Wang, C. et al. (2019) International Worm Meeting "Expression and functional studies of the DM-domain transcription factors reveal novel sexual dimorphisms."

WANG, C., Hobert, O.

[International Worm Meeting, 2019]

Understanding structural and functional differences in the brains of females and males is fundamental for explaining sexually dimorphic animal behaviors. Despite variations in sexual differentiation pathways among species, a common theme adapted across phyla is the use of the DM (Drosophila doublesex/C. elegans mab-3 domain) family transcription factors, making them ideal candidate genes for studying mechanisms behind sexual dimorphisms. The DM family genes are expanded extensively in various species and have been implicated in regulating the development of somatic gonads and differentiation of neurons. The C. elegans genome encodes 11 DM genes, two of which conserved in humans. Only a small subset of these genes have been characterized in detail before. By CRISPR/Cas9-mediated gfp tagging of all 11 endogenous DM genes, we discovered novel sexual dimorphisms in the C. elegans nervous system. In contrast to previously reported sexually dimorphic expression of DM genes in a few sensory neurons mostly born only in males and not hermaphrodites (somatic females), our CRISPR lines reveal that of the 294 neurons that are lineally and anatomically generated in both sexes (sex-shared), about 40% of them (or 24% of sex-shared neuron classes) express a DM gene exclusively in males. Importantly, these dimorphic neurons include not only sensory, but also inter- and motor neurons. Further, based on known connectome studies, some of these neurons generate sexually dimorphic synaptic connections with other sex-shared neurons, indicating potential roles of DM genes in regulating sexual dimorphisms on a synaptic level. Mutant analysis showed that mab-3, one of the DM genes, contributes to sexually dimorphic locomotory behaviors. Taken together, completion of this work using the conserved DM genes for studying neuronal sexual dimorphisms will shed light on our understanding on sex differences in the nervous system.

H Wang (1999) International C. elegans Meeting "Expression of amyloid precursor and related protein in C. elegans"

H Wang

[International C. elegans Meeting, 1999]

The major component of senile plaques in the brains of Alzheimer's disease patients is the amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Early-onset familial Alzheimer's disease has been shown to correlate with mutations in three genes, including the APP gene. We are studying a APP -related gene, apl-1 , in C . elegans . To identify genes that interact with apl-1 , transgenic lines in which an apl-1 cDNA is under the control of a neural-specific promoter (VAMP) were generated. These transgenic strains were confirmed by Western blot analysis to overexpress APL-1. The transgenic animals have several phenotypes, including sluggish movement and a flattened waveform. To identify genes that interact with apl-1 , we mutagenized transgenic animals with EMS, and looked for reversion of the sluggish phenotype. After screening about 3,000 haploid genomes, two strains that suppress the sluggish phenotype were isolated. The yn8 strain shows wild-type movement, while the yn9 strain shows hyperactive movement. These possible suppressor genes are currently being mapped. The putative suppressor mutation in the yn9 strain does not map to chromosome V or X. APL-1 does not contain the amyloid peptide. To determine whether production of amyloid peptide has any effect in C . elegans , transgenic strains containing a mouse APP cDNA under the control of the VAMP promoter were generated. The mAPP transgene is transcribed in transgenic animals as assayed by RT-PCR. Western blot analysis to determine whether the amyloid peptide is produced is underway. Several phenotypes have been characterized. The transgenic animals have an egg-laying defect, decrease in movement, and a longer defecation cycle. The huAPP cDNA was obtained recently, and similar studies will be performed.

Wang, Christopher L C et al. (2009) International Worm Meeting "Characterization of TEG-1 in C. elegans germ line."

Zhao, Lina, Schedl, Tim, Wilson-Berry, Laura, Wang, Christopher L C, Hansen, Dave

[International Worm Meeting, 2009]

The C. elegans germline is a polar tissue with distal cells mitotically dividing, while more proximal cells enter meiosis and differentiate into gametes. Results from biochemical and genetic studies indicate that the GLP-1/Notch signaling pathway promotes distal germ cell proliferation by antagonizing two redundant pathways, one containing GLD-1 and the other GLD-2, which each promote meiotic entry (1-3). Two genetic screens were performed to identify novel genes involved in regulating the proliferation vs. differentiation decision. In one screen, mutations that enhanced the over-proliferation phenotype of a weak glp-1(oz112oz120 gf) allele were identified. In the other screen, mutations that were synthetic tumorous with a gld-2 null allele were isolated. As a result, teg-1 (tumours enhancer of glp-1(gf)) was identified in both screens. The open reading frame of teg-1 encodes a protein of 370 amino acid residues. TEG-1-related proteins are found from yeast to human and contain GYF domain that is thought to play a role in protein-protein interactions (4). We have raised antibodies against TEG-1 peptides, which detect a band at about 65 kDa in N2 lysate, but not in a teg-1 null mutant lysate, indicating the antibodies are specific to TEG-1. Subcellular localization studies demonstrate that TEG-1 is found in all germline and somatic nuclei of the gonad, with stronger staining being detected in distal tip cell, proximal germ cells, and oocytes. Moreover, we found that TEG-1 co-immunoprecipitates with UAF-1, the large subunit of U2 small nuclear ribonucleoparitcle (snRNP) auxiliary factor (5), suggesting TEG-1 forms a complex with a subset of UAF-1. This finding is reminiscent the human TEG-1 homolog CD2BP2, which interacts with a tri-snRNP bridging protein that is a component of the spliceosome complex (6), and suggests C. elegans TEG-1 may also involve in pre-mRNA splicing. We are currently conducting experiments to identify potentially misspliced mRNAs in teg-1 mutants to better understand the mechanism of TEG-1in regulating the proliferation vs. meiotic entry decision in the germ line. 1. Hubbard, E.J. 2007. Dev. Dyn. 236: 3343-3357. 2. Kimble, J. and S.L. Crittenden. 2007. Annu. Rev. Cell Dev. Biol. 23:405-33. 3. Hansen, D. and T. Schedl. 2006. Curr. Top. Dev. Biol. 76:185-215. 4. Kofler, M.M. and C. Freund. 2006. FEBS J. 273:245-256. 5. Zorio, D.A. and T. Blumenthal. 1999. Nature 402:835-838. 6. Laggerbauer. B. et. al. 2005. RNA 11:598-608.

M Wang et al. (1999) International C. elegans Meeting "Vulval pattern formation revisited"

PW Sternberg, M Wang

[International C. elegans Meeting, 1999]

A lin-3 -encoded EGF-like growth factor produced by the anchor cell (AC) induces the vulval precursor cells (VPCs) to form a 3deg-3deg-2deg-1deg-2deg-3deg pattern during early L3. The granddaughters of the 1deg VPCs form an EFFE pattern; the granddaughters of the 2deg VPCs form an ABCD/DCBA pattern. These cells then undergo distinctive cell divisions and morphogenesis at late L3 and L4 to form a vulva. The precise formation of the final pattern is a multiple step process requiring the proper functioning of complex gene networks. We have first focused on the 1deg lineage pattern. At least two steps are involved in 1deg fate determination and patterning: first, induction to ensure a vulval fate and turning on early genes like egl-17 ; second, turning off early genes and differentially regulating the expression of late genes, such as cdh-3 , in E and F cells. By testing the response of VPCs to LIN-3 as a function of time, we demonstrate that VPC daughters are competent to respond to LIN-3. Truncation of LIN-3 signaling by AC ablation or overexpression of dominant negative RAS at different times indicates that a LIN-3 signal from the AC is required for proper induction at least until the end of the first VPC cell cycle or even later. If the level of LIN-3 signal is reduced by a lin-3 hypomorph mutation, the AC is required beyond the first VPC cell cycle. In addition to the induction step, AC signaling is also involved in the second step of 1deg patterning. Manipulation of LIN-3 signaling after the induction step (the early 4-cell stage of the VPCs) results in misspecification of the presumptive E and F cells. We are trying to decipher the signaling pathways that connect the initial VPC patterning to VPC progeny patterning. lin-39 is one connection between the ras signaling pathway and the downstream gene expression, cell fusion and cell cycle regulation. Careful examination of the second step of 1deg patterning suggests that it can be divided into substeps that are temporally and spatially regulated by a transcription factor network. For example, while turning off early gene expression requires lin-11 (LIM), keeping off early gene expression and regulating the differential expression of late genes require lin-11 and cog-1 (homeobox) in both E and F and particularly egl-38 (PAX) in F cells.

Yaming Wang et al. (2001) International C. elegans Meeting "Characterizing the C. elegans ortholog of STATs"

David E Levy, Yaming Wang

[International C. elegans Meeting, 2001]

STAT proteins are latent transcription factors that are activated by tyrosine phosphorylation in response to extracellular signals and have been identified in a variety of organisms. To study the biological function of the STAT gene in C. elegans and its underlying signal transduction pathway, we cloned the nematode ortholog of mammalian STAT genes. It encodes a protein of 82KD that contains a DNA binding domain, SH2 domain, and site for tyrosine phosphorylation conserved with its mammalian counterparts. Promoter::GFP studies and immunohistochemistry using rabbit antibody against the endogenous protein show that it is highly expressed in pharynx, intestine, body muscle and most of the neuronal cells. RNAi experiment failed to show any detectable phenotype. We then screened a Tc1 transposon insertion and deletion library using mut-2 strain MT3126, and obtained a mutant that has a 2.1kb genomic deletion in the gene, resulting in complete loss of protein expression. We have completed background cleaning and now are in the process of characterizing the mutant phenotype.

Duo Wang et al. (2001) International C. elegans Meeting "Osmoregulation by daf-2/ age-1 insulin pathway"

Duo Wang, Gary Ruvkun

[International C. elegans Meeting, 2001]

Water balance is an important variable for terrestrial and some marine animals. For example, there is exquisite regulation of thirst reflexes in mammals based on small percentage changes in hydration. We expected that water balance would be, just as food and temperature are, key environmental input to the decision to grow reproductively. Dauer larvae of C. elegans have distinct reprogrammed secretory-excretory systems, which play an important role in osmoregulation through all larval stages. So we tested whether dauer pathway, including daf-2 / insulin, daf-7/ TGF b , and daf-11 / guanyl cyclase pathways, mutants affect sensitivity to osmotic pressure. We found that worms with mutations in the daf-2 insulin pathway are specifically sensitive to high osmolarity compared with wild type animals (N2). Previous studies have suggested that signals through DAF-2 insulin receptor are mediated by AGE-1 (a PI-3 kinase), PDK-1 and AKT-1/2 (protein kinases); and DAF-16, a forkhead transcription factor, is the main target of the signals. Daf-2 (e1370) or age-1 (mg305) mutant worms on NGM plates with 200 mM NaCl showed 100% embryonic or L1 lethality while wild type (N2) animals were viable. Pdk-1 loss-of-function mutants were less sensitive to high osmolarity than daf-2 or age-1 mutants but were still more sensitive than N2. Interestingly, akt-1 loss-of-function mutants exhibited wild-type resistance to high osmolarity. The lethality of daf-2 or age-1 in response to high osmolarity can be suppressed by daf-16 (mgDf47) or rescued by neuronal-specific expression of an age-1 transgene, both of which are also able to suppress or rescue the daf-c and aging phenotype of the daf-2 or age-1 mutants. Thus relationship between dauer formation, longevity and osmoregulation might be indicated. Finally, similar results were obtained using various concentrations of KCl, Na 2 SO4, or sorbitol in NGM plates , indicating that the observed hypersensitivity is a general response to osmolarity rather than to toxic levels of a given salt constituent. In order to identify other genes involved in osmoregulation, we have performed a genetic screen for suppressors of daf-2 (e1370) lethality caused by high osmolarity. We have mutagenized 50,000 haploid genomes and have identified 13 independent mutations. Eleven are allelic to daf-16 and we are in the process of characterizing the other two mutants.

ZW Wang et al. (1999) International C. elegans Meeting "Function, Tissue Distribution, and Mutants of nSLO1"

M Nonet, O Saifee, ZW Wang, T Jegla, L Salkoff

[International C. elegans Meeting, 1999]

The slo-1 gene of C. elegans encodes a large-conductance, Ca 2+ -activated K + channel. Deduced amino acid sequence reveals a protein ( nSLO1 ) of 1141-residues which shares a high level of homology with the SLO1 proteins of mammals and Drosophila . Similar to these homologues, the nematode slo-1 gene is widely expressed in the nervous system and in many muscle types. This was shown by the expression pattern of a slo-1 promoter::GFP transgene. When nSLO1 was expressed in Xenopus oocytes, both single-channel and macroscopic currents could be recorded in inside-out membrane patches. The activity of the channel was Ca 2+ - and voltage-dependent; higher activity was seen with increasing cytoplasmic Ca 2+ concentrations and at more positive membrane potentials. Mutants of slo-1 were discovered in a screen for suppressors of a syntaxin mutant unc-64 ( e246 ). Homozygous e246 animals are behaviorally lethargic and uncoordinated. After subjecting e246 animals to EMS, animals showing a revertant phenotype were analyzed for new mutations. Six allelic mutants were thus identified and mapped to chromosome V in the vicinity of unc-80 where slo-1 resides. In the absence of e246 , animals homozygous for the new mutants are phenotypically jerky, and aldicarb-hypersensitive. However, in the genetic background of the e246 mutation, these mutants partially suppress the e246 lethargy. Introduction of the wild-type slo-1 gene (minigene) into the double mutants reinstated lethargy. Sequencing of the js118 mutant allele revealed a 270-bp deletion in slo-1 that removed the splice acceptor site of exon 16. As a result, exon 16 was absent; frame shift and premature termination occurred in the 17th exon. A large portion of the C-terminus, including the region conferring Ca 2+ -sensitivity, was therefore removed. These observations are in agreement with a hypothesis that the syntaxin mutation inhibits neuromuscular activity by reducing neurotransmitter release, whereas a loss of function mutation of the slo-1 gene counters this effect by decreasing K + conductance (and therefore increasing membrane excitability) at presynaptic and/or postsynaptic sites. Our results suggest that nSLO1 plays an important role in neuromuscular activities in C. elegans .

Xiaochen Wang et al. (2001) International C. elegans Meeting "Study of the function of phosphatidylserine receptor in C.elegans"

Ding Xue, Valerie A Fadok, Xiaochen Wang, Duncan Ledwich

[International C. elegans Meeting, 2001]

The recognition and clearance of apoptotic cells is an important step during apoptosis. In this process, it has been observed that apoptotic cells lose phospholipid asymmetry and expose phosphatidyserine (PS), an anionic phospholipid, on the outer surface of the plasma membrane. The exposed PS has been proposed to serve as a recognition marker for engulfing cells. How the exposed phosphatidylserine is recognized and mediates the corpse engulfment process is still largely unknown. Recently, a phosphatidylserine receptor (PSR) that appears to mediate specific recognition of PS on apoptotic cells by phagocytes has been identified in humans 1 . It is expressed on the surface of macrophages, fibroblasts and epithelial cells. When transfected into cultured cells, the PSR allows cultured B and T lymphocytes to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. This phosphatidylserine receptor is highly conserved throughout evolution. We have noted the presence of a homologous gene in C.elegans that shares high sequence homology with human PSR (46% identity). In order to investigate the possible role of the putative worm PSR homolog in the engulfment of dying cells during C. elegans programmed cell death, we are currently performing RNAi experiments and looking for deletions in the PSR homolog locus to examine the loss-of-function phenotypes of worm PSR. We have made several PSR::GFP fusions and are in the process of determining its expression pattern and localization in nematodes. These experiments will help us better understand the function of PSR in nematodes and also will likely provide additional important information about the mechanisms of cell corpse recognition and clearance in C.elegans . 1.Fadok, V.A. etal. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405,85-90(2000).

Wang BB et al. (1991) International C. elegans Meeting "An update on the mab-5 complex"

Kenyon CJ, Wang BB

[International C. elegans Meeting, 1991]

We are interested in determining whether C. elegans contains a complex of homeobox-containing genes similar to those found in Drosophila and in vertebrates. The order of the Drosophila homeotic selector genes in the Antennapedia and Bithorax complexes correlates with the order of their expression along the anterior-posterior axis of the embryo. Likewise, the vertebrate homologues of these genes are grouped into complexes and, as shown in the mouse, their chromosomal organization also correlates with their domains of expression. Previous work in our lab on mab-5, an Antp-like homeobox gene responsible for the normal specification of cells in the posterior body region, and more recent work by several labs on ceh-13, egl-5 and lin-39, suggest that a similar complex may exist in C. elegans ( discussed in Wang and Kenyon, WBG 11:5). The chromosomal positions and homeobox sequences of ceh-15, mab-5 and ceh-ll are analogous to the organization of homeobox genes in the Drosophila and vertebrate complexes. (WBG 11:5) We have now determined that ceh-13, a homeobox gene with lab-like homology (Schaller, et al., Nuc. Acids Res. 18:2033), is located to the left of ceh-15, also analogous to the organization in the Drosophila and vertebrate complexes. We are probing about 20 overlapping cosmids spanning the mab-5 region (mec-14 to egl-45) with degenerate homeobox-specific oligos described by Burglin, et al. (Nature 341:239) in order to identify more homeobox genes that may be a part of the mab-5 complex. Preliminary hybridization data suggest that there could be several additional Antp-like homeobox genes in the region.

Y Wang et al. (1999) International C. elegans Meeting "Molecular Cloning and Characterization of a STAT homologue in C. elegans"

DE Levy, Y Wang

[International C. elegans Meeting, 1999]

Though characterized by simplicity and directness in mammalian systems, JAK/STAT signaling pathway is involved in many different regulatory events, such as innate immune responses to viral or bacterial infection, T cell functions, and embryonic development. In Drosophila , a STAT homologue has been identified and shown to be essential for embryonic development. The fact that Dictyostelium discoideum has a STAT-like molecule to regulate its pre-stalk cell differentiation suggests the existence of STAT(s) in a wide variety of organisms. Here we report a C. elegans homologue of STATs. Nucleotide sequence analysis reveals that the 2.4kb cDNA contains a single ORF about 2.1kb which encodes a 80KD protein. With human STAT5b, ceSTAT shares 29% identity in SH2 domain, 37% in DNA binding domain. It also contains a GYIQ tyrosine phosphorylation motif which is conserved in all the STATs. Surprisingly, ceSTAT does not have an N-terminal domain which is highly conserved in mammalian STATs as well as in Drosophila STAT. Northern analysis of total RNA from mix-staged N2 worms shows a single mRNA of 2.4kb. When expressed in cell culture, ceSTAT can be tyrosine phosphorylated by mammalian kinases, resulting in its ability to bind DNA probe containing GAS element. Further, the C-terminus contains a transactivation domain that will function when fused to the GAL4 DNA binding domain. Initial studies of the function of ceSTAT involved injection of dominant-inhibitory dsRNA. We saw no phenotype in either injected animals, F1 or F2 progeny. This suggests that ceSTAT is probably not involved in worm developmental processes. To further determine the function of ceSTAT, we are currently isolating mutagenized worms carrying genomic mutations.