Left-right asymmetry: advances and enigmas.
Genesis. 2014 Jun;52(6):451-4
Authors: Halpern ME, Hobert O, Wright CV
PMID: 24943067 [PubMed - indexed for MEDLINE]
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.
PMID: 24510690 [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]
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
Authors: Pocock R, Hobert O
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]
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]
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]
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]