Douglas Portman et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Neural cell fate specification in the C. elegans male tail rays"

Douglas Portman, Renee Miller, Kwi Yeon Lee

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Understanding how multiple neural and glial cell types are generated from common precursor cells is a central problem in developmental neuroscience. We are using the rays, sensory organs in the C. elegans male tail, to characterize the genetic mechanisms that underlie these processes. Each of the 18 rays develops from a single ray neuroblast precursor. During L3 and L4, each ray neuroblast undergoes two rounds of asymmetric division to give rise to one programmed cell death and the three cells of each ray: the RnA and RnB sensory neurons and the Rnst structural cell. It has previously been shown that the atonal-family bHLH transcription factor LIN-32 is a critical mediator of ray development. In lin-32(null) males, nearly all rays are missing; lineage analysis suggests that this results from a failure to specify the ray neuroblast fate. Consistent with this, expression of lin-32::GFP in the male seam is first detectable in the ray neuroblasts. Thereafter, lin-32 expression remains strong only in the anterior branch of the ray sublineage, which gives rise to the RnA ray neuron. This raises the possibility that asymmetric lin-32 expression may also act to pattern neural fates among the progeny of the ray precursor cell. Consistent with this idea, we have found that weak alleles of the Wnt receptor lin-17 cause the generation of additional cells expressing lin-32::GFP and result in the development of additional RnA-like neurons. We have also found that the forced expression of lin-32 throughout the ray sublineage leads to defects in RnB development, suggesting that lin-32 is part of a mechanism that distinguishes the RnA and RnB cell fates. In addition, we are carrying out genetic screens using fluorescent ray neuron markers to identify additional mutants that disrupt ray development. Two new mutations, fs4 and fs5, cause defects in the expression of the RnB marker pkd-2::GFP without obvious morphological defects in the rays. This suggests that these mutations may affect a pathway specifically required for RnB development and/or differentiation. We are currently working toward the molecular identification of these genes.

Douglas Portman et al. (2005) International Worm Meeting "Specification of cell fates in the ray sublineage by the proneural gene lin-32 and the LIM-HD gene lim-7"

Douglas Portman, Kenneth Stewart, Kwi Yeon Lee

[International Worm Meeting, 2005]

Each of the 18 rays in the C. elegans male tail develops from a single ray neuroblast. In L3/L4 males, the ray neuroblast undergoes two rounds of asymmetric division to give rise to one programmed cell death and the three cells of each ray: the RnA and RnB sensory neurons and the Rnst structural cell. We are interested in the mechanism that governs the precise generation of these different but related cell types from a common precursor. We have previously shown that the bHLH transcription factor lin-32 is required for multiple steps of ray development. In the ray neuroblast, lin-32 specifies neural competence. Later, lin-32 is independently required for the proper differentiation of each branch of the ray sublineage. lin-32 expression is consistent with this model: activation of lin-32::GFP in the male seam is first detectable in the ray neuroblasts and continues until the final division of the ray sublineage. We have recently found that the expression of lin-32::GFP persists asymmetrically at the end of the sublineage, suggesting that the pattern of lin-32 expression may be important for patterning ray cell fates. However, other regulatory factors are likely to act downstream of or in parallel with lin-32 to create diversity in the ray lineage. The LIM-homeodomain gene lim-7 is an excellent candidate for such a factor. We identified this gene, the C. elegans Islet ortholog, in a microarray screen for new ray-expressed genes. lim-7::GFP is expressed transiently during the ray sublineage, in a more restricted pattern than lin-32, suggesting that it may act in a single ray cell type. In mosaic experiments using the lethal lim-7(tm674) deletion allele we have found that lim-7 is likely to be required for the expression of the tryptophan hydroxylase tph-1 in the RnB neurons. However, the expression of another RnB marker, pkd-2, is not affected, indicating that lim-7 does not control all aspects of RnB fate. To identify additional factors important for RnB specification, we are studying the cis-regulatory elements in a battery of seven genes, cwp-1 through -5, lov-1 and pkd-2, that are coexpressed in all RnB neurons but R6B. Though we have not yet identified common regulatory regions in by these genes, we have found that a short stretch of the first intron of pkd-2 is both necessary and sufficient for RnB expression. We are currently working to identify factors that act through this motif and to identify other RnB elements from genes in this battery. Together, we hope to assemble a more complete picture of the regulatory network that specifies neural subtype in the rays.

Douglas Portman et al. (2005) International Worm Meeting "The DM-domain gene dmd-3 regulates sex-specific C. elegans development in the tail spike, rays and gonad"

Douglas Portman, Grace Vangeison, Jeremy Rabinowitz

[International Worm Meeting, 2005]

The regulation of sexual dimorphism is a central issue in developmental biology. Recently, DM domain transcription factors in several species have been shown to control sex-specific characteristics in multiple cell types, much as Hox genes control position-specific characteristics. In a microarray screen for genes expressed in the male tail sensory rays, we identified a novel DM-domain gene, dmd-3, the third characterized member of this family in C. elegans. Reporters for dmd-3 show a striking pattern of male-specific expression in the rays, tail spike, hindgut, and gonad, consistent with a role in generating sexual dimorphism. In hermaphrodites, the only detectible dmd-3 expression is in the anchor cell.In support of this model, dmd-3(RNAi) and dmd-3(ok1327) males have phenotypes consistent with a partial sexual transformation. The most obvious of these is the persistence of a tail spike in the adult male. Normally, male tail retraction is accompanied by the fusion of the tail tip cells hyp8-11 in early L4. In contrast, we have found that hyp8-11 adopt a hermaphrodite-like fate in dmd-3 males and do not fuse. The male-specific expression of dmd-3 in hyp8-11 indicates that dmd-3acts cell autonomously to implement the sexual fate of these cells, perhaps by activating expression of the fusogen gene eff-1.dmd-3 males also have ray defects: the fan and rays are small, and nearly all ray RnA neurons ectopically express the dopaminergic markers dat-1 and cat-2. In wild-type males, expression of these genes is restricted to the RnA neurons of rays 5, 7 and 9, where their expression requires a TGF- signal. We believe that the dmd-3 phenotype may reflect a partial transformation of the ray into the postdeirid, a related but non-sex-specific sensory organ, for two reasons. First, we have found that the ectopic expression of dat-1 in dmd-3 males is independent of TGF- signaling, as it normally is in the PDE neuron of the postdeirid. Second, RnA axons in dmd-3 males often display aberrant anterior migration, similar to normal PDE axons. These results suggest that dmd-3 imparts male specificity in the rays by modifying a non-sex-specific sensory lineage.No obvious defects result from dmd-3(RNAi) or dmd-3(ok1327) in hermaphrodites. However, we have found that the expression of a DMD-3::YFP fusion protein in hermaphrodites results in the development of abnormal vulvae, pseudovulvae and multiple anchor cell-like cells. This suggests that dmd-3 may function to promote the development of the anchor cell. We speculate that dmd-3 is involved in distinguishing the anchor cell from the linker cell, its counterpart in the male gonad.

Douglas S Portman et al. (2002) East Coast Worm Meeting "Dissecting the C. elegans ray developmental pathway with DNA microarrays"

Scott W Emmons, Douglas S Portman

[East Coast Worm Meeting, 2002]

Nematode development provides an excellent opportunity to understand how spatial, temporal and sex-specific information is integrated to produce the variety of neuronal subtypes that compose the nervous system. In the C. elegans male tail, each of the eighteen sensory rays is composed of three cells (A- and B-type sensory neurons and an associated structural cell), all of which descend clonally from a single ray precursor cell. We have previously found that multiple steps of ray development require the functions of the bHLH transcription factors LIN-32 (the worm atonal ortholog) and HLH-2 (E/daughterless ortholog). To obtain a more complete view of the mechanisms that generate the three distinct cell types of each ray, we have undertaken a microarray-based approach to identify genes expressed in mature rays. We sought genes in two classes: (1) terminal differentiation genes that are informative about ray function and are useful as markers of cell fate, and (2) regulatory factors that may have roles in ray development. In collaboration with Stuart Kim, we have performed seven array hybridizations comparing mRNAs from adult males that completely lack rays (hlh-2; lin-32) to those that have eighteen wild-type rays as well as ectopic rays along the body (lin-22). Based on these data, we chose a number of genes with reproducibly high expression ratios and generated reporters to determine preliminary expression patterns. Of the 37 reporters we constructed, fifteen are expressed in some or all of the rays. Among the remaining non-ray genes, eleven reporters are expressed elsewhere in the nervous system; these may represent targets of lin-32 regulation in other neuronal lineages. Three additional reporters were expressed outside the nervous system, and the remaining seven showed no detectable expression. We conclude that our microarray-based approach successfully generated a dataset highly enriched for ray-expressed genes. Among the ray terminal differentiation genes we have identified, eight show strong reporter expression in most or all of the rays as well as a limited number of other cells. These genes include a beta-tubulin isoform expressed in all ray neurons, a TWIK-family potassium channel expressed in all A-type ray neurons, and a G-protein-coupled receptor expressed in most B-type ray neurons. We have also identified four genes encoding novel putative secreted proteins whose expression patterns are identical to those of the C. eleganspolycystin genes lov-1 and pkd-2, which have been shown by Barr and Sternberg to have important roles in male mating behavior. These four genes, expressed in the four CEMs, the hook neuron HOB and all B-type ray neurons except R6B, may also function in this process. In addition, we have identified a number of putative transcription factors that may be important for ray development. Reporters for hlh-10, encoding an eHAND-like bHLH protein, and ces-2, encoding a bZIP-domain protein, are expressed in several ray neurons (and other neurons as well) and may play roles in ray patterning. In addition, a reporter for the Zn-finger gene egl-46 is expressed in one neuron of each ray, and the LIM-homeodomain gene lim-7 appears to be expressed during the ray sublineage.

Douglas S Portman et al. (2002) West Coast Worm Meeting "Dissecting the C. elegans ray developmental pathway with DNA microarrays"

Scott W Emmons, Douglas S Portman

[West Coast Worm Meeting, 2002]

Nematode development provides an excellent opportunity to understand how spatial, temporal and sex-specific information is integrated to produce the variety of neuronal subtypes that compose the nervous system. In the C. elegans male tail, each of the eighteen sensory rays is composed of three cells (A- and B-type sensory neurons and an associated structural cell), all of which descend clonally from a single ray precursor cell. We have previously found that multiple steps of ray development require the functions of the bHLH transcription factors LIN-32 (the worm atonal ortholog) and HLH-2 (E/daughterless ortholog). To obtain a more complete view of the mechanisms that generate the three distinct cell types of each ray, we have undertaken a microarray-based approach to identify genes expressed in mature rays.

Douglas S Portman et al. (2001) International C. elegans Meeting "Neuronal fate specification by bHLH proteins in the C. elegans ray sublineage"

Douglas S Portman, Scott W Emmons

[International C. elegans Meeting, 2001]

The use of repeated sublineages is likely to be a common theme in development. In the nervous system, sublineages provide an ideal opportunity to examine the integration of mechanisms that specify general neuronal properties with those that specify the more restricted characteristics of subtype identity. We have been studying the sensory rays of the C. elegans male tail to characterize the regulatory network that controls selection of the ray precursor cells and establishes the four distinct fates of their progeny. During the male L3 stage, nine bilateral pairs of posterior seam cells are specified to become the ray precursor cells, or Rn cells. Through the execution of the ray sublineage, each Rn cell clonally gives rise to the three cell types of each ray (the ray structural cell Rnst and the ray neurons RnA and RnB) as well as a cell that undergoes programmed cell death. Superimposed on the ray sublineage are patterning mechanisms that generate differences between corresponding cells of different rays, including structural cell morphology and ray neuron neurotransmitter identity. The atonal -family bHLH transcription factor LIN-32 is expressed in all of the ray precursor cells, and is required to specify their fates as neuronal precursors. Because strong lin-32 alleles cause nearly complete ray loss, and ectopic expression of lin-32 can specify ectopic ray sublineages, lin-32 can be thought of as a ray proneural gene, acting early to implement commitment to the ray sublineage. However, lin-32 has later functions in ray development as well. By using weak lin-32 alleles along with ray-neuron-specific GFP markers, we have found that loss of lin-32 function can disrupt multiple steps in the ray sublineage, uncoupling cell fate specification in one branch from that of another. lin-32 function is also required for the terminally-differentiated characteristics of ray neurons and ray structural cells. These findings suggest that lin-32 has additional independent functions after the specification of the Rn cell, and are supported by recent results in Drosophila demonstrating that atonal functions in multiple steps of neuronal development as well. To better understand these multiple functions of lin-32 , we screened for suppressors and enhancers of the ray loss phenotype of a weak lin-32 allele and recovered two alleles of the gene hlh-2 . hlh-2 encodes CeE/Da, the worm member of the E/ daughterless family of general bHLH heterodimerization partners. Our hlh-2 mutations enhance the multiple ray defects caused by weak lin-32 alleles, and disrupt the formation of DNA-binding LIN-32:HLH-2 complexes in vitro . Together, these results indicate that lin-32 and hlh-2 are generally required throughout the ray sublineage for multiple fate-specification events, and imply that the LIN-32:HLH-2 complex has multiple, independent targets for different steps in ray development. To identify targets of the LIN-32:HLH-2 complex that implement ray cell fates, we are taking a microarray-based approach in collaboration with Stuart Kim (Stanford University). Using the hermaphrodite-lethal mutation dpy-28(y1ts) , we have compared whole-genome mRNA expression patterns in adult male populations lacking rays ( hlh-2; lin-32 mutants) to those with excess rays ( lin-22 mutants). This approach has thus far identified two new genes expressed in rays, the Onecut-homeodomain transcription factor ceh-39 and the G-protein coupled receptor C03A7.3. We are generating additional data in order to identify a more complete set of ray genes, and are using methods to identify sequence motifs enriched in the promoters of ray-specific genes. We plan to expand this analysis by profiling the gene expression patterns of wild-type animals from mid-L3 to adult, in order to be able to visualize changes in transcription during the ray sublineage.

Douglas S Portman et al. (2004) East Coast Worm Meeting "A genomic approach to the development and function of the C. elegans male tail rays"

Kwi Yeon Lee, Douglas S Portman, Daryl D Hurd, Nicole Juskiw, Carolyn Tyler, William R Mowrey, Hai Wu

[East Coast Worm Meeting, 2004]

The rays of the C. elegans male tail present an ideal opportunity to understand the genetic mechanisms by which distinct neural subtypes arise from neuroblast precursors. As sensory organs, the rays are also a model to address the means by which chemo- and mechanosensation regulate behavior. Each of the eighteen rays develops during L3 and L4 from a single ray precursor cell (Rn). Triggered by the action of the proneural bHLH gene lin-32 , each ray precursor executes the ray sublineage to generate two neurons of distinct subtype (RnA and RnB), the ray structural cell (Rnst), and a cell that undergoes programmed cell death. To better understand how the ray sublineage generates these distinct cell fates, we carried out microarray experiments designed to identify new ray-expressed genes. In these studies, we compared total patterns of gene expression between hlh-2 ; lin-32 and lin-22 adult males, which respectively lack and have supernumerary rays. By testing candidate ray genes with reporter constructs, we identified 17 genes whose ray expression was not previously known. Among these new ray-expressed genes are several transcription factors, including egl-46 , hlh-10 , lim-7 and an uncharacterized DM domain gene. These represent excellent candidates to act downstream of or in parallel with lin-32 to specify individual ray cell fates. We have found that mutations in egl-46 and hlh-10 do not seem to have an effect on ray development; lim-7 and the DM gene are currently being characterized. We also found a beta-tubulin isoform, tbb-4 , that is expressed in all ray neurons. Preliminary results indicate that this tubulin may be present in all ciliated sensory neurons and could contribute to the structure of the cilium itself. Finally, we have identified five novel genes, cwp-1 through -5 , that share the highly-specific expression pattern of lov-1 and pkd-2 , two genes known to be essential for male mating behavior. lov-1 , pkd-2 and the five cwp genes are expressed in all RnB neurons (except the R6Bs), the hook sensory neuron HOB and the male-specific CEM head sensory neurons. We are currently testing the possibility that the cwp genes function in a sensory pathway with lov-1 and pkd-2 . In addition, by defining minimal elements from these genes that are sufficient to drive RnB expression, we hope to characterize the regulatory mechanisms downstream of lin-32 that implement specific gene expression in the RnB neural subtype.

Douglas S Portman et al. (2001) International C. elegans Meeting "The C. elegans neurogenin-like gene ngn-1 has roles in axon outgrowth and guidance"

Oliver Hobert, Scott W Emmons, Douglas S Portman

[International C. elegans Meeting, 2001]

Neurogenins are a class of atonal -like bHLH transcription factors originally identified as vertebrate neuronal commitment factors. In some lineages, neurogenins act at the top of a cascade of bHLH factors required for neuronal development, and knockout mice lack certain classes of neurons. It has recently been proposed that neurogenins have roles in specifying both general neuronal properties and subtype-specific characteristics. We identified a worm neurogenin family member, ngn-1 (Y69A2AR.d), in a search for atonal -like genes that might function with lin-32 in male tail ray development. Inhibition of ngn-1 function by RNAi does not cause a ray-missing phenotype in males, as might be expected if ngn-1 functioned at the top of a ray-determination cascade. Interestingly, however, ngn-1 function seems to be required for the axonal guidance and connectivity of a subset of neurons in the worm. Interference with ngn-1 function by RNAi leads to a weak and variable Unc phenotype. To look more specifically at neuronal development, we have examined the effects of ngn-1(RNAi) on the expression of a variety of neuronal reporters. The most pronounced effect was seen with ttx-3::gfp , which is expressed in the AIY interneurons: ngn-1(RNAi) caused a highly-penetrant short-stop phenotype, in which the AIY axon extends anteriorly, but fails to turn dorsally to enter the nerve ring. Lower frequency defects have also been observed in glr-1::gfp -expressing neurons, in which ventral and ring axons sometimes wander or follow lateral or dorsal paths. Defects have also been observed in the RnB axons in the male tail, which show disorganization in the preanal ganglion region, where they synapse onto their target(s). We have seen no noticeable defects in other classes of neurons, including sensory neurons (as visualized by DiO staining) and GABAergic neurons (by unc-47::gfp expression). Moreover, we have not seen a reduction in the number of gfp-expressing cells with any of these markers, as might be expected if ngn-1 has a role in specifying the identity of neuronal precursors. To better understand how ngn-1 might function to specify axonal properties, we have constructed a full-length ngn-1::gfp reporter. With this construct, we have seen embryonic expression starting at about 120 min; by late gastrulation, expression is seen in 25-30 cells, at least some of which are likely to be AB descendants. Expression becomes restricted to ~10 head cells by the time of ventral enclosure; this expression persists until the three-fold stage. By hatching, no expression can be detected. A more complete characterization of this expression pattern is underway. The expression we have seen is consistent with two possibilities: ngn-1 could be acting cell-autonomously to promote neuronal characteristics in a restricted subset of neurons, including AIY, or it could act to specify earlier aspects of neuronal development in a pioneer cell(s) upon which the outgrowth and guidance of other axons depends. Further characterization of RNAi phenotypes and reporter expression should allow us to distinguish between these models.

Douglas S. Portman et al. (2007) International Worm Meeting "Wnt-regulated asymmetric expression of the Atonal/MATH-family factor LIN-32 patterns the progeny of the ray neuroblast."

Douglas S. Portman, Renee M. Miller

[International Worm Meeting, 2007]

Each sensory ray in the C. elegans male tale is made of three cells that descend from a single precursor, the ray neuroblast. Through two rounds of asymmetric division, the ray neuroblast gives rise to two sensory neurons (RnA and RnB), the ray structural cell (Rnst) and a programmed cell death (Rn.aap). This provides an excellent model in which to explore the genetic mechanisms that determine sex-specific neural and glial cell fates. The development of all three ray cell types requires the function of the atonal-class bHLH factor LIN-32. In lin-32 null mutants, very few rays develop; those that do form require the activity of HLH-2, the E/Daughterless-family factor that heterodimerizes with LIN-32. Consistent with this proneural-like role, lin-32 expression in the ray sublineage is first detectable in the common precursor of all three ray cells, the ray neuroblast Rn.a. Interestingly, however, once the Rn.a cell divides, lin-32 expression becomes asymmetric, persisting only in Rn.aa. This pattern repeats itself when Rn.aa divides, such that lin-32 remains on in Rn.aaa (which becomes the RnA neuron) but not its sister Rn.aap (the dying cell). This unexpected finding suggests that asymmetric lin-32 expression could be important for the patterning cell fates among the four progeny of each Rn.a cell. Recent results suggest that asymmetric lin-32 expression does indeed have an important function in ray development. In lin-17/Fz mutants, we find that both asymmetric lin-32 expression and ray cell fate is disrupted. These animals exhibit ectopic expression of lin-32 during the ray sublineage, indicating that Wnt signaling is necessary to restrict lin-32 to the anterior branch of the ray sublineage. Moreover, the RnA marker dat-1 is expressed in many additional cells in the lin-17 male tail, suggesting that ectopic lin-32 expression may promote RnA-like fate in the RnB neuron. To test this, we have expressed lin-32 throughout the late ray sublineage with a heat-shock promoter. In these animals, we observe loss of the RnB marker cwp-4 and some ectopic expression of dat-1. Together, these findings indicate that lin-32 acts not only as a proneural gene in the ray neuroblast but also to specify neural subtype among its descendants.

Douglas S Portman et al. (2003) International Worm Meeting "DNA microarrays identify genes expressed in the C. elegans male tail rays, including four novel genes coexpressed with the polycystins lov-1 and pkd-2"

Douglas S Portman, Scott W Emmons, Kwi Yeon Lee

[International Worm Meeting, 2003]

How do genetic networks control the patterns of gene expression that direct cell fate specification in the nervous system? We are using DNA microarrays to identify genes involved in development and function of the rays, sensilla in the C. elegans male tail used during mating. By comparing genome-wide expression profiles between adult males that differ in ray frequency and constructing reporter genes, we have identified a number of new ray-expressed genes, including the -tubulin isoform tbb-4, the K+ channel twk-21, the 7TM receptor srw-140 and several transcription factors. Further characterization of the roles and regulation of these genes is under way. Our microarrays have also identified four novel genes that share the characteristic male-specific expression patterns of lov-1 and pkd-2, the C. elegans polycystin genes. Barr et al. have previously shown that lov-1 and pkd-2 activities are required for multiple steps of male mating behavior (1,2). C. elegans PKD-2 is a TRP-family Ca2+ channel whose activity may be regulated by LOV-1 in response to sensory information. lov-1 and pkd-2 are expressed in the four CEM head sensory neurons, the hook neuron HOB and the B-type neurons of all of rays but ray 6. (In humans, mutations in the orthologous genes PKD1 and PKD2 cause autosomal dominant polycystic kidney disease, possibly by disrupting the function of renal epithelial cell cilia.) We have identified four genes (the cwp genes, coexpressed with polycystins) for which GFP reporters are expressed in patterns identical to lov-1 and pkd-2. Each cwp gene encodes a novel protein with a predicted signal sequence. These genes also have an unusual arrangement: cwp-1, cwp-2 and cwp-3 are located contiguously on the left arm of LG V, and cwp-4 is 207 kb to the right. Like LOV-1, the cwp-4 product contains an STP/mucin domain, suggesting that it is a target for O-glycosylation. The expression patterns of the cwp genes indicate that they may have roles in lov-1/pkd-2 signaling; their predicted extracellular localization suggests that this involvement could be in the early events of this pathway. The cwp genes also provide an opportunity to identify regulatory elements that specifically direct gene expression in the ray B-type neurons.