acid domain enables EXC-4 translocation from the cytosol to the lumenal membrane. The tubular architecture of this membrane requires EXC-4 for both its formation and maintenance.
PMID: 14684823 [PubMed - indexed for MEDLINE]
LIM homeobox gene-dependent expression of biogenic amine receptors in restricted regions of the C. elegans nervous system.
Dev Biol. 2003 Nov 1;263(1):81-102
Authors: Tsalik EL, Niacaris T, Wenick AS, Pau K, Avery L, Hobert O
Biogenic amines regulate a variety of behaviors. Their functions are predominantly mediated through G-protein-coupled 7-transmembrane domain receptors (GPCR), 16 of which are predicted to exist in the genome sequence of the nematode Caenorhabditis elegans. We describe here the expression pattern of several of these aminergic receptors, including two serotonin receptors (ser-1 and ser-4), one tyramine receptor (ser-2), and two dopamine receptors (dop-1 and dop-2). Moreover, we describe distinct but partially overlapping expression patterns of different splice forms of the ser-2 tyramine receptor locus. We find that each of the aminergic receptor genes is expressed in restricted regions of the nervous system and that many of them reveal significant overlap with the expression of regulatory factors of the LIM homeobox (Lhx) gene family. We demonstrate that the expression of several of the biogenic amine receptors is abrogated in specific cell types in Lhx gene mutants, thus establishing a role for these Lhx genes in regulating aspects of neurotransmission. We extend these findings with other cell fate markers and show that the lim-4 Lhx gene is required for several but not all aspects of RID motor neuron differentiation and that the lim-6 Lhx gene is required for specific aspects of RIS interneuron differentiation. We also use aminergic receptor gfp reporter fusions as tools to visualize the anatomy of specific neurons in Lhx mutant backgrounds and find that the development of the elaborate dendritic branching pattern of the PVD harsh touch sensory neuron requires the mec-3 Lhx gene. Lastly, we analyze a mutant allele of the ser-2 tyramine receptor, a target of the ttx-3 Lhx gene in the AIY interneuron class. ser-2 mutants display none of the defects previously shown to be associated with loss of AIY function.
PMID: 14568548 [PubMed - indexed for MEDLINE]
A transcriptional regulatory cascade that controls left/right asymmetry in chemosensory neurons of C. elegans.
Genes Dev. 2003 Sep 1;17(17):2123-37
Authors: Chang S, Johnston RJ, Hobert O
The molecular mechanisms of differential pattern formation along the left/right (L/R) axis in the nervous system are poorly understood. The nervous system of the nematode Caenorhabditis elegans displays several examples of L/R asymmetry, including the directional asymmetry displayed by the two ASE taste receptor neurons, ASE left (ASEL) and ASE right (ASER). Although bilaterally symmetric in regard to all known morphological criteria, these two neurons display distinct chemosensory capacities that correlate with the L/R asymmetric expression of three putative sensory receptor genes, gcy-5, expressed only in ASER, and gcy-6 and gcy-7, expressed only in ASEL. In order to understand the genetic basis of L/R asymmetry establishment, we screened for mutants in which patterns of asymmetric gcy gene expression are disrupted, and we identified a cascade of several symmetrically and asymmetrically expressed transcription factors that are sequentially required to restrict gcy gene expression to either the left or right ASE cell. These factors include the zinc finger transcription factor che-1; the homeobox genes cog-1, ceh-36, and lim-6; and the transcriptional cofactors unc-37/Groucho and lin-49. Specific features of this regulatory hierarchy are sequentially acting repressive interactions and the finely balanced activity of antagonizing positive and negative regulatory factors. A key trigger for asymmetry is the L/R differential expression of the Nkx6-type COG-1 homeodomain protein. Our studies have thus identified transcriptional mediators of a putative L/R-asymmetric signaling event and suggest that vertebrate homologs of these proteins may have similar functions in regulating vertebrate brain asymmetries.
PMID: 12952888 [PubMed - indexed for MEDLINE]
Two neuronal, nuclear-localized RNA binding proteins involved in synaptic transmission.
Curr Biol. 2003 Aug 5;13(15):1317-23
Authors: Loria PM, Duke A, Rand JB, Hobert O
While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.
PMID: 12906792 [PubMed - indexed for MEDLINE]
Functional mapping of neurons that control locomotory behavior in Caenorhabditis elegans.
J Neurobiol. 2003 Aug;56(2):178-97
Authors: Tsalik EL, Hobert O
One approach to understanding behavior is to define the cellular components of neuronal circuits that control behavior. In the nematode Caenorhabditis elegans, neuronal circuits have been delineated based on patterns of synaptic connectivity derived from ultrastructural analysis. Individual cellular components of these anatomically defined circuits have previously been characterized on the sensory and motor neuron levels. In contrast, interneuron function has only been addressed to a limited extent. We describe here several classes of interneurons (AIY, AIZ, and RIB) that modulate locomotory behavior in C. elegans. Using mutant analysis as well as microsurgical mapping techniques, we found that the AIY neuron class serves to tonically modulate reversal frequency of animals in various sensory environments via the repression of the activity of a bistable switch composed of defined command interneurons. Furthermore, we show that the presentation of defined sensory modalities induces specific alterations in reversal behavior and that the AIY interneuron class mediates this alteration in locomotory behavior. We also found that the AIZ and RIB interneuron classes process odorsensory information in parallel to the AIY interneuron class. AIY, AIZ, and RIB are the first interneurons directly implicated in chemosensory signaling. Our neuronal mapping studies provide the framework for further genetic and functional dissections of neuronal circuits in C. elegans.
PMID: 12838583 [PubMed - indexed for MEDLINE]
Characterization of Caenorhabditis elegans homologs of the Down syndrome candidate gene DYRK1A.
Genetics. 2003 Feb;163(2):571-80
Authors: Raich WB, Moorman C, Lacefield CO, Lehrer J, Bartsch D, Plasterk RH, Kandel ER, Hobert O
The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.
PMID: 12618396 [PubMed - indexed for MEDLINE]
New insights into the diversity and function of neuronal immunoglobulin superfamily molecules.
Annu Rev Neurosci. 2003;26:207-38
Authors: Rougon G, Hobert O
Immunoglobulin superfamily (IgSF) proteins are implicated in diverse steps of brain development, including neuronal migration, axon pathfinding, target recognition and synapse formation, as well as in the maintenance and function of neuronal networks in the adult. We provide here a review of recent findings on the diversity and the role of transmembrane and secreted members of IgSF proteins in the nervous system. We illustrate that the complexity of IgSF protein function results from various different levels of regulation including regulation of gene expression, protein localization, and protein interactions.
PMID: 12598678 [PubMed - indexed for MEDLINE]
Development and maintenance of neuronal architecture at the ventral midline of C. elegans.
Curr Opin Neurobiol. 2003 Feb;13(1):70-8
Authors: Hobert O, Bülow H
Work in flies, nematodes and vertebrates has shown that genes involved in axon patterning at the ventral midline are functionally conserved across phylogeny. Recent studies in Caenorhabditis elegans have implicated several new extracellular molecules, such as nidogen and heparan sulfate proteoglycans, in axonal tract formation at the midline. Furthermore, a conceptually new mechanism that regulates the maintenance of axon positioning at the midline has been described in C. elegans.
PMID: 12593984 [PubMed - indexed for MEDLINE]
Ezh2 controls B cell development through histone H3 methylation and Igh rearrangement.
Nat Immunol. 2003 Feb;4(2):124-31
Authors: Su IH, Basavaraj A, Krutchinsky AN, Hobert O, Ullrich A, Chait BT, Tarakhovsky A
Polycomb group protein Ezh2 is an essential epigenetic regulator of embryonic development in mice, but its role in the adult organism is unknown. High expression of Ezh2 in developing murine lymphocytes suggests Ezh2 involvement in lymphopoiesis. Using Cre-mediated conditional mutagenesis, we demonstrated a critical role for Ezh2 in early B cell development and rearrangement of the immunoglobulin heavy chain gene (Igh). We also revealed Ezh2 as a key regulator of histone H3 methylation in early B cell progenitors. Our data suggest Ezh2-dependent histone H3 methylation as a novel regulatory mechanism controlling Igh rearrangement during early murine B cell development.
PMID: 12496962 [PubMed - indexed for MEDLINE]
Identification of spatial and temporal cues that regulate postembryonic expression of axon maintenance factors in the C. elegans ventral nerve cord.
Development. 2003 Feb;130(3):599-610
Authors: Aurelio O, Boulin T, Hobert O
Patterns of gene expression are under precise spatial and temporal control. A particularly striking example is represented by several members of the zig gene family, which code for secreted immunoglobulin domain proteins required for maintaining ventral nerve cord organization in Caenorhabditis elegans. These genes are coordinately expressed in a single interneuron in the ventral nerve cord, known as PVT. Their expression is initiated at a precise postembryonic stage, long after PVT has been generated in mid-embryogenesis. We define spatial and temporal cues that are required for the precise regulation of zig gene expression. We find that two LIM homeobox genes, the Lhx3-class gene ceh-14 and the Lmx-class gene lim-6 are coordinately required for zig gene expression in PVT. Temporal control of zig gene expression is conferred by the heterochronic gene lin-14, a nuclear factor previously implicated in developmental timing in various contexts. Loss of the lim-6 and ceh-14 transcription factors and the developmental timer lin-14 cause not only a loss of zig gene expression but also lead to defects in the maintenance of ventral nerve cord architecture. Overriding the normal spatiotemporal control of zig gene expression through expression of one of the zig genes under control of heterologous promoters also causes axon patterning defects in the ventral nerve cord. Our findings illustrate the importance of spatial and temporal control of gene expression in the nervous system and, furthermore, implicate heterochronic genes in postmitotic neural patterning events.
PMID: 12490565 [PubMed - indexed for MEDLINE]
Behavioral plasticity in C. elegans: paradigms, circuits, genes.
J Neurobiol. 2003 Jan;54(1):203-23
Life in the soil is an intellectual and practical challenge that the nematode Caenorhabditis elegans masters by utilizing 302 neurons. The nervous system assembled by these 302 neurons is capable of executing a variety of behaviors, some of respectable complexity. The simplicity of the nervous system, its thoroughly characterized structure, several sets of well-defined behaviors, and its genetic amenability combined with its isogenic background make C. elegans an attractive model organism to study the genetics of behavior. This review describes several behavioral plasticity paradigms in C. elegans and their underlying neuronal circuits and then goes on to review the forward genetic analysis that has been undertaken to identify genes involved in the execution of these behaviors. Lastly, the review outlines how reverse genetics and genomic approaches can guide the analysis of the role of genes in behavior and why and how they will complement the forward genetic analysis of behavior.
PMID: 12486705 [PubMed - indexed for MEDLINE]
Introduction: behavioral genetics--the third century.
J Neurobiol. 2003 Jan;54(1):1-3
PMID: 12486696 [PubMed - indexed for MEDLINE]
Left-right asymmetry in the nervous system: the Caenorhabditis elegans model.
Nat Rev Neurosci. 2002 Aug;3(8):629-40
Authors: Hobert O, Johnston RJ, Chang S
PMID: 12154364 [PubMed - indexed for MEDLINE]
Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6346-51
Authors: Bülow HE, Berry KL, Topper LH, Peles E, Hobert O
Kallmann syndrome is a neurological disorder characterized by various behavioral and neuroanatomical defects. The X-linked form of this disease is caused by mutations in the KAL-1 gene, which codes for a secreted molecule that is expressed in restricted regions of the brain. Its molecular mechanism of action has thus far remained largely elusive. We show here that expression of the Caenorhabditis elegans homolog of KAL-1 in selected sensory and interneuron classes causes a highly penetrant, dosage-dependent, and cell autonomous axon-branching phenotype. In a different cellular context, heterologous C. elegans kal-1 expression causes a highly penetrant axon-misrouting phenotype. The axon-branching and -misrouting activities require different domains of the KAL-1 protein. In a genetic modifier screen we isolated several loci that either suppress or enhance the kal-1-induced axonal defects, one of which codes for an enzyme that modifies specific residues in heparan sulfate proteoglycans, namely heparan-6O-sulfotransferase. We hypothesize that KAL-1 binds by means of a heparan sulfate proteoglycan to its cognate receptor or other extracellular cues to induce axonal branching and axon misrouting.
PMID: 11983919 [PubMed - indexed for MEDLINE]
PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans.
Biotechniques. 2002 Apr;32(4):728-30
PMID: 11962590 [PubMed - indexed for MEDLINE]
Immunoglobulin-domain proteins required for maintenance of ventral nerve cord organization.
Science. 2002 Jan 25;295(5555):686-90
Authors: Aurelio O, Hall DH, Hobert O
During development, neurons extend axons along defined routes to specific target cells. We show that additional mechanisms ensure that axons maintain their correct positioning in defined axonal tracts. After termination of axonal outgrowth and target recognition, axons in the ventral nerve cord (VNC) of Caenorhabditis elegans require the presence of a specific VNC neuron, PVT, to maintain their correct positioning in the left and right fascicles of the VNC. PVT may exert its stabilizing function by the temporally tightly controlled secretion of 2-immunoglobulin (Ig)-domain proteins encoded by the zig genes. Dedicated axon maintenance mechanisms may be widely used to ensure the preservation of functional neuronal circuitries.
PMID: 11809975 [PubMed - indexed for MEDLINE]
The lin-11 LIM homeobox gene specifies olfactory and chemosensory neuron fates in C. elegans.
Development. 2001 Sep;128(17):3269-81
Authors: Sarafi-Reinach TR, Melkman T, Hobert O, Sengupta P
Chemosensory neuron diversity in C. elegans arises from the action of transcription factors that specify different aspects of sensory neuron fate. In the AWB and AWA olfactory neurons, the LIM homeobox gene lim-4 and the nuclear hormone receptor gene odr-7 are required to confer AWB and AWA-specific characteristics respectively, and to repress an AWC olfactory neuron-like default fate. Here, we show that AWA neuron fate is also regulated by a member of the LIM homeobox gene family, lin-11. lin-11 regulates AWA olfactory neuron differentiation by initiating expression of odr-7, which then autoregulates to maintain expression. lin-11 also regulates the fate of the ASG chemosensory neurons, which are the lineal sisters of the AWA neurons. We show that lin-11 is expressed dynamically in the AWA and ASG neurons, and that misexpression of lin-11 is sufficient to promote an ASG, but not an AWA fate, in a subset of neuron types. Our results suggest that differential temporal regulation of lin-11, presumably together with its interaction with asymmetrically segregated factors, results in the generation of the distinct AWA and ASG sensory neuron types. We propose that a LIM code may be an important contributor to the generation of functional diversity in a subset of olfactory and chemosensory neurons in C. elegans.
PMID: 11546744 [PubMed - indexed for MEDLINE]
A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans.
Development. 2001 Jun;128(11):1951-69
Authors: Altun-Gultekin Z, Andachi Y, Tsalik EL, Pilgrim D, Kohara Y, Hobert O
The development of the nervous system requires the coordinated activity of a variety of regulatory factors that define the individual properties of specific neuronal subtypes. We report a regulatory cascade composed of three homeodomain proteins that act to define the properties of a specific interneuron class in the nematode C. elegans. We describe a set of differentiation markers characteristic for the AIY interneuron class and show that the ceh-10 paired-type and ttx-3 LIM-type homeobox genes function to regulate all known subtype-specific features of the AIY interneurons. In contrast, the acquisition of several pan-neuronal features is unaffected in ceh-10 and ttx-3 mutants, suggesting that the activity of these homeobox genes separates pan-neuronal from subtype-specific differentiation programs. The LIM homeobox gene ttx-3 appears to play a central role in regulation of AIY differentiation. Not only are all AIY subtype characteristics lost in ttx-3 mutants, but ectopic misexpression of ttx-3 is also sufficient to induce AIY-like features in a restricted set of neurons. One of the targets of ceh-10 and ttx-3 is a novel type of homeobox gene, ceh-23. We show that ceh-23 is not required for the initial adoption of AIY differentiation characteristics, but instead is required to maintain the expression of one defined AIY differentiation feature. Finally, we demonstrate that the regulatory relationship between ceh-10, ttx-3 and ceh-23 is only partially conserved in other neurons in the nervous system. Our findings illustrate the complexity of transcriptional regulation in the nervous system and provide an example for the intricate interdependence of transcription factor action.
PMID: 11493519 [PubMed - indexed for MEDLINE]
Functions of LIM-homeobox genes.
Trends Genet. 2000 Feb;16(2):75-83
Authors: Hobert O, Westphal H
Homeobox genes play fundamental roles in development. They can be subdivided into several subfamilies, one of which is the LIM-homeobox subfamily. The primary structure of LIM-homeobox genes has been remarkably conserved through evolution. Have their functions similarly been conserved? A host of new data has been derived from mutational analysis in diverse organisms, such as nematodes, flies and vertebrates. These studies have revealed a prominent involvement of LIM-homeodomain proteins in tissue patterning and differentiation, and their function in neural patterning is evident in all organisms studied to date. Here, we summarize the recent findings on LIM-homeobox gene function, compare the function of these genes from different organisms and describe specific co-factor requirements.
PMID: 10652534 [PubMed - indexed for MEDLINE]