Carolyn Tyler et al. (2004) East Coast Worm Meeting "Transcriptional specification of neural subtype in the C. elegans male tail"

Douglas S Portman, Nicole Juskiw, Carolyn Tyler

[East Coast Worm Meeting, 2004]

Rays are C. elegans male tail sensory organs thought to transduce chemo- and mechanosensory cues important for mating behavior. Each of the eighteen rays contains two neurons, RnA and RnB, and a structural cell, Rnst, that descend from a single ray precursor cell, Rn. The RnB neurons of all rays (except ray 6), along with the hook HOB and head CEM neurons, express lov-1 and pkd-2 , genes that are required for the response of males to hermaphrodites and for the subsequent location of the hermaphrodite vulva 1 . In a microarray-based screen for new ray-expressed genes 2 , we identified five new genes, cwp-1 through -5 , that have a pattern of expression identical to that of lov-1 and pkd-2 . The highly specialized and restricted expression pattern of these seven genes provides an opportunity to understand how the differential regulation of gene expression that defines neuronal subtype is implemented. We speculate that lov-1 , pkd-2 and the five cwp genes share a common mechanism of transcriptional regulation. Using nested deletions and the pes-10 enhancer-element assay, we sought to define minimal regulatory regions sufficient to activate transcription in the RnB neurons. With this approach, we have identified small regions from cwp-1 , -2/3 , -5 and pkd-2 that are sufficient to drive GFP expression in the RnBs as well as the CEMs and HOB. We have observed some variability in expression among these constructs which might result from pes-10 context effects or from the lack of motifs necessary for robust expression. We also found that a small region upstream of cwp-4 can drive expression in the IL2 and URAD neurons as well as the RnB/HOB/CEM set. This suggests that this region may lack an element required for the function of the ARID protein CFI-1, which normally prevents expression of at least some of these genes in the IL2s and URADs 3 . We have not yet identified obvious sequence similarities in our minimal regions that could represent binding sites for a common RnB transcription factor. We are continuing to define the minimal requirements for RnB expression and will use bioinformatic and molecular strategies to identify the regulatory mechanisms that underlie the specific co-expression of this battery of genes. 1 M. M. Barr and P. W. Sternberg (1999) Nature 401 :386; M. M. Barr et al (2001) Curr Biol 11 :1341. 2 D. S. Portman and S. W. Emmons (2004) Dev Biol , in press. 3 S. Shaham and C. I. Bargmann (2002) Genes Dev 16 :972.

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.

Caroline A Spike et al. (2003) International Worm Meeting "Mutations that affect the assembly or stability of P granules"

Susan Strome, Caroline A Spike

[International Worm Meeting, 2003]

P granules are germ-cell-specific structures rich in RNA and protein that are required for proper germ cell development. Several protein components of P granules have been identified and ordered into a protein assembly pathway using molecular epistasis experiments. The constitutive P-granule protein PGL-1 is in the middle of this pathway, upstream of the eIF4E homolog IFE-1 and downstream of GLH-1, a protein similar to the Drosophila polar-granule protein VASA. Thus, GLH-1 is required to localize PGL-1, or a GFP::PGL-1 fusion protein expressed in the germline, to P granules. In order to identify new factors involved in P-granule assembly or stability, we have been conducting an F2 screen for mutations that phenocopy glh-1 loss-of-function mutations and prevent or reduce the accumulation of GFP::PGL-1 on P granules (thanks to Nicole Meyer for the GFP::PGL-1 strain). We have screened 5514 haploid genomes after EMS mutagenesis and found four mutations with reduced levels of GFP::PGL-1 on P granules. Three of these mutations map to the far left end of chromosome I and are likely to be mutations in the same gene. Our preliminary observations indicate that these mutations cause temperature-sensitive maternal-effect sterility as well as some maternal-effect lethality. pgl-1 and glh-1 mutations also cause temperature-sensitive maternal-effect sterility, reinforcing the idea that our new mutations may identify new components of P granules required for germline development as well as the appropriate localization of PGL-1. We will present a more detailed phenotypic analysis of these mutations at the meeting.

Anderton SM et al. (1996) East Coast Worm Meeting "Comparisons of serotonin containing neurons in freeliving nematodes: correlation (or not) of ventral cord neurons with vulval precursor cell patterns."

Anderton SM, Loer CM

[East Coast Worm Meeting, 1996]

We are interested in how patterns of serotonergic neurons are specified in the nematode. In C. elegans, six hypodermal cells (P38.p) are competent to produce vulva in the central body of the hermaphrodite. In this same body region in the ventral nerve cord (VNC) of the male, P38.aap normally divide to produce two neurons, one of which contains serotonin (CP neurons, P38.aapp). We wondered whether related species of worms that have a different number of vulval precursor cells than C. elegans might also alter the pattern of serotonergic neurons in the male. We first examined Pristionchus pactficus which in the hermaphrodite has only 4 Pn.p ectoblasts, P58.p; anterior and posterior Pn.p cells undergo programmed cell death (R. Sommer, personal communication). We found four serotonin-immunoreactive (JR) CP-like neurons in male Pristionchus VNC, correctly positioned to be P58.aapp. In the hermaphrodite VNC, we saw four cells, two anterior and two posterior to the vulva, and appearing to innervate the vulval region. These are likely to be 7C cells, only four of which (P58.aap) survive in Pristionchus (R. Sommer, pers. comm.). VC cells in C. elegans occasionally stain with serotonin antisera. With this simple picture in mind of a correlation between Pn.p cell fates in the hermaphrodite and Pn.aapp fate in the male, we examined several other species of worms with varying numbers of Pn.p cells (Sommer & Sternberg, 1995, Dev. Biol. 167: 61; Sommer & Sternberg, 1994, Science 265: 114). The simple-minded hypothesis did not hold up beyond Pristionchus. Panagrolaimus, also with P58.p cells in the hermaphrodite, had 8 CP-like somas in the male VNC (4 faint in the anterior, 4 bright in the posterior). Essentially the same pattern was seen in the male Rhabditella, in which the hermaphrodite has 5 Pn.p cells (P48.p). Both Pelodera and Teratorhabditis (each with P48.p in the hermaphrodite), looked like C. elegans, with 6 CP-like cells in the male VNC. Each of these species sometimes showed other faintly staining somas, paired with the CP-like neurons. Similar cells are sometimes seen in C. elegans and are believed to be CA neurons (Pn.aapa). We will present a complete pattern of serotoninIR cells in males and hermaphrodites for each of these species. We wish to thank Ralf Sommer for providing the non-C. elegans strains used and sharing unpublished information about them. Another undergraduate, Nicole Hinson, did the first staining with Pristionchus during a summer HHMI fellowship.

Nicole Saad et al. (2006) European Worm Meeting "Investigating the Role Of rnt-1 and bro-1, and their Mammalian Orthologues, In Seam Cell Proliferation in C. elegans"

Nicole Saad, Hiroshi KAGOSHIMA, Alison Woollard

[European Worm Meeting, 2006]

Nicole Saad, Hiroshi Kagoshima, and Alison Woollard. rnt-1 is the C. elegans orthologue of the Runx family of transcriptional regulators implicated in various human cancers. There are three Runx genes in humans; Runx1 is associated with acute myeloid leukaemia and is thought to act both as an oncogene and as a tumour suppressor. Runx2 is associated with cleidocranial dysplasia and Runx3 is postulated to be associated with gastric cancer. Previous work in the lab has identified rnt-1 as the defective gene in mab-2 mutants. Mab mutants have defects in the formation of the male tail, arising from seam cell lineages. mab-2/rnt-1 mutants have missing rays because there is a reduction in the number of ray precursor cells. The reduction in ray precursor cell number is a direct consequence of a reduction in the number of seam cells. The reduction in seam cell number in rnt-1 mutants is usually the result of a failure in particular cell divisions. Overexpressing rnt-1 results in an increase in seam cell number. This is compelling evidence that rnt-1 is a rate limiting regulator of cell proliferation.. The CBFbeta orthologue bro-1 has a similar function to rnt-1 in regulating seam cell proliferation. bro-1 deletion alleles have reduced numbers of seam cells and are missing rays in the male tail. bro-1 animals have similar cell proliferation defects to rnt-1 animals. CBFbeta has also been linked with leukaemia, and been shown to act as a binding partner for Runx factors. We are currently investigating bro-1 further. We would now like to explore whether over expression of bro-1 results in seam cell hyperplasia, and whether a synergistic effect is seen if rnt-1 and bro-1 are co-overexpressed. We are also testing whether rnt-1-induced seam cell hyperplasia is dependent on bro-1 function.. In order to assess the conservation of the Runx genes we are currently pursuing complementation experiments using mouse Runx cDNAs. Expression will be driven in worms using both the endogenous worm promoter and the hsp16.2 heatshock promoter to see if it is able to complement the function of the worm orthologue. We are also making transgenic worms expressing mammalian CBFbeta along with Runx1, Runx2 or Runx3, and testing these combinations for rescue of a rnt-1 mutant and induction of hyperplasia. C. elegans bro-1 is much more highly diverged from CBFbeta than rnt-1 is from Runx genes, therefore it is possible that mammalian Runx proteins will not function in C. elegans in the absence of CBFbeta. Therefore, we will also test interactions between bro-1 and mammalian Runx genes in yeast two-hybrid assays. We hope to present results from these experiments at the meeting.