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, 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 H et al. (2014) Lab Chip "On-demand optical immobilization of Caenorhabditis elegans for high-resolution imaging and microinjection."

Benian GM, Hwang H, Krajniak J, Lu H, Matsunaga Y

[Lab Chip, 2014]

This paper describes a novel selective immobilization technique based on optical control of the sol-gel transition of thermoreversible Pluronic gel, which provides a simple, versatile, and biocompatible approach for high-resolution imaging and microinjection of Caenorhabditis elegans.

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.

Hwang H et al. (2013) Biotechnol J "Microfluidic tools for developmental studies of small model organisms--nematodes, fruit flies, and zebrafish."

Lu H, Hwang H

[Biotechnol J, 2013]

Studying the genetics of development with small model organisms such as the zebrafish (Danio Rerio), the fruit fly (Drosophila melanogaster), and the soil-dwelling nematode (Caenorhabditis elegans), provide unique opportunities for understanding related processes and diseases in humans. These model organisms also have potential for use in drug discovery and toxicity-screening applications. There have been sweeping developments in microfabrication and microfluidic technologies for manipulating and imaging small objects, including small model organisms, which allow high-throughput quantitative biological studies. Here, we review recent progress in microfluidic tools able to manipulate small organisms and project future directions and applications of these techniques and technologies.

Hwang, H. et al. (2019) International Worm Meeting "The Role of NHR-49/PPARalpha in Modulation of Mitochondrial Function in Long-lived Germline-less Animals."

Shiva, S., Hwang, H., Ghazi, A., St.Croix, C.

[International Worm Meeting, 2019]

Aging and reproduction are closely associated biological processes. In Caenorhabditis elegans, removing germline stem cells (GSC) extends a lifespan and is accompanied by improved lipid homeostasis and stress resilience. This reproductive control of aging appears to be evolutionarily conserved. Previously, our lab identified the roles of conserved transcription regulators, DAF-16/FOXO3A, TCER-1/TCERG-1 and NHR-49/PPAR? in promoting germline-less longevity through modulation of lipid metabolic pathways. Especially, long-lived, germline-less glp-1 mutants exhibit increased expression of genes in fatty acid ?-oxidation, one of the main suppliers of electron equivalents for mitochondrial electron transport chain in oxidative phosphorylation. However, how mitochondria, a major site of cellular energy production and stress signaling, impact physiology upon germline removal is still largely unclear. In this study, we aim to investigate how removal of GSC, using a temperature-sensitive mutant glp-1, impacts mitochondrial functions such as respiration and stress response in a NHR-49 (functional homolog of vertebrate PPAR?) dependent manner. Using confocal microscopy image analysis, we found that muscles of young adult glp-1 mutants displayed reduced mitochondria mass compared to wild-type animals. This level remained constant with age unlike wild-type animals that displayed a decline in mitochondrial load. We also measured cellular respiration in young adult worms using a real-time bioenergetic analyzer. Basal respiration (BR) and spare respiratory capacity (SRC) was significantly lower in day1 adult glp-1 mutants than that in wild-type animals. This reduced respiration was enhanced in day5 adult glp-1 mutants and it required nhr-49. Interestingly, we further noted that SRC was maintained in glp-1 mutants following acute stress exposure, in contrast to loss of SRC exhibited by wild-type animals. As for wild-type, the reduction of SRC under stress was severe upon a functional loss of NHR-49. However, the maintenance of SRC in glp-1 mutants under stress was not abolished by a functional loss of NHR-49. Collectively, our data suggest that germline removal activates NHR-49/PPAR?, which allows enhanced mitochondrial respiratory activity during young adulthood but may not have a role in adaptation to environmental stressors.

Hwang H et al. (2016) Sci Rep "Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans."

Lu H, Hwang H, Matsunaga Y, Barnes DE, Ono S, Benian GM

[Sci Rep, 2016]

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

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.

Berninsone P et al. (2000) East Coast Worm Meeting "SQV-7, a protein involved in vulval invagination and embryonic development, transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose"

Hwang H-Y, Horvitz R, Berninsone P, Hirschberg C

[East Coast Worm Meeting, 2000]

Mutations in eight sqv (squashed vulva) genes (sqv-1 to 8) cause defects in vulval invagination and early embryonic development (1). SQV-1 and SQV-4 are similar to enzymes involved in nucleotide sugar metabolism (2), while SQV-3 and SQV-8 are similar to glycosyltransferases (3). SQV-7 is a multi-transmembrane protein resembling Golgi membrane nucleotide sugar transporters (4); these proteins specifically translocate nucleotide sugars from the cytosol into the lumen of the endoplasmic reticulum and Golgi apparatus where they are used as sugar donors by glycosyltransferases (4). Translocation is essential for glycosylation: yeast, protozoa and mammalian cell mutants defective in this function have a severe deficiency of the corresponding sugar in their glycoconjugates (4). Because GDP-mannose and UDP-glucose are the only nucleotide sugars transported into S.cerevisiae Golgi and ER vesicles in vivo and in vitro (5), this organism was used for expression of SQV-7 to determine its substrate specificity. We found that SQV-7 transports UDP-glucuronic acid, UDP-N-acetylgalactosamine and UDP-galactose in vitro, the first protein known to transport the former two nucleotide sugars. All three nucleotide derivatives are competitive, alternate, non-cooperative substrates, which suggests there is a single active site for all three substrates on SQV-7. Expression of the two mutant sqv-7 missense alleles results in significant reduction of the three transport activities. We hypothetize that in these mutants the biosynthesis of chondroitin, the major glycosaminoglycan in C. elegans (6), is more likely to be impaired than that of heparan sulfate since chondroitin polymerization requires both UDP-glucuronic acid and UDP-N-acetylgalactosamine while heparan sulfate polymerization does not require UDP-N-acetylgalactosamine. (1)Herman T. et al (1999) PNAS 96:968 (2)Hwang H. et al.(2000) East Coast C.elegans Meeting (3)Herman T. and Horvitz H. R. (1999) PNAS 96: 974 (4)Hirschberg C. et al (1998) Annu. Rev. Biochem.67: 49 (5)Berninsone P. et al (1997) J. Biol. Chem. 272: 12616 (6)Toyoda H. et al (2000) J. Biol. Chem. 275: 2269