The T-box gene tbx-2, the homeobox gene egl-5 and the asymmetric cell division gene ham-1 specify neural fate in the HSN/PHB lineage

Understanding how neurons adopt particular fates is a fundamental challenge in developmental neurobiology. To address this issue, we have been studying a Caenorhabditis elegans lineage that produces the HSN motor neuron and the PHB sensory neuron, sister cells produced by the HSN/PHB precursor. We have previously shown that the novel protein HAM-1 controls the asymmetric neuroblast division in this lineage. In this study we examine tbx-2 and egl-5, genes that act in concert with ham-1 to regulate HSN and PHB fate. In screens for mutants with abnormal HSN development, we identified the T-box protein TBX-2 as being important for both HSN and PHB differentiation. TBX-2, along with HAM-1, regulates the migrations of the HSNs and prevents the PHB neurons from adopting an apoptotic fate. The homeobox gene egl-5 has been shown to regulate the migration and later differentiation of the HSN. While mutations that disrupt its function show no obvious role for EGL-5 in PHB development, loss of egl-5 in a ham-1 mutant background leads to PHB differentiation defects. Expression of EGL-5 in the HSN/PHB precursor but not in the PHB neuron suggests that EGL-5 specifies precursor fate. These observations reveal a role for both EGL-5 and TBX-2 in neural fate specification in the HSN/PHB lineage.     

Otx-dependent expression of proneural bHLH genes establishes a neuronal bilateral asymmetry in C. elegans

Bilateral asymmetry in Caenorhabditis elegans arises in part from cell lineages that differ on the left and right sides of the animal. The unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that generates the MI neuron on the right and the e3D epithelial cell on the left. We isolated mutations in three genes that caused left-right symmetry in this normally asymmetric cell lineage by transforming MI into an e3D-like cell. These genes encode the proneural bHLH proteins NGN-1 and HLH-2 and the Otx homeodomain protein CEH-36. We identified the precise precursor cells in which ceh-36 and ngn-1 act, and showed that CEH-36 protein is asymmetrically expressed and is present in an MI progenitor cell on the right but not in its bilateral counterpart. This asymmetric CEH-36 expression promotes asymmetric ngn-1 and hlh-2 expression, which in turn induces asymmetric MI neurogenesis. Our results indicate that this left-right asymmetry is specified within the two sister cells that first separate the left and right branches of the cell lineage. We conclude that the components of an evolutionarily conserved Otx/bHLH pathway act sequentially through multiple rounds of cell division on the right to relay an initial apparently cryptic asymmetry to the presumptive post-mitotic MI neuron, thereby creating an anatomical bilateral asymmetry in the C. elegans nervous system.     

The myogenic potency of HLH-1 reveals wide-spread developmental plasticity in early C. elegans embryos

In vertebrates, striated muscle development depends on both the expression of members of the myogenic regulatory factor family (MRFs) and on extrinsic cellular cues, including Wnt signaling. The 81 embryonically born body wall muscle cells in C. elegans are comparable to the striated muscle of vertebrates. These muscle cells all express the gene hlh-1, encoding HLH-1 (CeMyoD) which is the only MRF-related factor in the nematode. However, genetic studies have shown that body wall muscle development occurs in the absence of HLH-1 activity, making the role of this factor in nematode myogenesis unclear. By ectopically expressing hlh-1 in early blastomeres of the C. elegans embryo, we show that CeMyoD is a bona fide MRF that can convert almost all cells to a muscle-like fate, regardless of their lineage of origin. The window during which ectopic HLH-1 can function is surprisingly broad, spanning the first 3 hours of development when cell lineages are normally established and non-muscle cell fate markers begin to be expressed. We have begun to explore the maternal factors controlling zygotic hlh-1 expression. We find that the Caudal-related homeobox factor PAL-1 can activate hlh-1 in blastomeres that either lack POP-1/TCF or that have down-regulated POP-1/TCF in response to Wnt/MAP kinase signaling. The potent myogenic activity of HLH-1 highlights the remarkable developmental plasticity of early C. elegans blastomeres and reveals the evolutionary conservation of MyoD function.     

MOM-5 frizzled regulates the distribution of DSH-2 to control C. elegans asymmetric neuroblast divisions

Asymmetric cell divisions produce all 302 neurons of the C. elegans hermaphrodite. Here, we describe a role for a C. elegans Dishevelled homolog, DSH-2, in an asymmetric neuroblast division. In dsh-2 mutants, neurons normally descended from the anterior neuroblast daughter of the ABpl/rpppa blast cell were frequently duplicated, while non-neuronal cells produced by the posterior daughter cell were often missing. These observations indicate that in the absence of dsh-2 function, the posterior daughter cell was transformed into a second anterior-like cell. Loss of mom-5, a C. elegans frizzled homolog, produced a similar phenotype. We also show that the DSH-2 protein localized to the cell cortex in most cells of the embryo. In the absence of MOM-5/Fz, DSH-2 was localized to the cytoplasm, suggesting that MOM-5 regulates asymmetric cell division by controlling the localization of DSH-2. Although all neurons in C. elegans are produced by an invariant pattern of cell divisions, our results indicate that cell signaling may contribute to asymmetric neuroblast division during embryogenesis.     

hlh-12, a gene that is necessary and sufficient to promote migration of gonadal regulatory cells in Caenorhabditis elegans,evolved within the Caenorhabditis clade

Specialized cells of the somatic gonad primordium of nematodes play important roles in the final form and function of the mature gonad. Caenorhabditis elegans hermaphrodites are somatic females that have a two-armed, U-shaped gonad that connects to the vulva at the midbody. The outgrowth of each gonad arm from the somatic gonad primordium is led by two female distal tip cells (fDTCs), while the anchor cell (AC) remains stationary and central to coordinate uterine and vulval development. The bHLH protein HLH-2 and its dimerization partners LIN-32 and HLH-12 had previously been shown to be required for fDTC specification. Here, we show that ectopic expression of both HLH-12 and LIN-32 in cells with AC potential transiently transforms them into fDTC-like cells. Furthermore, hlh-12 was known to be required for the fDTCs to sustain gonad arm outgrowth. Here, we show that ectopic expression of HLH-12 in the normally stationary AC causes displacement from its normal position and that displacement likely results from activation of the leader program of fDTCs because it requires genes necessary for gonad arm outgrowth. Thus, HLH-12 is both necessary and sufficient to promote gonadal regulatory cell migration. As differences in female gonadal morphology of different nematode species reflect differences in the fate or migratory properties of the fDTCs or of the AC, we hypothesized that evolutionary changes in the expression of hlh-12 may underlie the evolution of such morphological diversity. However, we were unable to identify an hlh-12 ortholog outside of Caenorhabditis. Instead, by performing a comprehensive phylogenetic analysis of all Class II bHLH proteins in multiple nematode species, we found that hlh-12 evolved within the Caenorhabditis clade, possibly by duplicative transposition of hlh-10. Our analysis suggests that control of gene regulatory hierarchies for gonadogenesis can be remarkably plastic during evolution without adverse phenotypic consequence.     

TRIM-9 functions in the UNC-6/UNC-40 pathway to regulate ventral guidance

TRIpartite Motif (TRIM) family proteins are ring finger domain-containing, multi-domain proteins implicated in many biological processes.Members of the TRIM-9/C-I subfamily of TRIM proteins, including TRIM-9, MIDI and MID2, have neuronal functions and are associated with neurological diseases. To explore whether the functions of C-I TRIM proteins are conserved in invertebrates, we analyzed Caenorhabditis elegans and Drosophila trim-9 mutants. C. elegans trim-9 mutants exhibit defects in the ventral guidance of hermaphrodite specific neuron (HSN) and the touch neuron AVM. Further genetic analyses indicate that TRIM-9 participates in the UNC-6-UNC-40 attraction pathway.Asymmetric distribution of UNC-40 during HSN development is normal in trim-9 mutants. However, the asymmetric localization of MIG-10,a downstream effector of UNC-40, is abolished in trim-9 mutants. These results suggest that TRIM-9 functions upstream of MIG-1 0 in the UNC40 pathway. Moreover, we showed that TRIM-9 exhibits E3 ubiquitin ligase activity in vitro and this activity is important for TRIM-9 function in vivo. Additionally, we found that Drosophila trim-9 is required for the midline attraction of a group of sensory neuron axons. Over-expression of the Netrin/UNC-6 receptor Frazzled suppresses the guidance defects in trim-9 mutants. Our study reveals an evolutionarily conserved function of TRIM-9 in the UNC-40/Frazzled-mediated UNC-6/Netrin attraction pathway.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.     

DSH-2 regulates asymmetric cell division in the early C. elegans somatic gonad

Like other organs, the C. elegans gonad develops from a simple primordium that must undergo axial patterning to generate correct adult morphology. Proximal/distal (PD) polarity in the C. elegans gonad is established early during gonadogenesis by the somatic gonad precursor cells, Z1 and Z4. Z1 and Z4 each divide asymmetrically to generate one daughter with a proximal fate and one with a distal fate. PD polarity of the Z1/Z4 lineages requires the activity of a Wnt pathway that activates the TCF/LEF homolog pop-1. How the gonadal pathway controlling pop-1 is regulated by upstream factors has been unclear, as neither Wnt nor Dishevelled (Dsh) proteins have been shown to be required. Here we show that the C. elegansdsh homolog dsh-2 controls gonadal polarity. As in pop-1 mutants, dsh-2 hermaphrodites have Z1 and Z4 lineage defects indicative of defective PD polarity and are missing gonadal arms. Males have an elongated but disorganized gonad, also with lineage defects. DSH-2 protein is expressed in the Z1/Z4 gonadal precursor cells. Asymmetric distribution of nuclear GFP::POP-1 in Z1 and Z4 daughter cells is reversed in dsh-2 mutants, with higher levels in distal than proximal daughters. dsh-2 and the frizzled receptor homolog lin-17 have a strong genetic interaction, suggesting that they act in a common pathway. We suggest that DSH-2 functions as an upstream regulator of POP-1 in the somatic gonad to control asymmetric cell division, thereby establishing proximal-distal polarity of the developing organ.