Hwang, Tom et al. (2015) International Worm Meeting "The Role of ZTF-15 in the meiotic synapsis checkpoint."

Chen, Pin-Hsi, Ye, Alice, Bhalla, Needhi, Hwang, Tom

[International Worm Meeting, 2015]

During meiosis I, homologous chromosomes must pair, synapse, and recombine to promote proper chromosome segregation. Defects in any of these processes can lead to incorrect chromosome segregation and aneuploid gametes. One mechanism that prevents aneuploidy during meiosis is the synapsis checkpoint, which activates apoptosis in the event of asynapsis. We identified ZTF-15 in an RNAi screen to identify new checkpoint components. Here we report that ztf-15 plays an integral role in the synapsis checkpoint in C. elegans. In genetic backgrounds that activate the synapsis checkpoint, loss of ZTF-15 reduces apoptosis to wildtype levels. Additional analysis indicates that ztf-15 is also required for timely pairing and synapsis of autosomes. These phenotypes are reminiscent of another synapsis checkpoint component, the conserved kinase PLK-2. To further interrogate a potential interaction between these two proteins, we performed two-hybrid analysis and found that PLK-2 and ZTF-15 interact. We also show that ZTF-15 is an in vitro substrate of PLK-2. Overall, these data suggest that ZTF-15 acts downstream of PLK-2 to promote synapsis checkpoint signaling. .

Hwang H-Y et al. (1997) International C. elegans Meeting "GENES INVOLVED IN VULVAL MORPHOGENESIS"

Horvitz HR, Hwang H-Y

[International C. elegans Meeting, 1997]

Mutations in sqv-1 to 7 (squashed vulva) and spe-2 result in indistinct separation between the anterior and posterior halves of the vulva from the late L3 stage onward (see abstract by Herman and Horvitz). Three of these genes (sqv-3, sqv-7, spe-2) have been cloned so far. The predicted proteins encoded by sqv-3 and sqv-7 are similar to proteins involved in glycosylation in other species. One model for the Sqv phenotype is that mutations in the sqv-1 to 7 and spe-2 genes result in abnormal adhesion between vulval cells with adhesive properties modulated by glycosylation. We are working toward cloning additional sqv genes. We have mapped sqv-1 to LGIV, between unc-24 and bnP3, and sqv-4 to LGV, between unc-42 and emo-1. We are mapping these genes further and will use transformation rescue to clone them to allow molecular studies of their roles in vulval development. In addition to causing vulval defects, sqv-1 to 7 and spe-2 cause maternal-effect embryonic lethality, suggesting that these genes have roles in other developmental processes. We plan to screen for suppressors of embryonic lethality. Some such suppressors may function in vulval morphogenesis.

Hwang H-Y et al. (1996) East Coast Worm Meeting "GENES INVOLVED IN VULVAL MORPHOGENESIS"

Hwang H-Y, Horvitz HR

[East Coast Worm Meeting, 1996]

The strongest alleles of sqv-1 to 7 (squashed vulva) and spe-2 result in indistinct separation between the anterior and posterior halves of the vulval invagination from the late L3 stage onward (see abstract by Herman and Horvitz). Three of these genes (sqv-3, sqv-7, spe-2) have been cloned so far. The predicted proteins encoded by sqv-3 and sqv-7 are similar to proteins involved in the glycosylation pathway in other species, suggesting glycosylation may play a part in vulval morphogenesis. The characterization of other sqv genes and of the interactions of the sqv genes with other genes should help elucidate the mechanisms of vulval morphogenesis. One gene that may interact with the sqv genes is lin-12. To find phenotypic differences among the sqv genes, we plan to examine the worms with mutations both in the sqv genes and in lin-12. The fates of six ventral hypodermal cells, P3.p, P4.p, P5.p, P6.p, P7.p, and P8.p are affected by lin-12. In wild-type animals, the vulva arises from the P(5-7).p cells; P6.p expresses the 1st fate, and P5.p and P7.p express the 2nd fate. Certain semi-dominant alleles of lin-12 cause P(3-8).p cells to express the 2nd fate, resulting in a multivulva phenotype. In worms lacking lin-12 function, the P(5-7).p cells all express the 1st fate, resulting in an abnormal protrusion at the normal position of the vulva. Because lin-12 semi-dominant and loss-of-function alleles each cause only certain classes of Pn.p cells to be generated, mutations in the sqv genes may cause distinct phenotypes in different lin-12 backgrounds. If so, we may be able to define the epistatic relationships among the sqv genes. We also plan to clone more sqv genes. We are now mapping these genes more precisely, which should allow us to use transformation rescue to clone them and to initiate molecular studies of their roles in vulval development. 1. Greenwald et al. (1983). Cell 34: 435-444.

Hwang, gyujin et al. (2015) International Worm Meeting "A Role for Peroxidasin PXN-1 in Aspects of C. elegans Development."

Hwang, gyujin, Cho, Jeong Hoon

[International Worm Meeting, 2015]

The Caenorhabditis elegans peroxidasins, PXN-1 and PXN-2, are extracellular peroxidases; pxn-2 is involved in muscleepidermal attachment during embryonic morphogenesis and in specific axon guidance. Here we investigate potential roles of the other homologue of peroxidasin, pxn-1, in C. elegans. A pxn-1 deletion mutant showed high lethality under heat-stress conditions. Using a transcriptional GFP reporter, pxn-1 expression was observed in various tissuesincluding neurons, muscles, and hypodermis. A translational fusion showed that PXN-1::GFP was secreted and localized in extracellular matrix, particularly along body wall muscles and pharyngeal muscles. Various neuronal developmental defects were observed in pxn-1 mutants and in pxn-1 over-expressing animals, including andedness, branching, breakage, tangling, and defasciculation. These results suggest that pxn-1, like other peroxidasins, plays an important role throughout development.

Hwang, H. et al. (2019) International Worm Meeting "Plasticity of ascr#3 avoidance behavior in C. elegans."

Hwang, H., Kim, K.

[International Worm Meeting, 2019]

Experiences or environmental cues can influence neuronal functions and behaviors. However, the neuronal and molecular mechanisms of behavioral plasticity are not fully understood. Caenorhabditis elegans secretes a mixture of small molecules referred as ascarosides that sense environmental conditions including population density and influence many aspects of development and behavior of C. elegans (Edison, 2009; Srinivasan et al., 2012). Wild-type hermaphrodites are repulsive to ascr#3(asc-?9, C9) while wild-type males are attractive to it (Macosko et al., 2009, Jang et al 2012). Previously, we have shown that ascr#3 avoidance behaviors are modulated by early experience of ascr#3 and feeding state, indicating that ascr#3 avoidance behaviors are plastic (Hong et al., 2017, Ryu et al., 2018). Here, we next investigated whether ascr#3 response is habituated by repeated exposure of ascr#3. We found that wild-type hermaphrodites exhibit normal ascr#3 avoidance to each exposure up to ten times, suggesting that ascr#3 avoidance behavior is not habituated. However, we found that the ascr#3-sensing ADL neurons shows decreased Ca2+ response to ascr#3 upon repeated exposure of ascr#3, indicating that adapted ADL responses to repeated ascr#3 exposure do not lead to habituation of ascr#3 avoidance behavior. In addition, we examined the effects of mating on ascr#3 avoidance and found that mated hermaphrodites appear to exhibit increased ascr#3 avoidance. Understanding how the behaviors are altered upon various experience or condition is the essential step to dissect molecular and neuronal mechanisms of behavioral plasticity.

Hwang, In-sok et al. (2013) International Worm Meeting "A CaMK signaling cascade modulates pheromone-mediated developmental plasticity."

Sengupta, Piali, Fang, Zhi, Kim, Kyuhyung, Hwang, In-sok

[International Worm Meeting, 2013]

Animals regulate their development in response to changing environmental conditions and their internal metabolic status. This developmental plasticity is mediated by changes in gene expression, but the exact mechanisms by which environmental signals are transduced and integrated with internal status to affect developmental programs are poorly understood. C. elegans responds to conditions of overcrowding, limited food and high temperature by arresting development as a dauer larva. High levels of secreted ascaroside pheromone serve as the primary signal to trigger entry into the dauer stage (Golden & Riddle, 1982; Jeong et al., 2005; Butcher et al., 2008). We and others previously showed that pheromone exposure down-regulates expression of a daf-7 TGF-b and a subset of putative G-protein coupled chemosensory receptor genes such as str-3 in the ASI neurons; this downregulation influences dauer formation (Nolan et al., 2002; Peckol et al., 1999; Kim et al., 2009). To identify the signaling pathways and genes required for this pheromone-regulated gene expression and dauer formation, we performed a genetic screen and also examined candidate genes (Kim et al., 2009). These analyses showed that the cmk-1 (CaM kinase I) and ckk-1(CaM kinase kinase) genes are required to downregulate str-3 GPCR expression upon pheromone exposure. As expected, cmk-1 mutants are also defective in the pheromone-mediated dauer decision. Additionally, we found that crh-1 (CREB) is required for str-3 expression even in the absence of pheromone. Surprisingly, CMK-1 and CRH-1 act non-autonomously in the ASE/AWC chemosensory neurons to regulate str-3 expression in the ASI neurons, suggesting that cmk-1 and/or crh-1 may act in these neurons to transmit environmental signals to ASI. The phenotypic analyses of double mutants indicate that the CMK-1 and CRH-1 may act in parallel pathways. Current experiments are aimed at identifying the sensory cues that regulate str-3 expression, and investigating the mechanisms by which information is transmitted from the AWC/ASE to the ASI neurons.

Hwang, Tom et al. (2013) International Worm Meeting "ZTF-15 is required for the meiotic synapsis checkpoint in C. elegans."

Hwang, Tom, Bhalla, Needhi, Ragle, Matt

[International Worm Meeting, 2013]

In order to achieve proper meiotic chromosome segregation, homologous chromosomes must pair and synapse in prophase I to facilitate crossover recombination. Defects in meiotic prophase events can result in programmed cell death (apoptosis) or cell cycle arrest, indicating that these events are monitored by checkpoint mechanisms to avoid the production of aneuploid gametes. In C. elegans, unsynapsed chromosomes activate germline apoptosis independently of a DNA damage checkpoint that monitors recombination. While independent of one another, both of these checkpoints require the pro-apoptotic factor, egl-1. In response to asynapsis or DNA damage, egl-1 is transcriptionally upregulated to promote apoptosis so that defective meiotic nuclei are removed and do not go on to form aneuploid gametes. When the DNA damage checkpoint is activated, the C. elegans ortholog of tumor suppressor p53, cep-1, is responsible for promoting transcription of egl-1. However, cep-1 is not required for the synapsis checkpoint. We are interested in how egl-1 transcription is upregulated when asynapsis occurs. In an RNAi screen to identify new checkpoint components, we discovered ztf-15 is required for the synapsis checkpoint. ZTF-15 contains 5 C2H2 zinc finger domains, a motif that often mediates binding to specific DNA sequences. We speculate that ZTF-15 is the synapsis checkpoint counterpart of cep-1 and that it acts as a transcription factor to promote egl-1 transcription in the event of asynapsis. To test this hypothesis, we plan to use MOSSci to fluorescently tag ZTF-15 as well as create an antibody that recognizes ZTF-15 to determine where it localizes in the C. elegans germline. If its localization is consistent with a role in apoptosis, we can further test ztf-15's relationship with egl 1 through use of qPCR and gel mobility shift assays. Through these experiments, we hope to elucidate how meiotic events are monitored by cell cycle checkpoints to protect against chromosome missegregation and aneuploidy, which can contribute to infertility, birth defects, and cancer predisposition.

H Hwang et al. (1999) International C. elegans Meeting "Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis"

H Hwang, HR Horvitz

[International C. elegans Meeting, 1999]

Eight sqv ( sq uashed v ulva) genes ( sqv-1 to 8 ) have a mutant vulval morphogenetic phenotype that is defined by reduced separation between the anterior and posterior halves of the vulva in the L4 (1). SQV-3 and SQV-8 are glycosyltransferase-like proteins, and SQV-7 is similar to a putative nucleotide-sugar transporter (2). We have previously reported the cloning of sqv-1 and sqv-4 (3). Both SQV-1 and SQV-4 are similar to enzymes involved in nucleotide-sugar metabolism. These molecular identities suggest that glycosylation is important in shaping the vulva during development. We have now determined biochemically that SQV-4 is a UDP-glucose dehydrogenase. Recombinant SQV-4 expressed in E. coli can reduce NAD in the presence of UDP-glucose, suggesting that UPD-glucose is being converted to UDP-glucuronate. Like known UDP-glucose dehydrogenases, SQV-4 appears to act specifically on UDP-glucose, as it fails to reduce NAD using any of the many other nucleotide-sugars tested. Mutant recombinant SQV-4 derived from either of the two genetically identified mutant alleles does not show detectable activity. Preliminary antibody staining using a rabbit polyclonal antibody directed against a GST::SQV-4 indicates that SQV-4 is localized to the vulval cells and several other tissues, including seam cells. This result agrees with the expression of SQV-4::GFP fusion protein and supports a model in which SQV-4 acts in the vulval cells during vulval development. We also are trying to determine the functions of the other four cloned sqv genes using biochemical assays and heterologous complementation. Lastly, we are mapping and cloning the three remaining sqv genes. 1. Herman, T. et al. (1999). PNAS 96 : 968-973. 2. Herman, T. and Horvitz, H.R. (1999). PNAS 96 : 974-979. 3. Hwang, H. and Horvitz, H.R. (1998) East Coast C. elegans meeting, p. 103.

B Hwang et al. (1999) International C. elegans Meeting "Regulation of the C. elegans epidermal growth factor homolog LIN-3"

K Verbrugghe, N Moghal, B Hwang, J Liu, P Sternberg

[International C. elegans Meeting, 1999]

LIN-3, an epidermal growth factor in Caenorhabditis elegans , is involved in multiple developmental processes. Much is known about the downstream pathways from LIN-3 and its receptor LET-23, but little is understood about the mechanisms that regulate LIN-3. We are taking several approaches to understand the mechanisms regulating LIN-3. First, anti-LIN-3 antibodies and epitope-tagged LIN-3 have been generated to study its tissue-specific expression and putative regulated processing events. Second, the cis-acting elements of lin-3 which govern its anchor cell-specific and vulval cell-specific expression are being sought by deletion analysis of lin-3 regulatory regions. Finally, a genetic screen looking for mutations that suppress the vulvaless phenotype caused by decreased LIN-3 function has identified a potential candidate regulator of lin-3 as well as downstream signaling molecules. One mutation, rok(sy340) suppresses lin-3 in a semi-dominant manner and maps to LG X. sy340 is epistatic to let-23(sy97) and let-60(n2034) , but not to lin-45(sy96) , suggesting that rok(sy340) may encode a product working between Ras and Raf. Another mutation, prl-1 (positive regulator of lin-3 ), has been characterized by three dominant gain of function alleles. The spectrum of suppression by prl-1 suggests that prl-1 acts at the same level or upstream of let-23 . Gonad ablation abolished the suppression by prl-1 , suggesting that PRL-1 acts through the anchor cell to induce vulval cells. Intragenic revertants of prl-1 are being sought to isolate loss of function mutants of prl-1 , which will be valuable for addressing the function of prl-1 in the regulation of lin-3 .

Hwang H-Y et al. (1998) East Coast Worm Meeting "NUCLEOTIDE-SUGAR BIOSYNTHESIS AND GLYCOSYLATION MODULATE VULVAL MORPHOGENESIS"

Hwang H-Y, Horvitz HR

[East Coast Worm Meeting, 1998]

To analyze the process of vulval morphogenesis, Herman and Horvitz (Cold Spring Harbor Quant. Biol. 62, 353, 1997) identified 25 mutants defective in vulval invagination but normal in vulval cell lineages. These mutants define eight genes, sqv-1 to 8 (sqv, squashed vulva), the primary phenotype of which is reduced separation between the anterior and posterior halves of the vulva in the L4. Some of the mutants have additional defects: strong alleles of all sqv genes cause maternal-effect lethality, and sqv-3 mutations cause male tail morphogenetic defects. Herman and Horvitz reported that three of the sqv genes encode proteins involved in glycosylation. SQV-3 is similar to a family of glycosyltransferases that add sugars onto glycoproteins and glycolipids. SQV-8 is similar to a UDP-glucuronosyltransferase that adds glucuronic acid to glycoproteins. SQV-7 is similar to LPG2, a Leishmania donovani putative Golgi GDP-mannose transporter. We have cloned and characterized two additional sqv genes. sqv-1 encodes a 467 amino acid protein similar to a group of nucleotide-sugar biosynthetic enzymes, including UDP-glucose epimerases and dTDP-glucose dehydratases. sqv-4 encodes a 478 amino acid protein similar to UDP-glucose dehydrogenases, which convert UDP-glucose to UDP-glucuronic acid. The expression patterns of sqv-1::GFP and sqv-4::GFP suggest that both genes are expressed in many tissues, including gut, neurons and the vulva. sqv-4 cDNA expressed under heat-shock promoters can rescue the Sqv-4 mutant phenotype: heat-shock treatment of L1 and L2 larvae rescues the vulval defect but not the maternal-effect lethality, while heat-shock treatment of L3 and L4 larvae and adults can rescue maternal-effect lethality but not necessarily the vulval defect. We propose that SQV-1 and SQV-4 synthesize nucleotide sugars that are transported into the Golgi by nucleotide-sugar transporter(s), including SQV-7, then used as substrates for glycosyltransferases including SQV-3 and SQV-8. We speculate that glycosyltransferases encoded by sqv-3 and sqv-8 and possibly other yet-to-be-cloned sqv genes act on a small set of glycoconjugates, some of which modify cell-cell or cell-matrix interactions to modulate the vulval invagination process. The maternal-effect lethality of the sqv mutants may be caused by a defect in a biological process that shares components of a glycosylation pathway involved in vulval invagination. We are currently studying the biochemical properties of the cloned SQV proteins and searching for the target(s) of SQV-mediated glycosylation.