W Zhang et al. (1999) International C. elegans Meeting "Analysis of several mutations that disrupt vulval development"

M Han, W Zhang, A Mapes

[International C. elegans Meeting, 1999]

We have conducted ts and sterile mutant screens for mutations that disrupt various steps of vulval development. Analyses of several mutations will be reported. ku260 and ku272 . Mutations have similar vulval phenotype likely due to defects in VPC generation. Mutants display either an everted vulva (Evl or Pvl) or fail entirely to differentiate vulval tissue. Nomarski analysis indicates that such mutants are often missing more than one Pn.p cell, resulting in either abnormal or no vulval invaginations. Using an unc-47::GFP as a marker, we observed that many GABAergic neurons in the ventral cord are absent (approximately 50% missing). ku260 homozygotes are Unc, likely due to the missing VD neurons. Both ku260 and ku272 mutants have an abnormal somatic gonad and contain no oocytes, leading to a sterile phenotype. ku260 was mapped to LGIV between unc-24 and unc-129 and is uncovered by eDf19 . Pools of cosmids are being injected in order to rescue the mutant phenotype. ku260 fails to complement evl-24(ar124) which was previously isolated by Seydoux and Greenwald as a class III Evl mutation(1). ku272 has been mapped to right arm of LGI. ku259, ku255 and ku270 . These mutations appear to cause defects in both vulval lineage and morphogenesis and display an Evl phenotype (subtle for ku270 ). We also detect a low to moderate percentage of double invaginations and protrusions in ku259 and ku255 mutant worms. Nomarski analysis indicates that the abnormal or double invaginations observed are at least partly due to lineage defects. All three mutations also cause a sterile phenotype (incomplete for ku270 ). ku259 was mapped to LGIII between dpy-1 and daf-2 . DNA-mediated transformation is currently being performed to identify the affected gene. ku255 was mapped between unc-13 and nob-3 on LGI and failed to complement evl-17(ar94) (1). ku270 has been mapped to the right of lin-25 on LGV. The cloning of both genes is in progress. 20-30% of ku273 homozygous mutants are Egl and some contain two or three vulval protrusions. Nomarski analysis reveals that the Egl and Muv phenotype are caused by defects in vulval lineage and morphogenesis. We are currently mapping this gene to a chromosomal location. (1) Seydoux, G., C. Savage, and Greenwald. I. (1993). Dev. Biol. 157,423-436.

Shenghyuan Zhang et al. (2001) International C. elegans Meeting "Suppressors that recover daf-7::gfp expression in tph-1(mg280)"

Shenghyuan Zhang, Ji Ying Sze

[International C. elegans Meeting, 2001]

tph-1 encodes a tryptophan hydroxylase, the key enzyme for serotonin biosynthesis. Our previous studies show that daf-7::gfp expression is reduced 6-fold in the deletion mutant tph-1(mg280). This suggests that serotonin may normally up-regulate daf-7 expression. To isolate genes that upregulate daf-7 expression in response to serotonin signaling, we have been screening mutagenized tph-1(mg280) worms for suppressors that recover daf-7::gfp expression. We have clonally screened 3,870 haploid genomes and isolated 41 suppressors, 15 of them are being back-crossed. DiI staining of back-crossed suppressors showed a normal dye filling pattern, suggesting that the mutations do not grossly compromise the integrity of the chemosensory neurons. Further examination of the back-crossed suppressors has led us to classify the suppressors into two classes. The first class of the suppressors shows a strong daf-7::gfp expression only in L2 and L3 tph-1(mg280). The second class of the suppressors shows a strong daf-7::gfp expression even in the adults. The results of genetic and molecular characterization of the suppressors will be discussed.

Zhang Y et al. (1991) International C. elegans Meeting "THE ANALYSIS OF UNC-104 GENE EXPRESSION."

Zhang Y, Otsuka AJ

[International C. elegans Meeting, 1991]

Mutations in the unc-104 gene of the nematode C. elegans result in uncoordinated and slow movement. The unc-104 gene was cloned by using transposon unc-104 alleles [A.J.Otsuka et-122 (1991)]. An open reading frame which encodes a 1584-amino acid protein was discovered by DNA sequence analysis of unc-104 cDNAs. This protein has a sequence similar to the kinesin heavy chain motor domain. The greatest similarities are in the amino-terminal ATPase and microtubule- binding domain. However, since the remainder of the protein sequence has less similarity to kinesin and it was found recently that unc-116 product has greater similarity to Drosophila kinesin. Therefore,the unc-104 gene product may belong to a novel group of kinesin-like proteins. This motor protein might be used for axonal transport of neuronal components, including synaptic vesicles. Northern blot and in situ hybridization at various developmental stages of the worm are underway in order to understand whether or not this gene product exists only in the adult worm or throughout the development of the hybridizations may determine whether or not unc-104 is expressed predominantly in the nervous system. The results can further reveal the functions of this gene product.

Zhang M et al. (1997) International C. elegans Meeting "POSSIBLE INTERACTION BETWEEN PAR-3 AND PAR-5"

Kemphues KJ, Zhang M

[International C. elegans Meeting, 1997]

Six par genes play an important role in the generation of polarity in the one-cell embryo. As all those genes have been or are close to being cloned, we have begun to investigate possible interactions among the PAR proteins. We think PAR-5(W. Wang et al., 1996 East Coast Worm Meeting abstract 2) and PAR-3 might interact for two reasons: 1.In par-5 mutant, the PAR-3 is mislocalized. 2.PAR-5 was identified as a member of 14-3-3 protein family and PAR-3 was found to have the RSXpSXP consensus motif that has been shown to be a binding site for 14-3-3 proteins. We subcloned the par-5 gene and two different-sized fragments of par-3 into the yeast 2-hybrid vectors and detected a strong interaction when par-3 fragments were fused with Gal4 activation domain while par-5 was fused with the binding domain. We detected a weak interaction when they were cloned in the other direction. Thus, these two gene products interact in yeast. We are currently using coimmunoprecipitation to determine whether PAR-3 and PAR-5 can interact in worms. (Thanks to Dr. Andy Golden for providing the anti-peptide par-5 antiserum). We are also testing the ability to produce par-5 phenocopies by injecting peptides containing the RSXpSXP motif into wild type worms. Furthermore, we are trying to rescue par-3 mutants with par-3 mutated at the RSXpSXP site and comparing the rescue result with that using wild type par-3.

Zhang M et al. (1995) International C. elegans Meeting "SITE-SELECTED INSERTION OF Tc5 INTO src-1"

Collins JJ, Thacker CM, Zhang M

[International C. elegans Meeting, 1995]

We have islolated a Tc5 insertion in src-1, a worm homolog of the vertebrate proto-oncogene c-src. The vertebrate gene encodes a cytoplasmic tyrosine kinase implicated in the control of cell proliferation and differentiation. Activating mutations in c-src lead to cell transformation and neoplasia. Major questions remain about the normal function of c-src and how it is altered by oncogenic mutations. The identification of src-1 (Thacker and Capecchi; 1989 C. elegans Meeting p.247), provides an opportunity to use genetics to investigate these problems. Our efforts to isolate a Tc1 insertion in src-1, using the sib selection/PCR protocol devised by Rushforth and Anderson, were confounded by high frequency somatic insertion of Tc1 in this region. So we adapted the method and isolated a Tc5 insertion in src-1. The insertion is in an intron in the region of the gene that encodes the kinase domain. Homozygous src-1::Tc5 mutants exhibit no obvious phenotypic defects. We are testing the assumption that Tc5 is spliced out along with the intron, generating normal src-1 message. We are also screening for deletion derivatives to isolate and characterize src-1 knock-out worms. Tc5 offers certain advantages over Tc1 in the sib selection /PCR approach. Most notably, Tc5 exhibits little or no somatic insertion and its low copy number tends to reduce PCR artifacts and mispriming events. Tc5 generates deletions upon excision like Tc1; we are currently investigating its suitability for gene replacement approaches.

Y Zhang et al. (1999) International C. elegans Meeting "Phenotypic analysis of two C. elegans endocytosis mutants rme-1 and rme-8"

D Hirsh, L Pedraza, B Grant, Y Zhang

[International C. elegans Meeting, 1999]

Yinhua Zhang, Barth Grant, Laura Pedraza and David Hirsh, Dept. of Biochemistry and Molecular Biophysics, Columbia University, New York, NY10032 We wish to complement the classical cell biology studies on receptor-mediated endocytosis in animal cells with a genetic analysis in C. elegans . We isolated mutants defective in yolk-protein uptake using our YP170-GFP assay (see abstract by Grant et al). Here we describe two endocytosis mutants: rme-1 which appears to function in the endosome, and rme-8 which appears to function early in endocytosis. rme-1 mutants have a mild block in YP170-GFP uptake, but have normal brood size and viability. rme-1 worms also form large vacuoles in their gut cells and to a lesser degree in hypodermal cells. In wildtype gut cells, RME-1 is present on small vesicles with a punctate pattern. These vesicles take up a low level of GFP from the body cavity when it is secreted from muscle. In one missense mutant, RME-1 is also present on the surface of large vacuoles. The large vacuoles accumulate GFP to a higher level than do wild type vesicles. One model would be that the large vacuoles are derived from the small vesicles and reflect a defect in endosome sorting which results in a mild reduction of YP170-GFP uptake by rme-1 mutants. Currently we are testing this model. RME-1 contains a conserved region of 555 amino acids which has 75% identity to its mammalian homologs, indicating they function similarly in mammalian cells. rme-8 is a ts lethal with a strong block in YP170-GFP uptake at all temperatures. rme-8 is required at all developmental stages; shifting mutant larvae to 25C causes arrested development. Arrested larvae fail to shed their old cuticles. Shifting adult hermaphrodites to 25C causes endoreplication in oocytes and degeneration of the gonad and intestine. The yolk receptor (RME-2) and alpha-adaptin (a component in clathrin coated pit) co-localize with yolk in discrete dots at 15C and in large patches at 25C. This colocalization suggests that the rme-8 mutation blocks the internalization step of endocytosis at the plasma membrane. rme-8 maps to a 300kb genomic interval between gld-1 and lin-10 ; we are further mapping it in order to clone it and identify its function in endocytosis.

Yue Zhang et al. (2001) International C. elegans Meeting "The C.elegans Mi-2 chromatin remodelling proteins LET-418 and CHD-3 have essential developmental functions"

Yue Zhang, Frederic Guerri, Myriam Passannante, Fritz Mueller, Chantal Wicky

[International C. elegans Meeting, 2001]

The Mi-2 protein is the central component of the recently isolated NuRD (nucleosome remodeling and histone deacetylase) complex .Although the NuRD complex has been the subject of extensive biochemical analyses,little is yet known about its biological function. Therefore we have initiated an analysis of the two C.elegans Mi-2 homologues LET-418 and CHD-3. We found that they play essential roles during development and that the two proteins possess both shared and unique functions during vulval cell determination. LET-418 is involved in antagonizing theRTK/Ras /MAP kinase pathway via the synMuv pathway, whereas LET-418 and CHD-3 together appear to be important for the proper execution of the LIN-12 Notch dependent secondary cell fate of P5.p and P7.pvulval precursor cells,. Currently, we are investigating a possible connection between let-418, chd-3 and the lin-12 Notch signalling pathway during vulva development. Furthermore,we have started an yeast two hybrid screen to identify proteins interacting with both C.elegans Mi-2 homologs . The results will be discussed.

H Zhang et al. (1999) International C. elegans Meeting "sop-1, -2 and -3: New Components That Regulate C. elegans Hox Gene Expression"

SW Emmons, H Zhang

[International C. elegans Meeting, 1999]

During male tail development, the Hox genes mab-5 and egl-5 are required for the seam cell V6 to generate five pairs of sensory organs, called rays. How mab-5 and egl-5 expression is initiated in V6 and how this expression pattern is dynamically regulated throughout development is largely unknown. pal-1 , which encodes a Drosophila caudal homolog, is required for mab-5 expression in V6. Neighboring seam cells send inhibitory signals to inhibit mab-5 expression. The nature of these inhibitory signals is unknown, but Wnt signaling, as well as pal-1, can overcome them (1). To understand how Hox genes are spatially and temporally regulated, we performed a screen for suppressors of pal-1 ( e2091) , a regulatory mutant, and identified three new genes, sop-1 , - 2 and -3 ( s uppressor o f p al-1 ), that are involved in regulating mab-5 and egl-5 expression in V6 and elsewhere. Mutations in all three genes are recessive and strongly suppress the pal-1(e2091) ray defect in a mab-5- dependent manner. By mosaic analysis, we found that sop-1 cannot suppress the ray phenotype of pal-1(ct224) , a null allele. This suggests that sop-1(bx92) may suppress pal-1(e2091) by restoring pal-1(+) activity in V6. We have cloned sop-1 by transformation rescue, and found that it encodes a 3520-AA protein that contains a Q-rich region at its C terminus. Mutations in the human sop-1 homolog, HOPA , have been implicated in mental retardation (2). sop-2 mutants have pleiotropic ray and vulval phenotypes that might be explained by the ectopic expression of Hox genes. By using mab-5::gfp and egl-5::gfp reporter genes, we have found that Hox genes are ectopically expressed in sop-2 mutants, including massive expression in the head neurons, possibly accounting for a lethal phenotype at 25degC. The molecular characterization of sop-2 is in progress. bar-1 , a b -catenin homolog, is required for ray production in sop-3; pal-1 mutants. This suggests that sop-3 acts to prevent inappropriate expression of the Wnt pathway. No previously known components of the Wnt pathway are present in the small genetic interval containing sop-3 . (1) CP Hunter et al. (1999) Development 126 , 805-814 (2) RA Philibert et al. (1998) Molecular Psychiatry 3 , 303-309.

Yun Zhang et al. (2001) International C. elegans Meeting "Generation of cell-type-specific RNA and gene expression profiles for Mechanosensation Neurons in C. elegans"

Yun Zhang, Thomas Delohery, Marty Chalfie, Charles Ma

[International C. elegans Meeting, 2001]

One of the great advantages of C. elegans as an experimental system is the ability to characterize mutant phenotypes at the level of single type of cells. With the sequencing of the genome, the use of DNA microarrays offers the opportunity to look at the genome-wide transcriptional activity under different experimental conditions. Unfortunately, array experiments using whole worm RNA do not afford the resolution needed to look, for example, at the differences in expression between specific wild type and mutant nerve cells. We have developed a technique to generate cell-type-specific RNA to use with cDNA microarrays to study gene expression profiles for a single type of C. elegans cells and have applied it to study the differentiation and function of the embryonic touch receptor neurons (ALM and PLM). While saturation mutagenesis for touch insensitive mutants have identified several genes needed for touch cell development and function, those screens could not have identified redundant genes, pleiotropic genes or genes that mutate to a touch super-sensitive phenotype. DNA array data should reveal such genes as well as provide candidates for as yet uncloned mec genes. To identify mec-3 -dependent genes expressed in the touch cells, we collected late-stage embryos from strains with integrated GFP arrays: either P mec-18 gfp in a wild-type background for differentiated touch cells or P mec-3 gfp in a mec-3 (e1338) background for mec-3 mutant cells that should have become touch cells. Cells from dissociated embryos were cultured overnight (many extended processes in culture) and the GFP-positive cells were enriched 100-fold by fluorescence-activated cell sorting. Typical post-sort values from cell sorting are 4x10 6 cells with 40-50% being GFP positive. mRNAs from these cells were linearly amplified 10 6 -fold using a modified Eberwine's method, labeled and applied to cDNA microarrays (in Stuart Kim's lab) representing virtually all of the C. elegans genes. The reliability and sensitivity of the expression profiles is suggested by the finding that 6 of the 10 known mec-3 -dependent genes were among the top 15 genes and 8 out of 10 known mec-3 -dependent genes (including mec-3 itself) had ratios above 4.0 (the two other genes had ratios higher than 2.0). In addition, the profile also provides several groups of interesting candidates besides unknown function genes with ratios equal to or higher than ratios of known mec-3 -dependent genes: 1) the gene with the highest ratio maps to the same position as the previously uncloned mec-17 gene, which is needed for the maintenance of touch cell fate (we have confirmed that this gene is expressed exclusively in the touch cells and have identified two missense mutations in mec-17 (u265) ), 2) two other genes map to the area of mec-15 and a single mutation on X that disrupts touch cell microtubules, 3) several genes that contribute to the T-complex chaperonins, which are needed for the formation of tubulins, 4) several channel genes in addition to mec-4 and mec-10 and 5) genes for G-protein coupled receptors and transcription factors. We are also using RNAs from sorted and unsorted cells to identify touch-cell-enriched genes that are not mec-3 -dependent.

Zhang H et al. (1997) International C. elegans Meeting "GENETIC SCREEN FOR NEW COMPONENTS OF THE MALE RAY DEVELOPMENT PATHWAY"

Zhang H, Emmons SW

[International C. elegans Meeting, 1997]

A hierarchy of regulatory events during postembryonic development of the posterior seam cells plays an important role in male ray development. A posterior seam cell sends a signal to its anterior neighbor to inhibit production of ray(s). Some genes can antagonize this inhibitory signal to allow V5 and V6 to produce rays in wild type males. For example, the pal-1 gene acts autonomously in V6 to override the inhibitory signal from the posterior seam cell T and allows V6 to produce rays 2- 6. One possible downstream target of pal-1 is the Hox gene mab-5. The nature of the inhibitory signal is unknown. In order to identify the signal from T and to identify possible components of the mab-5 regulatory pathway, we performed a screen for suppressors of pal-1(e2091). Nineteen mutations defining 15 genes were obtained from about 4000 genomes screened. Molecular characterization of two genes identified in this screen, sop-1 (suppressor of pal-1) and sop-2, is in progress. The effect of sop-1 mutations is limited to V6, and usually results in normal development of V6 rays. This suggests that the sop-1 mutation acts early in the hierarchy, restoring a normal cascade of regulatory events. Thus sop-1 is a candidate for a component of the inhibitory T signalling pathway. In contrast to sop-1, the sop-2 phenotype is more pleiotropic, possibly because it causes ectopic expression of Hox genes during later developmental stages. In sop-2, 7% of male sides have rays 2 and 3 transformed to rays 4, 5, 6. This is probably due to ectopic egl-5 expression in the anterior branch of the V6 lineage. About 30% of hermaphrodite alae are discontinuous in sop-2 mutant, and this is also dependent on Hox genes.