Y Fung Wong et al. (2003) International Worm Meeting "Two novel zinc finger proteins act as regulators of ray lineage and determine ray identity"

King L Chow, Y Fung Wong, S Hong Ho

[International Worm Meeting, 2003]

Zinc finger-containing transcription factors have been documented in the regulation of gene transcription in a number of developmental models. We have identified two zinc finger proteins, W06H12.1 and Y40B1A.4, both of which have three conserved C2H2 zinc finger domains. These proteins appear to be essential for the differentiation of the male tail region where nine pairs of sensory rays with distinct identities reside. We previously reported that mab-21 is required for the specification of the ray 6 identity. In a yeast two-hybrid screen with MAB-21 protein bait, W06H12.1 was first identified. W06H12.1 product has 380 amino acids with three C2H2 zinc finger domains and two putative nuclear localization signal sequences. RNAi experiments showed that knock-down of this gene resulted in ray fusion and occasional Mab-21 phenotype. By reporter assay with a 1.5kb promoter region in transgenic animals, W06H12.1 was found to be expressed in hypodermis, seam from embryonic to larval stage. In adults, expression in the hermaphrodite vulva and male tail ray structural cells were also observed. We further demonstrated that the W06H12.1 promoter region driving a mab-21 cDNA could rescue mab-21 mutant effectively, suggesting that the two genes have overlapping temporal and spatial expression patterns and may act together in the ray 6 determination process. W06H12.1 protein have been overexpressed in bacteria and the purified protein will be used for identify target DNA binding sites in the genome. The results of this experiment may lead to the identification of target genes important of the ray 6 structural cell differentiation. Y40B1A.4 obtained in the same genetic screen appeared to play a different role in ray differentiation. Male animals subjected to RNAi treatment showed extensive ray loss phenotype, as observed in mutant lin-32 and mab-22 males. In addition, it displayed a slightly uncoordinated and dumpy phenotype. Using specific gfp markers of the ray structural cells and neuronal cells, we showed that the ray loss phenotype was caused primarily by abnormal ray cell lineage and potential defective structural cell function. The characterization of Y40B1A.4 expression profile is in progress. The analysis of this gene and its relationship with other genes controlling ray formation may yield new insight into the detail control of ray cell assembly process.

Robyn Lints et al. (2003) International Worm Meeting "Progressive anteroposterior refinement of C. elegans sensory ray neurotransmitter phenotype in the V6 lineage ray neurons."

Robyn Lints, C Li, S W Emmons, K Kim, L Jia

[International Worm Meeting, 2003]

In simple animals that exhibit little experience-dependent plasticity in the nervous system, the developmental genetic programs that invest neural cells with their heterogeneous properties play a major role in shaping animal behavior. We are exploiting the simplicity of the C. elegans male sensory rays to identify the patterning mechanisms underlying diversity in ray neuron neurotransmitter phenotype. The rays are serially homologous structures but each has a unique identity overlaying the basic reiterated lineage pattern. Each ray contains two ultra-structurally distinct neuron types, RnA and RnB (where n stands for rays 1 to 9). Across the rays, however, RnA and RnB neurons differ in their neurotransmitter expression profiles. In previous studies we identified developmental regulators that establish the wild type pattern of dopaminergic fate among the RnA neurons. Here we investigate the mechanisms underlying the complex patterning of RnB neurons, which we have determined produce either serotonin (5HT), FMRFamide-related neuropeptides (FaRPs) from multiple genes, or a combination of both. We find that no single genetic program is used consistently to specify a given neurotransmitter phenotype and that axial position determines the particular genetic program used. In rays descended from the seam cell V6 (rays 2 to 6), we have identified three genetic activities that regulate neurotransmitter phenotype and ray-specific morphology, Abdominal B-like Hox gene egl-5, the DBL-1 (TGF-beta) pathway and mab-18 (Pax-6). egl-5 is expressed in V6 lineage branches leading to rays 3 to 6 but not ray 2. In egl-5 mutants, ray 6 is not generated and R3B to R5B neurons are transformed to a R2B neurotransmitter fate. Within the EGL-5-expressing rays, the DBL-1 pathway and mab-18 function in lineages that produce R5B and R6B, respectively, to differentiate these neurons from R3B by suppressing a potential to produce 5HT and by inducing the expression of specific FaRP-encoding genes. These data, supported by observations on RnA neuron patterning and ray morphology, suggest that wild type patterns of V6 ray identity are derived through the progressive anteroposterior refinement of a ground state identity corresponding to that of ray 2, the most anterior V6 ray. The delineation of this ground state identity may represent a deconstruction of the evolutionary process in which an ancestral ray identity, initially expressed by all V6 rays, was modified by successive layers of regulation in specific ray lineages.

Ray A et al. (1987) International C. elegans Meeting "changes in protein phosphorylation associated with dauer larva recovery."

Ray A, Riddle DL

[International C. elegans Meeting, 1987]

Ray, Sneha et al. (2021) International Worm Meeting "Specificity in Glia-Neuron Interactions"

Singhvi, Aakanksha, Ray, Sneha

[International Worm Meeting, 2021]

Nervous systems consist of two cell types, neurons and glia, that physically and molecularly interact. One site where glia associate closely with neurons is the neuron receptive endings (NREs), where neurons receive input. Glia can actively regulate NRE shape and function, with consequences on neuron activity and animal behavior. Each glia can associate with multiple NREs. However, whether or how a single glia regulates different associated NREs remains a crucial outstanding question. Unlike most organisms, C. elegans has invariant glia-neuron cell-cell contacts and each cell is born of invariant lineages. This makes it a uniquely tractable system to investigate specificity of individual glia-NRE interactions. To examine this, we focused on the C. elegans amphid sheath (AMsh) glia, which interacts with the NREs of 12 different sensory neurons, including the thermosensory AFD neuron, odor-sensing AWC neuron, and taste-sensing ASE neuron. Ablation of AMsh glia impacts most associated NRE shapes and/or function. We found that AMsh glia localizes the cation chloride cotransporter KCC-3 to a molecular micro-domain only around AFD-NRE, hinting at non-uniform interaction between AMsh glia and different NREs. Indeed, while AMsh glial KCC-3 regulates AFD NRE shape and associated thermosensory behavior, it notably does not affect ASE or AWC NRE shape, or ASE-mediated behavior. To probe how this specificity is achieved, we sought to determine how AMsh KCC-3 localizes to a glial micro-domain. Surprisingly, our genetic and laser ablation studies reveal that the AFD neuron does not recruit KCC-3 localization. Instead, disrupting microtubule-based cilia, present on all amphid NREs, is required to localize glial KCC-3 around AFD-NRE. We are currently investigating the role of individual amphid NREs on KCC-3 localization. Our structure-function studies also identify a 89 amino-acid sequence on KCC-3 that guides its localization. Current studies aim to localize this further and determine interacting partners. These studies show that C. elegans AMsh glia regulate different NREs differently by localizing specific regulatory molecules to single NRE contact sites, and aim to shed light on the underlying molecular mechanism.

Fitch DHA et al. (1995) International C. elegans Meeting "EVOLUTION OF RAY PATTERN IN RHABDITIDAE"

Fitch DHA, Emmons SW

[International C. elegans Meeting, 1995]

To identify what genes and cellular mechanisms change during the evolution of morphology, we are studying the evolution of the nematode male tail. One highly variable morphological feature is the number and arrangement of the rays. We compared development of the rays in ten species and found that variable ray position may result from changes in genes encoding cell recognition or adhesion proteins, or the pattern forming genes that regulate them. Analysis of epidermal cell arrangements with the MH27 adherens junction antibody revealed remarkable conservation at early stages. All but one species have 9 rays, with rays 5 and 7 lying dorsally. In one species the ray 8 cell group is absent. A laterally placed phasmid in two species has been classically mistaken for a tenth ray. The species differences in the order and spacing of rays correlate with apparent changes in cell associations, resulting in movement of ray cells away from their initial positions. What did the ancestral male tail look like? To reconstruct the steps that have led to present day tail morphologies, we analyzed 36 morphological and developmental characters in the context of a phylogeny based on 18S rRNA gene sequences. The ancestral nematode may have had relatively uniformly spaced rays, with rays 5 and 7 opening on the dorsal surface and the remaining rays extending to the edge of the fan. Several evolutionary changes in ray development and morphology are similar to mutant phenotypes in C. elegans, and hence point to genes in which changes may have been important for ray pattern evolution.

Zhang YH et al. (1996) East Coast Worm Meeting "THE ROLE OF egl-5 IN RAY DEVELOPMENT"

Emmons SW, Ferreira HB, Zhang YH

[East Coast Worm Meeting, 1996]

In the male tail, the lateral hypodermal cell V6 has two cell lineage branches, an anterior V6.pap branch and a posterior V6.ppp branch, from which 5 sensory rays are generated. V6.pap produces rays 2 and 3 and V6.ppp produces rays 4, 5, and 6. These rays differ in position and morphology. We are interested in understanding how these ray identities are specified. In particular, we are studying the role of the posterior HOX gene egl-5 in specification of V6 rays. Gene dosage studies as well as the nature of the null phenotype implicate egl-5 in specification of rays 2-6. Recently, Salser and Kenyon (1) have analyzed the expression of a second HOX gene, mab-5, in seam lineages. They found that in the postembryonic lineage derived from the next most anterior seam cell, V5, mab-5 specifies the properties of alternative lineage branches by being turned on in posterior branches and off in anterior branches after asymmetric cell division. The function of egl-5 in specification of cell fates in the V6 lineage could be explained if egl-5 follows a similar rule of expression and is necessary and sufficient for cell fates in posterior branches of the V6 lineage. Indeed we find that an egl-5 GFP reporter gene is expressed in the V6.ppp branch and not in the V6.pap branch. Later it is expressed at a higher level in lineage branches leading to rays 5 and 6, and at a lower level in the branch leading to ray 4. We are presently testing antibodies made to a bacterial EGL-5 fusion protein to determine the levels of EGL-5 protein in these lineages. We also plan to test the effect of ectopic expression from a heat shock-driven transgene. The expression pattern of mab-5 in the late V6 seam lineages is complementary to that of egl-5. Whereas egl-5 is expressed in V6.ppp branches leading to rays 4, 5, and 6, mab-5 is expressed primarily in V6.pap branches leading to rays 2 and 3, with some weaker expression overlapping with egl-5 in the V6.ppp branch leading to ray 4. This pattern appears to be inconsistent with the genetic observation that generation of all V6 rays depends on mab-5 function, including rays 5 and 6 where mab-5 is not expressed. We propose that egl-5 is responsible for generation of rays in these two posteriormost V6 lineage branches, and that mab 5 turns on egl 5. This proposal, however, contradicts the observation that ray 5 persists in an egl-5 mutant. This could be explained if one function of egl-5 is to repress mab-5, so that in an egl-5 mutant mab-5 expression turns on ectopically in the ray 5 branch. These models are presently being tested by gene expression studies in various mutant backgrounds. They suggest a mutually antagonistic interaction between mab-5 and egl-5. They also demonstrate the caution that must be exercised in interpreting mutant phenotypes when such mutually antagonistic gene pairs are involved. 1. Salser, S. J. and Kenyon, C. (1996) A C. elegans Hox gene sitches on, off, on and off again to regulate proliferation, differentiation and morphogenesis. Development 122, 1651-1661.

Emmons SW et al. (1995) International C. elegans Meeting "THE GENETIC BASIS OF RAY IDENTITY"

Emmons SW, Zhang YH, Chow K-L

[International C. elegans Meeting, 1995]

The sensory rays in the C. elegans male tail are serially homologous: each is comprised of the same three cell types, and each is generated by repetition of identical ray sublineages. Yet the rays differ from each other in subtle aspects of their structure, in neurotransmitter usage, in position within the epidermis, and in expression of unknown morphogenetic determinants that allow them to assemble independently of their neighbors. Laser ablation studies showed that the pattern of ray identities is not established by signals between neighboring ray cells or by positional signals along the body. Instead, the potential to generate subsets of rays is progressively restricted during the cell lineages leading to the rays. A number of genetic observations are explained by a model in which egl-5 expression is the primary determinant of identities 2-6 according to the following pattern: OFF:2, ON:3, OFF:4, ON: 5 and 6. This model explains the results of gene dosage studies with the HOM-C genes mab-5 and egl-5, as well as the egl-5 null phenotype. Signals from neighboring seam cells may influence egl-5 expression in the V6 lineage, since ablation of T.ap in a pal-1 background results in posterior transformation of ray identities (2,3 to 4,5,6). mab-5 appears to influence the identities of rays 2-6 indirectly, possibly by inhibiting egl-5. Rays 5 and 6 are further distinguished by expression of mab-18, which is regulated in part by a TGFb signalling pathway.

Sutherlin ME et al. (1991) International C. elegans Meeting "A SPECTRUM OF RAY LOSS MUTATIONS"

Schirmacher H, Sutherlin ME, Chow K-L, Emmons SW, Kassem IAA, Zhang YH

[International C. elegans Meeting, 1991]

We have continued to characterize ray loss mutants in order to identify all genes necessary for male tail peripheral sense organ development. We group these genes into two general classes on the basis of their phenotypes: EARLY GENES are required for events leading to the generation of ray cell groups, and LATE GENES are involved in the assembly of the ray cell groups into rays and the subsequent function of the rays. The genes are provisionally grouped as follows, judging by the severity of the mutant phenotypes and pending lineage analysis. EARLY GENES bx66ts males exhibit grossly abnormal tail morphology and the absence of lateral alae at the nonpermissive temperature of 25 C. Hermaphrodites grown at 25 C also have abnoqmal alae and are sterile. At 16 C, both males and hermaphrodites are morphologically wild type. The TSP of bx66ts is from the Ll stage onwards, thus bx66ts may be an allele of a gene required for all lateral epidermal (seam) cell divisions, including those that divide to produce the ray cell groups. bx40ts 1 is sterile at 25 C, and exhibits a maternal effect, even for the Mab phenotype, which resembles mab-9(el245)11 (club tail phenotype) at 25 C. bx68ts 111 is also a maternal effect mutation that results in variable ray loss; hermphrodites grown at 25 C are Egl. bx27ts 1 is a mutation that results in variable ray loss and swollen bursa at all temperatures. bx27ts also confers a temperature sensitive lethal phenotype. Development arrests at the stage at which the animals are shifted from permissive (16 C) to nonpermissive temperature (25 C). mab-24(e2169)111 results in variable V5 and V6- derived (rays 1-6) ray loss and ray mispositioning. Both sexes are Unc; animals are forward coilers and are slightly uncoordinated when backing. LATE GENES mab-22(bxS9ts)111 appears to affect the assembly of the rays from the constituent ray cell groups. bxS9ts results in extensive ray loss at 25 C, but not at 16 C. The TSP corresponds to the time of structural cell-cuticle association (papilla formation) just prior to morphogenesis. Lineage analysis and immunofluorescence visualization of hypodermal cell boundaries with the monoclonal antibody, MH27 ( provided by R. Waterston) show no defects in division pattern, cell morphology, or cell movements up until the point of papilla formation. When papillae do form, often very thin processes are attached to the cuticle instead of rays. EM reconstructions of sectioned animals will identify the nature of the thin processes.

Daryl Hurd et al. (2007) International Worm Meeting "Tubulins in male tail ray sensory cilia."

Renee Miller, Daryl Hurd, Douglas Portman

[International Worm Meeting, 2007]

The dendritic ends of many sensory neurons terminate with primary (non-motile) cilia, which are microtubule-based cellular extensions critical for the sensory functions of these cells. Though primary cilia occur in a variety of cell types in animals, little is known about the specific tubulins that comprise the axoneme underlying their characteristic architecture and function. Recent genomic approaches have identified two of nine alpha-tubulins, tba-6 and tba-9, and one of six beta-tubulins, tbb-4 as likely components of the microtubule cytoskeleton in ciliated worm neurons. To study the expression of these tubulin genes, we created and analyzed transcriptional and translational YFP fusions. We found that these tubulins are expressed in subsets of ciliated sensory neurons in the head of both males and hermaphrodites. We also observed that all three are co-expressed in the RnA and RnB neurons of the male tail, where they accumulate in the ciliated tips of dendrites. To determine the cellular role of these tubulins, we obtained loss of function mutant alleles and constructed all pair-wise combinations of double mutants and the triple mutant. We observed no gross abnormalities in the anatomy, fertility or behavior of single, double or triple mutant hermaphrodites. However, certain single mutants, all of the double mutant combinations and the triple mutant showed significant reductions in the efficiency of male mating behavior. Mutant males were selectively impaired in the initial response to contact step of mating, which is known to rely on RnB neuronal function. In addition, we have analyzed the structure of the male tail ray cilia by observing the localization of an OSM-6::GFP fusion protein in all genotypes. We found that elimination of certain combinations or of all three of these tubulins caused a marked reduction in the accumulation of OSM-6::GFP in male tail ray cilia. These observations indicate that tba-6, tba-9, and tbb-4 are specifically required for the structure and function of the primary cilia in male tail ray neurons.

Hahn AC et al. (1997) International C. elegans Meeting "GENETIC MAPPING OF A RAY IDENTITY GENE"

Hahn AC, Emmons SW

[International C. elegans Meeting, 1997]

Each of the 9 pairs of sensory rays of the C. elegans male tail possesses a unique identity. Ray identity serves to define the morphologic characteristics of the ray as well as to maintain separation between cells of different rays during assembly. Transformation of a ray's identity is manifested as relocation and fusion with rays of similar identity. Factors known to be involved in determination of ray identity include the Hox genes mab-5 and egl-5, which act in a dosage-dependent, mutually opposing fashion to specify identity in certain rays. The mechanism by which these two genes act is unknown, as are the factors that regulate their activity. To investigate these issues, the properties of several additional genes involved in ray identity specification are being investigated. We are mapping one of these genes, mab-26 (bx80sd). The homozygous mutant is characterized by relocations and fusions among all rays, as well as by deformities throughout the body. bx80 is likely to be a loss-of-function allele, since a wild-type copy of mab-26 carried on a free duplication partially rescues the homozygous mutant phenotype. By several 3-factor crosses mab-26 has been mapped 0.1cM to the right of dpy-9 on the left arm of LG IV. dpy-9 has not been cloned; however dpy-9 has been mapped to within 0.1cM of daf-1, a cloned gene. These data specify a region narrow enough to begin rescue attempt by cosmid microinjection.