InteractiveFly: GeneBrief

unc-4: Biological Overview | References

The Drosophila ventral nerve cord (VNC) is composed of thousands of neurons born from a set of individually identifiable stem cells. The VNC harbors neuronal circuits required to execute key behaviors, such as flying and walking. Leveraging the lineage-based functional organization of the VNC, this study investigated the developmental and molecular basis of behavior by focusing on lineage-specific functions of the homeodomain transcription factor, Unc-4. Unc-4 was found to function in lineage 11A to promote cholinergic neurotransmitter identity and suppress the GABA fate. In lineage 7B, Unc-4 promotes proper neuronal projections to the leg neuropil and a specific flight-related take-off behavior. It was also uncovered that Unc-4 acts peripherally to promote proprioceptive sensory organ development and the execution of specific leg-related behaviors. Through time-dependent conditional knock-out of Unc-4, it was found that its function is required during development, but not in the adult, to regulate the above events (Lacin, 2020).

How does a complex nervous system arise during development? Millions to billions of neurons, each one essentially unique, precisely interconnect to create a functional central nervous system (CNS) that drives animal behavior. Work over several decades shows that developmentally established layers of spatial and temporal organization underlie the genesis of a complex CNS. For example, during spinal cord development in vertebrates, different types of progenitor cells arise across the dorso-ventral axis and generate distinct neuronal lineages in a precise spatial and temporal order. The pMN progenitors are located in a narrow layer in the ventral spinal cord and generate all motor neurons. Similarly, twelve distinct pools of progenitors that arise in distinct dorso-ventral domains generate at least 22 distinct interneuronal lineages. Within each lineage, neurons appear to acquire similar identities: they express similar sets of transcription factors, use the same neurotransmitter, extend processes in a similar manner and participate in circuits executing a specific behavior (Lacin, 2020).

The adult Drosophila ventral nerve cord (VNC), like the vertebrate spinal cord, also manifests a lineage-based organization. The cellular complexity of the VNC arises from a set of segmentally repeated set of 30 paired and one unpaired neural stem cells (Neuroblasts [NBs]), which arise at stereotypic locations during early development. These individually identifiable NBs undergo two major phases of proliferation: the embryonic phase generates the functional neurons of the larval CNS, some of which are remodeled to function in the adult, and the post-embryonic phase generates most of the adult neurons. The division mode within NB lineages adds another layer to the lineage-based organization of the VNC. Each NB generates a secondary precursor cell, which divides via Notch-mediated asymmetric cell division to generate two neurons with distinct identities. After many rounds of such cell divisions, each NB ends up producing two distinct hemilineages of neurons, termed Notch-ON or the 'A' and Notch-OFF or the 'B' hemilineage. This paper focuses only on postembryonic hemilineages, which from this point on in the paper are refered to as hemilineages for simplicity. Within a hemilineage, neurons acquire similar fates based on transcription factor expression, neurotransmitter usage, and axonal projection. Moreover, neurons of each hemilineage appear dedicated for specific behaviors. For example, artificial neuronal activation of the glutamatergic hemilineage 2A neurons elicit specifically high frequency wing beating, while the same treatment of the cholinergic hemilineage 7B neurons leads to a specific take-off behavior. Thus, hemilineages represent the fundamental developmental and functional unit of the VNC (Lacin, 2020).

Previous work has mapped the embryonic origin, axonal projection pattern, transcription factor expression, and neurotransmitter usage of essentially all hemilineages in the adult Drosophila VNC (see Lacin, 2019; Shepherd, 2019). Here, this study leverages this information to elucidate how a specific transcription factor, Unc-4, acts within individual hemilineages during adult nervous system development to regulate neuronal connectivity and function, and animal behavior. Unc-4, an evolutionarily conserved transcriptional repressor, is expressed post-mitotically in seven of the 14 cholinergic hemilineages in the VNC: three 'A' -Notch-ON- hemilineages (11A, 12A, and 17A) and four 'B' -Notch-OFF- hemilineages (7B, 18B, 19B, and 23B). For four of the Unc-4+ hemilineages (7B, 17A, 18B, and 23B), the neurons of the sibling hemilineage undergo cell death. For the remaining three (11A, 12A, and 19B), the neurons of the sibling hemilineage are GABAergic (Lacin, 2019). Unc-4 expression in these hemilineages is restricted to postmitotic neurons and it appears to mark uniformly all neurons within a hemilineage during development and adult life (Lacin, 2014; Lacin, 2016; Lacin, 2020 and references therein).

This study generated a set of precise genetic tools that allowed uncovering of lineage-specific functions for Unc-4: in the 11A hemilineage, Unc-4 drives the cholinergic identity and suppresses the GABAergic fate; in the 7B hemilineage, Unc-4 promotes correct axonal projection patterns and the ability of flies to execute a stereotyped flight take-off behavior. This study also found that Unc-4 is expressed in the precursors of chordotonal sensory neurons and required for the development of these sensory organs, with functional data indicating Unc-4 functions in this lineage to promote climbing, walking, and grooming activities (Lacin, 2020).

Using precise genetic tools, this study dissected the function of the Unc-4 transcription factor in a lineage-specific manner. Within the PNS, Unc-4 function is needed for the proper development of the leg chordotonal organ and walking behavior; whereas in the CNS, Unc-4 dictates neurotransmitter usage within lineage 11A and regulates axonal projection and flight take-off behavior in lineage 7B. Below, are discussed three themes arising from this work: lineage-specific functions of individual transcription factors, an association of Unc4+ lineages with flight, and the lineage-based functional organization of the CNS in flies and vertebrates (Lacin, 2020).

Seven neuronal hemilineages express Unc-4 in the adult VNC, but the phenotypic studies revealed a function for Unc-4 in only two of them: in the 11A hemilineage, Unc-4 promotes the cholinergic fate and inhibits the GABAergic fate, while in the 7B hemilineage, Unc-4 ensures proper flight take-off behavior likely by promoting the proper projection patterns of the 7B interneurons into the leg neuropil. Why was no loss-of-function phenotype detected for Unc4 in most of the hemilineages in which it is expressed? A few reasons may explain this failure. First, the phenotypic analysis was limited: Neuronal projection patterns and neurotransmitter fate were detected, but not other molecular, cellular, or functional phenotypes. Unc-4 may function in other lineages to regulate other neuronal properties that were not assayed, such as neurotransmitter receptor expression, channel composition, synaptic partner choice, and/or neuronal activity. In addition, as this analysis assayed all cells within the lineage, it would have missed defects that occur in single cells or small groups of cells within the entire hemilineage. Second, Unc-4 may act redundantly with other transcription factors to regulate the differentiation of distinct sets of neurons. Genetic redundancy among transcription factors regulating neuronal differentiation is commonly observed in the fly VNC. Thus, while the research clearly identifies a role for Unc-4 in two hemilineages, it does not exclude Unc-4 regulating more subtle cellular and molecular phenotypes in the other hemilineages in which it is expressed. Similarly, pan-neuronal deletion of Unc-4 specifically in the adult did not lead to any apparent behavioral defect even though Unc-4 expression is maintained in all Unc-4+ lineages throughout adult life, suggesting that Unc-4 function is dispensable in mature neurons after eclosion under standard lab conditions. Future work will be required to ascertain whether Unc-4 functions during adult life or in more than two of its expressing hemilineages during development. Nonetheless, this work shows that Unc-4 executes distinct functions in the 7B and 11A lineages. The Hox transcription factors, Ubx, Dfd, Scr, and Antp, have also been shown to execute distinct functions in different lineages in the fly CNS, suggesting transcription factors may commonly drive distinct cellular outcomes in the context of different lineages. What underlies this ability of one transcription factor to regulate distinct cellular events in different neuronal lineages? The ancient nature of the lineage-specific mode of CNS development likely holds clues to this question. The CNS of all insects arises via the repeated divisions of a segmentally repeated array of neural stem cells whose number, ~30 pairs per hemisegment, has changed little over the course of insect evolution. Within this pattern, each stem cell possessing a unique identity based on its position and time of formation. Each stem cell lineage has then evolved independently of the others since at least the last common ancestor of insects, approximately 500 million years ago. Thus, if during evolution an individual transcription factor became expressed in multiple neuronal lineages after this time, it would not be surprising that it would execute distinct functions in different neuronal lineages. The lineage-specific evolution of the CNS development in flies, worms, and vertebrates may explain why neurons of different lineages that share specific properties, for example, neurotransmitter expression, may employ distinct transcriptional programs to promote this trait (Lacin, 2020).

Although Unc-4 appears to have distinct functions in different lineages, this study found that an association with flight is a unifying feature among most Unc4+ interneuron lineages and motor neurons. All Unc-4+ hemilineages in the adult VNC except the 23B hemilineage heavily innervate the dorsal neuropils of the VNC, which are responsible for flight motor control and wing/haltere related behaviors, including wing-leg coordination. For example, hemilineages 7B, 11A, and 18B regulate flight take-off behavior and 12A neurons control wing-based courtship singing (Harris et al., 2015; Shirangi et al., 2016). In addition, most Unc-4+ motor neurons are also involved with flight - these include MN1-5, which innervate the indirect flight muscles, as well as motor neurons that innervate the haltere and neck muscles, which provide flight stabilization. Since Unc-4 is conserved from worms to humans, it is likely that Ametabolous insects, like silverfish, which are primitively wingless, also have unc-4. It has yet to be determined, though, whether in such ametabolous insects the same hemilineages express Unc-4, and hence this pattern was in place prior to the evolution of flight. This would suggest that there was some underlying association amongst this set of hemilineages that may have been exploited in the evolution of flight. Alternatively, Unc-4 may be lacking in these hemilineages prior to the evolution of flight but then its expression may have been acquired by these hemilineages as they were co-opted into a unified set of wing-related behaviors (Lacin, 2020).

The adult fly VNC is composed of 34 segmentally repeated hemilineages, which are groups of lineally related neurons with similar features for example, axonal projection and neurotransmitter expression. These hemilineages also appear to function as modular units, each unit appears responsible for regulating particular behaviors, indicating the VNC is assembled via a lineage-based functional organization. The vertebrate spinal cord exhibits similar organization: lineally-related neurons acquire similar fates ('cardinal classes') and function in the same or parallel circuits. The similarity of the lineage-based organization in insect and vertebrate nerve/spinal cords raises the question whether they evolved from a common ground plan or are an example of convergent evolution. Molecular similarities in CNS development between flies and vertebrates support both CNS's arise from a common ground plan. For example, motor neuron identity in both flies and vertebrates, use the same set of transcription factors: Nkx6, Isl, and Lim3. Moreover, homologs of many transcription factors expressed in fly VNC interneurons, such as Eve and Lim1, also function in interneurons of the vertebrate spinal cord. Whether any functional/molecular homology is present between fly and vertebrate neuronal classes awaits comparative genome-wide transcriptome analysis and functional characterization of neuronal classes in the insect VNC and vertebrate spinal cord (Lacin, 2020).

Strong purifying selection on the Odysseus gene in two clades of sibling species of the Drosophila montium species subgroup

The Odysseus (OdsH) gene was duplicated from its ancestral neuron-expressed gene, unc-4, and then evolved very rapidly under strong positive Darwinian selection as a speciation gene causing hybrid-male sterility between closely related species of the Drosophila simulans clade. Has OdsH also experienced similar positive selection between Drosophila sibling species other than those of the simulans clade? This study cloned and sequenced OdsH and unc-4 from two clades of the Drosophila montium species subgroup, the Drosophila lini and the Drosophila kikkawai clades. The ratios of Ka/Ks for OdsH were remarkably low between sibling species of these two clades, suggesting that OdsH has been subjected to strong purifying selection in these two clades (Wen, 2006).

Gene duplication and speciation in Drosophila: evidence from the Odysseus locus

The importance of gene duplication in evolution has long been recognized. Because duplicated genes are prone to diverge in function, gene duplication could plausibly play a role in species differentiation. However, experimental evidence linking gene duplication with speciation is scarce. This study shows that a hybrid-male sterility gene, Odysseus (OdsH), arose by gene duplication in the Drosophila genome. OdsH has evolved at a very high rate, whereas its most immediate paralog, unc-4, is nearly identical among species in the Drosophila melanogaster subgroup. The disparity in their sequence evolution is echoed by the divergence in their expression patterns in both soma and reproductive tissues. It is suggested that duplicated genes that have yet to evolve a stable function at the time of speciation may be candidates for "speciation genes," which is broadly defined as genes that contribute to differential adaptation between species (Ting, 2004).

A novel Drosophila paired-like homeobox gene related to Caenorhabditis elegans unc-4 is expressed in subsets of postmitotic neurons and epidermal cells

A novel Drosophila paired-like homeobox gene, DPHD-1, has been isolated. The homeodomain of DPHD-1 showed 85% amino-acid identity with that of the C. elegans Unc-4 protein. Whole-mount in situ hybridization of embryos and third-instar larvae revealed that the DPHD-1 mRNA is specifically localized in subsets of postmitotic neurons in the central nervous system (CNS) and in the developing epidermis with a segmentally repeated pattern. Double staining with a posterior compartment marker, an anti-Engrailed antibody, showed that DPHD-1 expressing neurons in the CNS were present in the posterior compartment, whereas DPHD-1 expression in the epidermis was restricted to the anterior compartment in each segment. This temporal and spatial expression pattern suggests that DPHD-1 may play a role in determining the distinct cell types in each segment (Tabuchi, 1998).

Identification of avoidance genes through neural pathway-specific forward optogenetics

Understanding how the nervous system bridges sensation and behavior requires the elucidation of complex neural and molecular networks. Forward genetic approaches, such as screens conducted in C. elegans, have successfully identified genes required to process natural sensory stimuli. However, functional redundancy within the underlying neural circuits, which are often organized with multiple parallel neural pathways, limits one's ability to identify 'neural pathway-specific genes', i.e. genes that are essential for the function of some, but not all of these redundant neural pathways. To overcome this limitation, a 'forward optogenetics' screening strategy was developed in which natural stimuli are initially replaced by the selective optogenetic activation of a specific neural pathway. This strategy was used to address the function of the polymodal FLP nociceptors mediating avoidance of noxious thermal and mechanical stimuli. According to expectations, mutations were identified in 'general' avoidance genes that broadly impact avoidance responses to a variety of natural noxious stimuli (unc-4, unc-83, and eat-4) and mutations were identified that produce a narrower impact, more restricted to the FLP pathway (syd-2, unc-14 and unc-68). Through a detailed follow-up analysis, this study further showed that the Ryanodine receptor UNC-68 acts cell-autonomously in FLP to adjust heat-evoked calcium signals and aversive behaviors. As a whole, this work (i) reveals the importance of properly regulated ER calcium release for FLP function, (ii) provides new entry points for new nociception research and (iii) demonstrates the utility of the forward optogenetic strategy, which can easily be transposed to analyze other neural pathways (Marques, 2019).

Although epigenetic control of stem cell fate choice is well established, little is known about epigenetic regulation of terminal neuronal differentiation. This study found that some differences among the subtypes of Caenorhabditis elegans VC neurons, particularly the expression of the transcription factor gene unc-4, require histone modification, most likely H3K9 methylation. An EGF signal from the vulva alleviated the epigenetic repression of unc-4 in vulval VC neurons but not the more distant nonvulval VC cells, which kept unc-4 silenced. Loss of the H3K9 methyltransferase MET-2 or H3K9me2/3 binding proteins HPL-2 and LIN-61 or a novel chromodomain protein CEC-3 caused ectopic unc-4 expression in all VC neurons. Downstream of the EGF signaling in vulval VC neurons, the transcription factor LIN-11 and histone demethylases removed the suppressive histone marks and derepressed unc-4. Behaviorally, expression of UNC-4 in all the VC neurons caused an imbalance in the egg-laying circuit. Thus, epigenetic mechanisms help establish subtype-specific gene expression, which are needed for optimal activity of a neural circuit (Zheng, 2013).

UNC-4 antagonizes Wnt signaling to regulate synaptic choice in the C. elegans motor circuit

Coordinated movement depends on the creation of synapses between specific neurons in the motor circuit. In C. elegans, this important decision is regulated by the UNC-4 homeodomain protein. unc-4 mutants are unable to execute backward locomotion because VA motor neurons are mis-wired with inputs normally reserved for their VB sisters. It wa proposed that UNC-4 functions in VAs to block expression of VB genes. This model is substantiated by the finding that ectopic expression of the VB gene ceh-12 (encoding a homolog of the homeodomain protein HB9) in unc-4 mutants results in the mis-wiring of posterior VA motor neurons with VB-like connections. This study shows that VA expression of CEH-12 depends on a nearby source of the Wnt protein EGL-20. The results indicate that UNC-4 prevents VAs from responding to a local EGL-20 cue by disabling a canonical Wnt signaling cascade involving the Frizzled receptors MIG-1 and MOM-5. CEH-12 expression in VA motor neurons is also opposed by a separate pathway that includes the Wnt ligand LIN-44. This work has revealed a transcriptional mechanism for modulating the sensitivity of specific neurons to diffusible Wnt ligands and thereby defines distinct patterns of synaptic connectivity. The existence of comparable Wnt gradients in the vertebrate spinal cord could reflect similar roles for Wnt signaling in vertebrate motor circuit assembly (Schneider, 2012).

Distinct mechanisms for delimiting expression of four Caenorhabditis elegans transcription factor genes encoding activators or repressors

Regulatory transcription factors operate in networks, conferring biological robustness that makes dissection of such gene control processes difficult. The nematode Caenorhabditis elegans is a powerful molecular genetic system that allows the close scrutiny needed to understand these processes in an animal, in vivo. Strikingly lower levels of gene expression were observed when a gfp reporter was inserted into C. elegans transcription factor genes, in their broader genomic context, in comparison to when the reporter was fused to just the promoter regions. The lower level of expression is more consistent with endogenous levels of the gene products, based on independent protein and transcript assays. Through successive precise manipulations of the reporter fusion genes, elements essential for the lower level of expression were localised to the protein-coding region. With a closer focus on four transcription factor genes, the expression of both genes encoding transcriptional activators was found to be restricted by a post-transcriptional mechanism while expression of both genes encoding transcriptional repressors was delimited by transcriptional repression. An element through which the transcriptional repression acts for unc-4 was localised to a 30 base-pair region of a protein-encoding exon, with potentially wider implications for how homeobox genes operate. The hypothesis that the distinction in mechanisms delimiting expression of the two types of transcription factor genes, as observed in this study, may apply more widely is raised. This leads to observations concerning the implications of these different mechanisms on stochastic noise in gene expression and the consequent significance for developmental decisions in general (Bamps, 2011).

The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans

The T-box transcription factor mab-9 has been shown to be required for the correct fate of the male-specific blast cells B and F, normal posterior hypodermal morphogenesis, and for the correct axon migration of motor neurons that project circumferential commissures to dorsal muscles. In this study, an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9:gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat-shock promoter has the opposite effect, causing repression of mab-9 in various cells. mab-9 expression in unc-37 mutants was also found elevated in DA motor neurons, consistent with known roles for UNC-37 as a co-repressor with UNC-4. These results identify mab-9 as a novel target of the UNC-4/UNC-37 repressor complex in motor neurons, and suggest that mis-expression of mab-9 may contribute to the neuronal wiring defects in unc-4 and unc-37 mutants (Jafari, 2011).

UNC-4 represses CEH-12/HB9 to specify synaptic inputs to VA motor neurons in C. elegans

In C. elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, a cell-specific microarray strategy was used that has identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. Expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord (Von Stetina, 2007).

Transcription factor cascades define the structure of the vertebrate motor circuit by regulating the differentiation of specific neurons that contribute to this network. A striking feature of these pathways is the frequent use of negative gene regulation to produce distinct fates between neurons generated from adjacent progenitor domains. This study shows that a similar mechanism of repression involving conserved transcriptional components distinguishes the fates of C. elegans motor neurons born as sisters from a common mother cell. These results also offer a strikingly new finding, an explicit link between this biological strategy and the choice of presynaptic partners, a developmental decision of critical importance to motor neuron function. A model is presented of transcriptional regulation of synaptic specificity in C. elegans and the possibility that related schemes may also define wiring in the vertebrate spinal cord is discussed (Von Stetina, 2007).

C. elegans mutants in the unc-4 homeodomain gene display a strong backward movement defect that results from the miswiring of VA class motor neurons with inputs normally reserved for VB motor neurons. Intriguingly, other aspects of VA cell fate (i.e., axon trajectory and process placement) are unchanged, suggesting that UNC-4 functions to control only the synaptic fate of this cell type. This study shows that this change in synaptic specificity depends in part on misexpression of the VB-specific transcription factor, CEH-12/HB9, in VA motor neurons. Normally, UNC-4 functions with UNC-37/Groucho to block ceh-12/HB9 expression in the VAs. Because HB9 is also believed to function as a transcriptional repressor in other organisms, it is proposed that ectopic CEH-12/HB9 in unc-4 and unc-37 mutants triggers miswiring by turning off genes that specify VA inputs. It is possible that ectopic CEH-12/HB9 also activates VB genes that drive the creation of VB-type inputs. These results provide strong genetic evidence for at least one additional pathway downstream from UNC-4 that functions in parallel to CEH-12/HB9. The relative contributions of these pathways to VA input specificity are biased along the anterior-posterior (A/P) axis with ectopic CEH-12 selectively driving the creation of VB inputs to posterior VA motor neurons in unc-4 mutants and the presumptive parallel pathway imposing VB inputs to anterior VAs. Finally, a third set of VB genes, glr-4, del-1, and acr-5 are negatively regulated by unc-4 but have no detectable role in the VA miswiring defect. These cell surface proteins and ion channel components could be indicative of physiologically important differences in the excitability or signaling capacity of VA versus VB motor neurons. In the future, it will be interesting to determine if ectopic ceh-12 expression contributes to the observed depletion of synaptic vesicles in unc-4 mutant neurons (Von Stetina, 2007).

Although ceh-12 is required for the imposition of VB-type inputs to posterior VA motor neurons in unc-4 mutants, inputs to most VB motor neurons apparently do not depend on ceh-12 activity. Two lines of evidence support this conclusion: (1) ceh-12 knock-out mutants do not show an obvious forward movement defect as would be expected if VB motor neurons were miswired; (2) the elimination of ceh-12 activity in these mutants does not perturb the creation of gap junctions between most VBs and AVB command interneurons. These data are consistent with the proposal that ceh-12 functions in parallel to a redundant pathway in VB motor neurons that is sufficient to retain VB-type inputs (Von Stetina, 2007).

This work describes the use of a GFP-tagged UNC-7S marker protein for visualizing gap junctions between specific neuron pairs in the C. elegans motor circuit. This assay has provided an unprecedented opportunity to score gap junction specificity in the light microscope in multiple animals and in a variety of different mutant backgrounds. These experiments indicate that the innexin, UNC-7S, is expressed in AVB command interneurons for assembly into gap junctions with B-class motor neurons. Genetic and physiological data suggest that these gap junctions are likely to be heterotypic, and also include the innexin UNC-9. The ectopic gap junctions between AVB and A-class motor neurons that appear in unc-4 mutants may have a similar subunit composition, since unc-9 is the most abundant innexin transcript expressed in A-class motor neurons. It follows that UNC-9 is also a likely candidate for assembly into gap junctions between VA and AVA command interneurons in wild-type animals. Gap junctions with AVB tend to be located on the motor neuron soma, whereas gap junctions with AVA are more often distributed along the length of the motor neuron partner. Thus, unc-4 may orchestrate the assembly of UNC-9 into gap junctions at particular locations within A-class motor neurons and with selected presynaptic partners. Although gap junctions have been previously thought to provide a largely developmental role in the generation of neural networks in higher vertebrates, recent evidence suggests that these 'electrical' synapses are also important for neural function in adult nervous systems. This view is consistent with ultrastructural and immunochemical data showing that gap junctions are widely distributed in the mature mammalian brain and spinal cord. Since the mechanisms that control the specificity of gap junction assembly in the vertebrate CNS are unknown, the discovery of downstream genes that regulate gap junction placement in C. elegans could provide targets for molecular studies in more complex nervous systems. Moreover, the joint regulation by unc-4 (or ceh-12) of the specificity of chemical and electrical synapse formation is indicative of a common nexus for pathways controlling the assembly of both types of synapses (Von Stetina, 2007).

These findings indicate that ceh-12 conspires with at least one additional pathway in VA motor neurons to control input specificity. unc-4 regulation of ceh-12 is restricted to VA motor neurons in the posterior region of the ventral nerve cord. Because anterior VA motor neurons are also miswired in unc-4 mutants, it is proposed that the presumptive downstream pathway functioning in parallel to ceh-12 may be selectively derepressed in anterior VAs. Other unc-4-regulated genes should be represented in the microarray profile of unc-37 mutant VA motor neurons. One plausible candidate in this data set that could function in parallel to ceh-12 is cog-1, the C. elegans homolog of the homeodomain transcription factor, Nkx6. In Drosophila, dHB9 and Nkx6 act together in ventrally projecting motor neurons to repress dorsal motor neuron traits. COG-1 regulates a similar decision in the C. elegans nervous system by preventing ASER sensory neurons from adopting characteristics normally reserved for ASEL. Potential COG-1 interactions with CEH-12 are suggested by the observation that cog-1::GFP is also expressed in VA and VB motor neurons. cog-1 and other candidate unc-4 target genes in the microarray data set that function in parallel to ceh-12 may be revealed by RNA interference (RNAi) tests currently underway to detect genes that enhance ceh-12-dependent suppression of the Unc-4 phenotype (i.e., improved backward locomotion). Conversely, RNAi of transcripts that are depleted in the unc-37 microarray data set and therefore potentially repressed by ectopic ceh-12 should result in an Unc-4 like movement defect if these genes are required for specifying VA-type inputs (Von Stetina, 2007).

The results showing that ceh-12 preserves VB motor neuron fate by repressing VAB-7/Eve, parallels earlier observations that HB9 regulates motor neuron differentiation in flies, birds, and mammals. In Drosophila, dHB9 is expressed in a subset of ventrally projecting motor neurons where it represses the dorsal motor neuron determinant, Eve, and blocks the adoption of a dorsal axon trajectory. Eve, in turn, opposes ventral fates in dorsal motor neurons by reciprocally repressing dHB9 in a Groucho-dependent mechanism. Interestingly, HB9 is also restricted to ventrally projecting motor neurons in the vertebrate spinal cord where it acts to prevent expression of markers for interneurons arising from the adjacent V2 progenitor domain. In this case, ectopic expression of HB9 in V2 neuroblasts is sufficient to drive expression of motor neuron markers as well as impose motor neuron-like morphological characteristics (i.e., ventral axonal projections). This dual function of HB9 to block as well as activate expression of motor neuron-specific traits is similar to the finding that CEH-12 inhibits VA motor neuron differentiation while simultaneously promoting a specific VB trait. Together, these observations suggest that the key role of HB9 function in motor neuron differentiation is evolutionarily ancient. In this regard, it is noted that the UNC-4 homolog, UNCX4.1, is strongly expressed in the V3 neural progenitor domain immediately adjacent to the MN region in which HB9 resides. It will be interesting to determine if UNCX4.1 functions in the V3 domain to block HB9 expression (Von Stetina, 2007).

Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior

Egg-laying behavior in Caenorhabditis elegans is activated by signaling through the G-protein G(rho)q and inhibited by signaling through a second G-protein, G(rho)o. Activation of egg laying depends on the serotonergic hermaphrodite-specific neurons (HSNs), but the neurotransmitter(s) and cell(s) that signal to inhibit egg laying are not known. Mutants for G-protein signaling genes have well characterized defects in egg laying. This study presents an analysis of mutants for other genes reported to lack inhibition of egg laying. Of the nine strongest, six have morphological defects in the ventral-type C (VC) neurons, which synapse onto both the HSNs and the egg-laying muscles and are thus the third cell type comprising the egg-laying system. Laser-ablating VC neurons could also disrupt the inhibition of egg laying. The remaining three mutants (unc-4, cha-1, and unc-17) are defective for synthesis or packaging of acetylcholine in the VCs. The egg-laying defects of unc-4, cha-1, and unc-17 were rescued by VC-specific expression of the corresponding cDNAs. In addition, increasing synaptic acetylcholine by reducing acetylcholinesterase activity, with either mutations or the inhibitor aldicarb, decreased egg laying. Finally, a knock-out for the HSN-expressed receptor G-protein-coupled acetylcholine receptor 2 (GAR-2) was found to show a partial defect in the inhibition of egg laying and fails to respond to aldicarb. These results show that acetylcholine released from the VC neurons inhibits egg-laying behavior. This inhibition may be caused, in part, by acetylcholine signaling onto the HSN presynaptic terminals, via GAR-2, to inhibit neurotransmitter release (Bany, 2003).

The C. elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory

Locomotory activity is defined by the specification of motoneurone subtypes. In the nematode, C. elegans, DA and DB motoneurones innervate dorsal muscles and function to induce movement in the backwards or forwards direction, respectively. These two neurone classes express separate sets of genes and extend axons with oppositely directed trajectories; anterior (DA) versus posterior (DB). The DA-specific homeoprotein UNC-4 interacts with UNC-37/Groucho to repress the DB gene, acr-5 (nicotinic acetylcholine receptor subunit). The C. elegans even-skipped-like homoedomain protein, VAB-7, coordinately regulates different aspects of the DB motoneurone fate, in part by repressing unc-4. Wild-type DB motoneurones express VAB-7, have posteriorly directed axons, express ACR-5 and lack expression of the homeodomain protein UNC-4. In a vab-7 mutant, ectopic UNC-4 represses acr-5 and induces an anteriorly directed DB axon trajectory. Thus, vab-7 indirectly promotes DB-specific gene expression and posteriorly directed axon outgrowth by preventing UNC-4 repression of DB differentiation. Ectopic expression of VAB-7 also induces DB traits in an unc-4-independent manner, suggesting that VAB-7 can act through a parallel pathway. This work supports a model in which a complementary pair of homeodomain transcription factors (VAB-7 and UNC-4) specifies differences between DA and DB neurones through inhibition of the alternative fates. The recent findings that Even-skipped transcriptional repressor activity specifies neurone identity and axon guidance in the mouse and Drosophila motoneurone circuit points to an ancient origin for homeoprotein-dependent mechanisms of neuronal differentiation in the metazoan nerve cord (Esmaeili, 2002).

Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons

Motor neuron function depends on neurotransmitter release from synaptic vesicles (SVs). The UNC-4 homeoprotein (Drosophila homolog: Unc-4) and its transcriptional corepressor protein UNC-37 regulate SV protein levels in specific C. elegans motor neurons. C. elegans UNC-4 is expressed in four classes (DA, VA, VC, and SAB) of cholinergic motor neurons. Antibody staining reveals that five different vesicular proteins (putative vesicular acetylcholine transporter UNC-17, choline acetyltransferase, Synaptotagmin, Synaptobrevin, and RAB-3) are substantially reduced in unc-4 and unc-37 mutants in these cells; nonvesicular neuronal proteins (Syntaxin, UNC-18, and UNC-11) are not affected, however. Ultrastructural analysis of VA motor neurons in a null unc-4 mutant confirms that SV number in the presynaptic zone is reduced (~40%) whereas axonal diameter and synaptic morphology are not visibly altered. Because the UNC-4-UNC-37 complex has been shown to mediate transcriptional repression, it is proposed that these effects are performed via an intermediate gene. These results are consistent with a model in which this unc-4 target gene ('gene-x') functions at a post-transcriptional level as a negative regulator of SV biogenesis or stability. Experiments with a temperature-sensitive unc-4 mutant show that the adult level of SV proteins strictly depends on unc-4 function during a critical period of motor neuron differentiation. unc-4 activity during this sensitive larval stage is also required for the creation of proper synaptic inputs to VA motor neurons. The temporal correlation of these events may mean that a common unc-4-dependent mechanism controls both the specificity of synaptic inputs as well as the strength of synaptic outputs for these motor neurons (Lickteig, 2001).

The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification

The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. This study reports the expression and functional analysis of cnd-1, a C. elegans NeuroD homolog. cnd-1 expression was first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes were expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibited multiple defects in the ventral cord motor neurons. First, the number of motor neurons was reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. These data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family (Hallam, 2000).

UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans

The UNC-4 homeoprotein and the Groucho-like corepressor UNC-37 specify synaptic choice in the Caenorhabditis elegans motor neuron circuit. In unc-4 mutants, VA motor neurons are miswired with inputs from interneurons normally reserved for their lineal sisters, the VB motor neurons. This study shows that UNC-4 and UNC-37 function together in VA motor neurons to repress VB-specific genes and that this activity depends on physical contact between UNC-37 and a conserved Engrailed-like repressor domain (eh1) in UNC-4. Missense mutations in the UNC-4 eh1 domain disrupt interactions between UNC-4 and UNC-37 and result in the loss of UNC-4-dependent repressor activity in vivo. A compensatory amino acid substitution in UNC-37 suppresses specific unc-4 alleles by restoring physical interactions with UNC-4 as well as UNC-4-dependent repression of VB-specific genes. It is proposed that repression of VB-specific genes by UNC-4 and UNC-37 is necessary for the creation of wild-type inputs to VA motor neurons. The existence of mammalian homologs of UNC-4 and UNC-37 indicates that a similar mechanism could regulate synaptic choice in the vertebrate spinal cord (Winnier, 1999).

Paired-related murine homeobox gene expressed in the developing sclerotome, kidney, and nervous system

We isolated a murine homeobox containing gene, Uncx4.1. The homeodomain sequence exhibits 88% identity to the unc-4 protein at the amino acid level. In situ hybridization analysis revealed that Uncx4.1 is expressed in the paraxial mesoderm, in the developing kidney, and central nervous system. The most intriguing expression domain is the somite, where it is confined to the caudal part of the newly formed somite and subsequently restricted to the caudal domain of the developing sclerotome. In the central nervous system, Uncx4.1 is detected in the developing spinal cord, hindbrain, mesencephalon, and telencephalon. The temporal and spatial expression pattern suggests that Uncx4.1 may play an important role in kidney development and in the differentiation of the sclerotome and the nervous system (Mansouri, 1997).

The Groucho-like transcription factor UNC-37 functions with the neural specificity gene unc-4 to govern motor neuron identity in C. elegans

Groucho and Tup1 are members of a conserved family of WD repeat proteins that interact with specific transcription factors to repress target genes. Mutations in WD domains of the Groucho-like protein, UNC-37, affect a motor neuron trait that also depends on UNC-4, a paired type homeodomain protein that controls neuronal specificity in Caenorhabditis elegans. In unc-4 mutants, VA motor neurons assume the pattern of synaptic input normally reserved for their lineal sister cells, the VB motor neurons; the loss of normal input to the VAs produces a distinctive backward movement defect. Substitution of a conserved residue (H to Y) in the fifth WD repeat in unc-37(e262) phenocopies the Unc-4 movement defect. Conversely, an amino acid change (E to K) in the sixth WD repeat of UNC-37 is a strong suppressor of unc-37(e262) and of specific unc-4 missense mutations. UNC-4 expression in the VA motor neurons specifies the wild-type pattern of presynaptic input. UNC-37 is also expressed in the VAs and unc-37 activity in these neurons is sufficient to restore normal movement to unc-37(e262) animals. It has been proposed that UNC-37 and UNC-4 function together to prevent expression of genes that define the VB pattern of synaptic inputs and thereby generate connections specific to the VA motor neurons. The WD repeat domains of UNC-37 and of the human homolog, TLE1, are functionally interchangeable in VA motor neurons which suggests that this highly conserved protein domain may also specify motor neuron identity and synaptic choice in more complex nervous systems. It is unknown whether UNC-4 and UNC-37 act in parallel pathways to regulate separate sets of target genes, or whether the two regulate a common set of target genes (Pflugrad, 1997).

Identification of novel paired homeodomain protein related to C. elegans unc-4 as a potential downstream target of MASH1

A novel paired homeodomain protein, PHD1, most closely related to C. elegans unc-4, has been identified by a differential RT-PCR method. Unc-4 has no paired domain (See Drosophila Paired) and is thus grouped separately from paired-homeodomains into a prd-like class. PHD1 is expressed in a narrow layer adjacent to the ventricular zone of the dorsal spinal cord, immediately following expression of MASH1 but preceding overt neuronal differentiation. Some cells coexpressing MASH1 and PHD1 can be seen, suggesting that these two genes are sequentially activated within the same lineage. In the olfactory sensory epithelium, PHD1 expression not only follows but is dependent upon MASH1 function, suggesting that PHD1 acts downstream of MASH1. A sequential action of bHLH and paired homeodomain proteins is apparent in other neurogenic lineages and may be a general feature of both vertebrate and invertebate neurogenesis (Saito, 1996).

Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input

In the nematode, Caenorhabditis elegans, VA and VB motor neurons arise from a common precursor cell but adopt different morphologies and synapse with separate sets of interneurons in the ventral nerve cord. A mutation that inactivates the unc-4 homeodomain gene causes VA motor neurons to assume the VB pattern of synaptic input while retaining normal axonal polarity and output; the disconnection of VA motor neurons from their usual presynaptic partners blocks backward locomotion. This study shows that expression of a functional unc-4-beta-galactosidase chimeric protein in VA motor neurons restores wild-type movement to an unc-4 mutant. It is proposed that unc-4 controls a differentiated characteristic of the VA motor neurons that distinguishes them from their VB sisters, thus dictating recognition by the appropriate interneurons. These results show that synaptic choice can be controlled at the level of transcription in the post-synaptic neuron and identify a homeoprotein that defines a subset of cell-specific traits required for this choice (Miller, 1995).

Dominant unc-37 mutations suppress the movement defect of a homeodomain mutation in unc-4, a neural specificity gene in Caenorhabditis elegans

The unc-4 gene of Caenorhabditis elegans encodes a homeodomain protein that defines synaptic input to ventral cord motor neurons. unc-4 mutants are unable to crawl backward because VA motor neurons are miswired with synaptic connections normally reserved for their sister cells, the VB motor neurons. These changes in connectivity are not accompanied by any visible effects upon neuronal morphology, which suggests that unc-4 regulates synaptic specificity but not axonal guidance or outgrowth. In an effort to identify other genes in the unc-4 pathway, a selection scheme was devised for rare mutations that suppress the Unc-4 phenotype. Four, dominant, extragenic, allele-specific suppressors of unc-4(e2322ts), a temperature sensitive allele with a point mutation in the unc-4 homeodomain, were isolated. The data indicate that these suppressors are gain-of-function mutations in the previously identified unc-37 gene. The loss-of-function mutation unc-37(e262) phenocopies the Unc-4 movement defect but does not prevent unc-4 expression or alter VA motor neuron morphology. These findings suggest that unc-37 functions with unc-4 to specify synaptic input to the VA motor neurons. It is proposed that unc-37 may be regulated by unc-4. Alternatively, unc-37 may encode a gene product that interacts with the unc-4 homeodomain (Miller, 1993).

Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons

Identification of the genes orchestrating neurogenesis would greatly enhance understanding of this process. This study sought genes defining synaptic specificity by identifying mutations that alter synaptic connectivity in the motor circuitry in the nematode C. elegans. Electron microscopy of serial sections were used to reconstruct the ventral nerve-cords of uncoordinated (unc) mutants that have distinctive locomotory choreographies. This study describes the phenotype of mutations in the unc-4 gene in which a locomotory defect is correlated with specific changes in synaptic input to a subset of the excitatory VA motor neurons, normally used in reverse locomotion. The circuitry alterations do not arise because of the inaccessibility of the appropriate synaptic partners, but are a consequence of changes in synaptic specificity. The VA motor neurons with altered synaptic inputs are all lineal sisters of VB motor neurons; the VA motor neurons without VB sisters have essentially the same synaptic inputs as in wild-type animals. The normal function of the wild-type allele of unc-4 may thus be to invoke the appropriate synaptic specificities to VA motor neurons produced in particular developmental contexts (White, 1992).

C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons

The creation of neural circuits depends on the formation of synapses between specific sets of neurons. Little is known, however, of the molecular mechanisms governing synaptic choice. A mutation in the unc-4 gene alters the pattern of synaptic input to one class of motor neurons in the Caenorhabditis elegans ventral nerve cord. In unc-4(e120), the presynaptic partners of VA motor neurons are replaced with interneurons appropriate to motor neurons of the VB class. This change in neural specificity is not accompanied by any detectable effects on neuronal morphology or process extension. The absence of a functional unc-4 gene product accounts for the mutant phenotype. The unc-4 gene encodes a homeodomain protein and thus is likely to function as a transcription factor. The limited effect of the unc-4 null mutation on cell fate may mean that unc-4 regulates the expression of a small number of target genes and that the products of these genes are directly involved in the choice of synaptic partners (Miller, 1992).

Search PubMed for articles about Drosophila

Bamps, S., Wirtz, J. and Hope, I. A. (2011). Distinct mechanisms for delimiting expression of four Caenorhabditis elegans transcription factor genes encoding activators or repressors. Mol Genet Genomics 286(2): 95-107. PubMed ID: 21655972

Bany, I. A., Dong, M. Q. and Koelle, M. R. (2003). Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior. J Neurosci 23(22): 8060-8069. PubMed ID: 12954868

Esmaeili, B., Ross, J. M., Neades, C., Miller, D. M. and Ahringer, J. (2002). The C. elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory. Development 129(4): 853-862. PubMed ID: 11861469

Hallam, S., Singer, E., Waring, D. and Jin, Y. (2000). The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate specification. Development 127(19): 4239-4252. PubMed ID: 10976055

Jafari, G., Appleford, P. J., Seago, J., Pocock, R. and Woollard, A. (2011). The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans. Mech Dev 128(1-2): 49-58. PubMed ID: 20933597

Lacin, H., Zhu, Y., Wilson, B. A. and Skeath, J. B. (2014). Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system. Development 141(5): 1011-1021. PubMed ID: 24550109

Lacin, H. and Truman, J. W. (2016). Lineage mapping identifies molecular and architectural similarities between the larval and adult Drosophila central nervous system. Elife 5: e13399. PubMed ID: 26975248

Lacin, H., Chen, H. M., Long, X., Singer, R. H., Lee, T. and Truman, J. W. (2019). Neurotransmitter identity is acquired in a lineage-restricted manner in the Drosophila CNS. Elife 8. PubMed ID: 30912745

Lacin, H., Williamson, W. R., Card, G. M., Skeath, J. B. and Truman, J. W. (2020). Unc-4 acts to promote neuronal identity and development of the take-off circuit in the Drosophila CNS. Elife 9. PubMed ID: 32216875

Lickteig, K. M., Duerr, J. S., Frisby, D. L., Hall, D. H., Rand, J. B. and Miller, D. M., 3rd (2001). Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons. J Neurosci 21(6): 2001-2014. PubMed ID: 11245684

Mansouri, A., Yokota, Y., Wehr, R., Copeland, N. G., Jenkins, N. A. and Gruss, P. (1997). Paired-related murine homeobox gene expressed in the developing sclerotome, kidney, and nervous system. Dev Dyn 210(1): 53-65. PubMed ID: 9286595

Marques, F., Saro, G., Lia, A. S., Poole, R. J., Falquet, L. and Glauser, D. A. (2019). Identification of avoidance genes through neural pathway-specific forward optogenetics. PLoS Genet 15(12): e1008509. PubMed ID: 31891575

Miller, D. M. and Niemeyer, C. J. (1995). Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input. Development 121(9): 2877-2886. PubMed ID: 7555714

Miller, D. M., Niemeyer, C. J. and Chitkara, P. (1993). Dominant unc-37 mutations suppress the movement defect of a homeodomain mutation in unc-4, a neural specificity gene in Caenorhabditis elegans. Genetics 135(3): 741-753. PubMed ID: 7904971

Miller, D. M., Shen, M. M., Shamu, C. E., Burglin, T. R., Ruvkun, G., Dubois, M. L., Ghee, M. and Wilson, L. (1992). C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons. Nature 355(6363): 841-845. PubMed ID: 1347150

Pflugrad, A., et al. (1997). The Groucho-like transcription factor UNC-37 functions with the neural specificity gene unc-4 to govern motor neuron identity in C. elegans. Development 124: 1699-1709. PubMed Citation: 9165118

Saito, T., Lo, L., Anderson, D. J. and Mikoshiba, K. (1996). Identification of novel paired homeodomain protein related to C. elegans unc-4 as a potential downstream target of MASH1. Dev Biol 180(1): 143-155. PubMed ID: 8948581

Schneider, J., Skelton, R. L., Von Stetina, S. E., Middelkoop, T. C., van Oudenaarden, A., Korswagen, H. C. and Miller, D. M., 3rd (2012). UNC-4 antagonizes Wnt signaling to regulate synaptic choice in the C. elegans motor circuit. Development 139(12): 2234-2245. PubMed ID: 22619391

Shepherd, D., Sahota, V., Court, R., Williams, D. W. and Truman, J. W. (2019). Developmental organization of central neurons in the adult Drosophila ventral nervous system. J Comp Neurol 527(15): 2573-2598. PubMed ID: 30919956

Tabuchi, K., Yoshikawa, S., Yuasa, Y., Sawamoto, K. and Okano, H. (1998). A novel Drosophila paired-like homeobox gene related to Caenorhabditis elegans unc-4 is expressed in subsets of postmitotic neurons and epidermal cells. Neurosci Lett 257(1): 49-52. PubMed ID: 9857963

Ting, C. T., Tsaur, S. C., Sun, S., Browne, W. E., Chen, Y. C., Patel, N. H. and Wu, C. I. (2004). Gene duplication and speciation in Drosophila: evidence from the Odysseus locus. Proc Natl Acad Sci U S A 101(33): 12232-12235. PubMed ID: 15304653

Von Stetina, S. E., Fox, R. M., Watkins, K. L., Starich, T. A., Shaw, J. E. and Miller, D. M., 3rd (2007). UNC-4 represses CEH-12/HB9 to specify synaptic inputs to VA motor neurons in C. elegans. Genes Dev 21(3): 332-346. PubMed ID: 17289921

Wen, S. Y., Shimada, K., Kawai, K. and Toda, M. J. (2006). Strong purifying selection on the Odysseus gene in two clades of sibling species of the Drosophila montium species subgroup. J Mol Evol 62(5): 659-662. PubMed ID: 16612548

White, J. G., Southgate, E. and Thomson, J. N. (1992). Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons. Nature 355(6363): 838-841. PubMed ID: 1538764

Winnier, A. R., Meir, J. Y., Ross, J. M., Tavernarakis, N., Driscoll, M., Ishihara, T., Katsura, I. and Miller, D. M. (1999). UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans. Genes Dev 13(21): 2774-2786. PubMed ID: 10557206

Zheng, C., Karimzadegan, S., Chiang, V. and Chalfie, M. (2013). Histone methylation restrains the expression of subtype-specific genes during terminal neuronal differentiation in Caenorhabditis elegans. PLoS Genet 9(12): e1004017. PubMed ID: 24348272

date revised: 15 September 2020

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