Simske, Jeffrey et al. (2015) International Worm Meeting "Glucose intolerance during C. elegans embryogenesis."

Dong, Yi, Simske, Jeffrey

[International Worm Meeting, 2015]

Glycogen debranching enzyme AGL-1 and AMP kinase signaling are required for normal C. elegans embryogenesis. One remarkable phenotype of both agl-1 and AMPK alpha subunit (aak-2) mutants is the dramatic enhancement of embryonic arrest phenotypes following glucose feeding. For agl-1, lethality is 18% when grown on NGM/OP50 and 99% when supplemented with 0.2% glucose. For aak-2 mutants, there essentially is no lethality on NGM/OP50 and as high as 77% lethality on glucose, depending on the regimen of starvation recovery and dietary restriction. agl-1 sensitivity to glucose requires daf-2, since mutations in daf-2 suppress the agl-1 glucose-dependent embryonic lethality. The role of insulin signaling pathway genes in embryonic development is further supported by the observations that certain daf-2 alleles alone or in daf-16 double mutants, display embryonic defects [1]. Interestingly, over-expression of a daf-16::gfp reporter increases agl-1 embryonic lethality (but only a modest shift of daf-16::gfp to the nucleus is observed in agl-1 mutants). To determine whether glucose intolerance is exclusive to agl-1 and aak-2, other mutants in carbohydrate metabolism were tested for embryonic arrest phenotypes. In one case, mutations in glycogen phosphorylase (tm5211) increased the penetrance of embryonic arrest when grown on glucose or under dietary restriction conditions, reinforcing the importance of maternal diet in embryonic development. To better understand glucose tolerance during embryogenesis, a genetic screen was carried out to identify mutants that display increased embryonic lethality in the presence of elevated glucose. So far, two gin (for Glucose INtolerance) alleles, gin-1(cv10) and gin-2(cv11) have been isolated and both display increased penetrance and severity of embryonic arrest phenotypes in the presence of 0.2% glucose. gin-1(cv10) is 73% viable on NGM/OP50 and 30% viable with glucose supplement. gin-2(cv11) is 21% viable on NGM/OP50 and 0.17% viable with glucose. Complementation tests indicate gin-1 and gin-2 are not agl-1 alleles. gin-1 and gin-2 hermaphrodites grown with glucose recover and lay a higher fraction of hatching eggs when transferred to NGM/OP50, similar to agl-1 mutants. The gin-2 mutant appears to have both a maternal and zygotic requirement. gin-2 embryonic arrest on normal OP50 occurs more frequently during morphogenesis, whereas in embryos grown with glucose, cell divisions cease prior to morphogenesis and intracellular inclusion bodies increase in size and number. Further study of these gin alleles and additional screens are in progress in an effort to characterize the pathways required for glucose tolerance during embryogenesis.1. Gems, D., et al., Genetics, 1998. 150(1): p. 129-55.

Simske, Jeffrey S. (2009) International Worm Meeting "The role of Glycogen Debranching Enzyme during C. elegans development and aging."

Simske, Jeffrey S.

[International Worm Meeting, 2009]

In a screen for enclosure defective mutants, the partially penetrant, maternal-effect cold-sensitive allele jc11 was identified. 82 and 19 percent of jc11 animals display mutant phenotypes at 15 and 25 degrees, respectively, and a single maternal or zygotic copy of wild type jc11 is sufficient for gene function. Using 4D microscopy jc11 embryos are observed to arrest at various developmental stages including pre-morphogenesis, enclosure, and elongation. Animals that successfully complete elongation often have morphogenetic defects including tail and snout abnormalities. jc11 was genetically inseparable from the lin-7 allele used in the genetic screen and a minimal genomic region containing the neighboring gene R06A4.8 was sufficient for the rescue of jc11 phenotypes. RNAi against R06A4.8 phenocopies jc11. R06A4.8 is predicted to encode glycogen debranching enzyme (agl-1). The R06A4.8 locus in jc11 animals was completely sequenced and a four base pair deletion was found in the c-terminus of agl-1. This deletion is predicted to result in a frame shift and early termination, resulting in the truncation the final 253 residues of the protein, adding 22 extraneous residues prior to a stop codon. The jc11 mutation is predicted to eliminate the glycogen binding domain of debranching enzyme. jc11 was also phenocopied by treatment with MOR-14 (N-Methyl-1-Deoxynojirimycin), an inhibtor that specifically targets the a-1,6-glucosidase activity of glycogen-debranching enzyme. Overexpression of AGL-1::GFP results in increased debranching activity based on reduced glycogen staining with carminic acid in both embryos and adults expressing AGL-1::GFP. Recently, debranching enzyme was shown to interact with the b1 subunit of the energy sensor AMP-activated kinase, while binding of AMPK b1 directly to glycogen depends on the a-1,6-branch; AMPK binding to glycogen branches in turn functions to inhibit AMPK activity [1, 2]. Loss of agl-1 function in C. elegans is predicted to result in the formation of a ''limit dextrin'' glycogen molecule in which outer glucose chains are digested by phosphorylase to within four glucose residues of the a-1-6 branch, resulting in a glycogen molecule likely to bind to and inhibit AMPK activity. The prediction that AMPK activity is inhibited in agl-1 mutants is supported by initial experiments indicating that jc11 animals surviving to adulthood have shorter life spans, similar to animals lacking AMPK activity. The role of glycogen debranching enzyme in the integration of energy and lifespan signals via AMPK is being further investigated. 1.Sakoda, H., et al.,. Am J Physiol Endocrinol Metab, 2005. 289(3): p. E474-81. 2.McBride, A., et al., Cell Metab, 2009. 9(1): p. 23-34.

Simske, Jeffrey S. (2011) International Worm Meeting "Glycogen debranching enzyme AGL-1 regulates embryogenesis and lifespan through AMP Kinase dependent and independent pathways."

Simske, Jeffrey S.

[International Worm Meeting, 2011]

Metabolic status generally affects embryogenesis and lifespan. agl-1, which encodes the single C. elegans glycogen debranching enzyme, is involved in embryogeneis and lifespan. The hypomorphic allele jc11 and the more severe putative null allele tm4809 are both partially penetrant, cold-sensitive, maternal-effect alleles that result in embryonic arrest at various stages. Hatching 'escaper' animals have shortened lifespans and suppress the longer lifespan of daf-2 mutants at 20 deg C, and overexpression of AGL-1::GFP results in lifespan extension. AGL-1 affects embryogenesis through regulation of the energy sensor AMP-activated kinase (AMPK). AMPK is a heterotrimeric protein with a catalytic a subunit encoded by aak-1 or aak-2, and regulatory subunits b (aakb-1, aakb-2) and g (5 genes). The b subunit functions as a glycogen sensor and in one vertebrate system, the b1 subunit interacts directly with glycogen debranching enzyme. Strong binding of AMPK b1 to glycogen was shown to depend on the branch structure; binding in turn functions to inhibit AMPK activity. Loss of agl-1 function is predicted to result in the formation of a 'limit dextrin' glycogen molecule with digestion stalled at branch points, and well-suited to bind to and inhibit AMPK activity. Supporting this model are the observations that AMPK activity is reduced in agl-1 mutants, overexpression of aak-2 or the aakb-1 glycogen-binding domain suppresses agl-1 phenotypes, and compounds thought to activate AMPK, such as metformin and resveratrol also suppress agl-1 phenotypes. Finally, AGL-1 and the AAKB-1 co-immunoprecipitate from worm extracts, suggesting that AGL-1 and AAKB-1 physically interact to regulate energy metabolism and lifespan via glycogen. Dietary supplement with cornstarch (mixed linear and branched polysaccharides) bypasses agl-1 phenotypes, whereas supplement with pure amylopectin (branched polysaccharides alone) not only fails to bypass, but enhances agl-1 phenotypes. Supplement with various mono or di-saccharides does not suppress agl-1 phenotypes. These findings support the hypothesis that branched carbohydrates generally inhibit AMPK activation. Interestingly, dietary supplement with glucose and glycerol dramatically enhance agl-1 phenotypes, increasing penetrance of the most severe agl-1 phenotypes to nearly 100%, even at permissive temperatures. Glycogen levels are higher in glucose supplemented animals, while AMPK inactivation is not further reduced and enhancement of agl-1 by glucose is not suppressed by AMPK overexpression, suggesting there are AMPK-independent metabolic functions for agl-1. Genetic suppressors of glucose-enhanced agl-1 phenotypes have been identified, and will be presented.

Jeffrey Simske et al. (2007) International Worm Meeting "egl-38 regulation of actomyosin during enclosure and elongation."

Jeffrey Simske, Deborah Schulman

[International Worm Meeting, 2007]

The pathways leading from cell specification to the generation of organ, tissue and animal form are not well understood. In C. elegans, specification of epidermal cells is followed by the migration of these cells to seal the embryo (enclosure) and constriction of these cells to generate the vermiform morphology (elongation). In a previous screen for enclosure defective mutants, we identified a maternal effect temperature-sensitive allele, jc1(WBPaper00023223). We determined that jc1 is an allele of the C. elegans Pax/2/5/8 homologue egl-38. egl-38(jc1) displays the egl-38 Unc and Egl phenotypes, fails to complement egl-38(sy294) and is rescued by cosmids that include the entire egl-38 locus and regulatory sequences. egl-38::GFP reporters that include only egl-38and the immediate surrounding sequences show expression in some embryonic epidermal cells and neuroblasts, suggesting that egl-38 may be required in both cell types during enclosure. We analyzed a previously isolated, strong allele of egl-38, s1775 and found that introduction of a single s1775 allele from males into wild-type hermaphrodites results in enclosure and elongation defects, indicating that s1775 is a semi-dominant, zygotic allele. Furthermore, progeny of jc1/Df animals have a fully penetrant embryonic lethal phenotype and display a higher percentage of enclosure phenotypes than jc1/jc1 progeny or s1775. These findings suggest that jc1 and s1775 are non-null alleles of egl-38. To determine how egl-38 regulates enclosure and elongation, we tested various cell junction and cell morphological markers in egl-38(jc1) animals and discovered that egl-38 is required for the expression of some proteins involved in actomyosin contraction. One such protein is VAB-9, an adherens junction component and a putative tetra-span integral membrane protein. Surprisingly, we found that both vab-9 and egl-38 were required for the junctional localization of the myosin phosphatase MEL-11. Furthermore, mutations in vab-9 suppress the semi-dominant enclosure and elongation phenotypes of mel-11(it26). In parallel with these experiments, we carried out a screen for suppressors of the egl-38(jc1)phenotype and identified three extragenic suppressor, one of which results in fully penetrant elongation defects in an otherwise wild-type background. This allele, cv1, is incompletely semi-dominant; cv1/+ larvae display some tail tip morphogenetic defects. We are currently cloning cv1 and analyzing genetic doubles between vab-9, egl-38, cv1 and previously identified mutants involved in actomyosin contraction during enclosure and elongation. These experiments will order egl-38 and vab-9 activity relative to genes involved in actomyosin contraction.

Jeffrey S Simske (2005) International Worm Meeting "Interaction Between VAB-9 and the PAR Complex During Junctional Assembly"

Jeffrey S Simske

[International Worm Meeting, 2005]

VAB-9 is a tetraspan integral membrane cell junction protein required downstream of HMR-1 (cadherin) for normal morphology. The vertebrate VAB-9 orthologue TM4SF10, and a TM4SF10::GFP fusion protein functionally substitute for VAB-9 in C. elegans, and localize at adherens junctions in all epithelia. In kidney podocytes, which have highly specialized cell junctions called slit diaphragms, TM4SF10 is transiently expressed during development, localizes at cell contacts following cadherin engagement, and diminishes concurrent with initiation of the expression Nephrin (a marker and critical transmembrane protein in the mature slit diaphragm). PAR-6 co-IPs with TM4SF10::GFP from a Triton X-100 soluble fraction of polarized epithelial MDCK cells; PAR-3 and other tight junction proteins do not co-IP. Furthermore, only a small fraction of TM4SF10 co-precipitates with the adherens junction complex. Together these results suggest that VAB-9 may act at a late step in junctional assembly. To order VAB-9 functions relative to the PAR complex during junctional assembly, the following experiments were carried out. First, temporal and spatial expression of VAB-9 relative to PAR-3 was determined. In intestinal and pharyngeal cells, PAR-3 and PAR-6 were detected and localized apically prior to VAB-9 localization. Second, the localization of PAR-3 (in the intestine and somatic gonad) and PAR-6::GFP expressed under ajm-1 promoter control (in the pharynx, intestine, epidermis, and somatic gonad) was scored in vab-9 animals, and was found localized similar to wild type. Third, a kinase-dead version of PKC-3 (PKCkn) was expressed using either the ajm-1 or heat shock promoters during enclosure. (Similarly altered vertebrate atypical PKC proteins are known to disrupt junctional assembly in cultured epithelial cells) Expression of this construct resulted in embryonic lethality and body and tail morphology defects. In some cases, disruption of the annular distribution of VAB-9 was observed; suggesting PKC-3 regulates VAB-9 localization. Somewhat unexpectedly, PKC-3kn protein localized to nuclei, whereas PKC-3 was cytoplasmic. In some vertebrate cell types, atypical PKC proteins are found in both cytoplasm and nucleus, but in previous experiments showing atypical PKCkn effect on junctional assembly in vertebrate epithelial cells, atypical PKCkn remained cytoplasmic (1,2). I am currently investigating the effects of cytoplasmic forms of PKC-3kn or altered PAR-6 proteins on cell junction assembly. In addition, I am testing whether vertebrate PAR6B (cytoplasmic) or PAR6A (junctional) interact directly with TM4SF10. 1. J Biol Chem 276, 13015-24 (2001). 2. J Cell Sci 115, 3565-73. (2002).

Jeffrey S Simske et al. (2001) International C. elegans Meeting "Analysis of VAB-9, a Putative Integral Membrane, Cell Junction Protein"

Jeffrey S Simske, Jeff Hardin

[International C. elegans Meeting, 2001]

vab-9 encodes a putative four-pass transmembrane protein with a predicted topology similar to the claudin family of tight junction proteins. vab-9 mutants have tail and body shape defects and are variably Dpy and Egl. We previously reported that VAB-9-GFP localizes to the cellular junctions of all epithelia, and to the cell membrane of larval body wall muscles. Using VAB-9 antibodies we have determined that in embryos VAB-9 co-localizes with HMP-1 ( a -catenin) at adherens junctions, is apical to JAM-1 ( j unction- a ssociated m olecule 1), and is basal to PAR-3 and PAR-6. These results suggest there are three distinct regions of protein localization at the epithelial cell-cell junction and that VAB-9 is most closely associated with the adherens junction protein HMP-1. One hypothesis is that each protein localization domain represents a functionally distinct region of the epithelial cell junction. JAM-1 and DLG-1 (the homologue of Drosophila Discs large) are thought to maintain the tightness of the paracellular space, a function commonly attributed to septate or tight junctions (see abstract by Koppen, et. al. ). Thus, JAM-1 and DLG-1 may define a septate junction-like region in C. elegans epithelia and this region may be distinct from the adherens junction. It was surprising therefore that VAB-9, which is similar to the tight junction claudin proteins, did not co-localize with JAM-1. To determine the functional relationship between these genes, we constructed double mutants. vab-9 mutations enhance the phenotypes of both jam-1 and dlg-1(RNAi) embryos. Specifically, jam-1 and dlg-1(RNAi) embryos arrest at two-fold and do not rupture, whereas vab-9; jam-1 and vab-9; dlg-1(RNAi) animals rupture prior to this stage. HMP-1 and PAR-3 are properly localized in vab-9; jam-1 animals, indicating there are no general defects in epithelial polarity in these embryos. One possibility is that VAB-9 and DLG-1/JAM-1 localize independently to distinct regions of the lateral membrane that together regulate epithelial tightness. Consistent with this hypothesis, we find that let-413 mutations, which disrupt junctional localization of DLG-1 and JAM-1, do not similarly disrupt the localization of HMP-1 and VAB-9. We previously reported that vab-9 is required for the organization of the circumferential actin filaments in epithelial cells required for coordinated elongation. Thus, VAB-9 may be a novel junctional protein with multiple roles in regulating cell morphology and adhesion. Surprisingly, we found that VAB-9 localizes along the muscle quadrant tracks in embryos. In contrast, other epithelial cell-junction proteins ( e.g. HMP-1, JAM-1 and DLG-1) are not expressed along muscle quadrants. Co-staining with muscle markers suggested that this muscle quadrant-associated VAB-9 is expressed by the hypodermis. We tested this possibility (and the possibility that vab-9 has a role in muscle morphogenesis), by using Andy Fire's tissue-specific RNAi technique. RNAi of vab-9 in the hypodermis eliminated all VAB-9 expression in the embryo. These embryos arrest during elongation and ultimately are paralyzed. This phenotype is more severe than that of the four existing alleles of vab-9 , which are viable and fertile. We are currently examining the localization of muscle components in these embryos. VAB-9 localization along muscle quadrants, at muscle cell membranes and at epithelial cell junctions suggests that VAB-9 may have a general role in cell adhesion.

Jeffrey S Simske (2002) Mid-west Worm Meeting "VAB-9 is a claudin-like adherens junction protein that regulates hypodermal morphology and adhesion"

Jeffrey S Simske

[Mid-west Worm Meeting, 2002]

To understand how hypodermal cell shape and adhesion regulate organismal morphology in C. elegans, I am analyzing the gene vab-9. vab-9 mutants have tail and body shape defects resulting from defects in either the attachment or distribution of circumferential actin filaments at the adherens junctions. In the normal embryo, circumferential actin filament bundles are evenly spaced along the anterior-posterior axis; their contraction results in the elongation of the embryo. VAB-9 is a putative four-pass transmembrane protein homologous to canine BCMP1 (Brain cell membrane protein 1) and similar to the PMP22/Claudin family of proteins. Claudins are the major protein components of tight junctions and mediate calcium independent cell adhesion. Despite the similarity of VAB-9 to the tight junction claudin proteins, VAB-9 localizes to the adherens junctions of all C. elegans epithelia. VAB-9 localization is dependent on other adherens junction components. In the absence of HMR-1 (cadherin), VAB-9 localization at the adherens junction is completely missing, whereas in the absence of HMP-1 (a -catenin) or HMP-2 (b -catenin), VAB-9 localizes at junctions, but the distribution about the periphery of the junction is disrupted. 3D time-lapse analysis of VAB-9-GFP localization in hmr-1(RNAi) and hmp-1(RNAi) animals using multi-photon excitation microscopy was carried out by Mathias Kppen and Jeff Hardin at the University of Wisconsin. In hmr-1(RNAi) animals, VAB-9-GFP apparently fails to reach the plasma membrane, whereas in hmp-1(RNAi) animals, initial VAB-9-GFP junctional localization is normal, but the distribution about the periphery of adherens junctions is rapidly lost. Ultimately, in hmp-1(RNAi) animals VAB-9-GFP becomes concentrated into small, isolated puncta at the junction. Interestingly, HMR-1 localization is almost identical to VAB-9 in hmp-1 and hmp-2 mutants. In contrast to these findings, mutations in vab-9 do not obviously disrupt the localization of HMR-1, HMP-1, or HMP-2. Together, these results suggest that VAB-9 is closely associated with the adherens junction and that VAB-9 affects actin organization by regulating the interaction between actin and the cadherin-catenin complex. Alternatively, VAB-9 may mediate interactions with the actin cytoskeleton distinct from those with cadherin and catenins. VAB-9 similarity to PMP22/Claudin proteins suggests that VAB-9 may have a role in cell adhesion, however, vab-9 mutants alone have no obvious cell adhesion defects. Nevertheless, vab-9 mutations enhance the cell adhesion defects of ajm-1 mutants and dlg-1(RNAi) animals. Transmitting electron microscopy (carried out by Paul Sims and Jeff Hardin, University of Wisconsin) revealed that large gaps are present between hypodermal cells of vab-9; ajm-1 animals. These gaps extend along the entire apical-basal axis of the hypodermal cells and are more extensive than the gaps in the apical junctions of ajm-1 mutants alone. Since VAB-9 is localized apical to DLG-1 and AJM-1, one possibility is that VAB-9 and DLG-1/AJM-1 independently regulate hypodermal cell adhesion. To determine how VAB-9 is associated with the cytoskeleton, I am currently searching for VAB-9-interacting proteins. To test directly for the ability of VAB-9 to mediate cell-cell adhesion, I am expressingVAB-9 in L fibroblasts. I thank the Hardin Lab (experiments), the Priess Lab (lab space, reagents, advice), John White (multi-photon excitation microscopy) and David Hall (TEM advice).

Shao, Xiangqiang et al. (2013) International Worm Meeting "An In Vivo Analysis of Critical Functional Domains of a-catenin in C. elegans."

Shao, Xiangqiang, Hardin, Jeff, Simske, Jeffrey, Audhya, Anjon

[International Worm Meeting, 2013]

Stable intercellular adhesions are critical for normal embryonic development in metazoans, during which tissue integrity is maintained as cells undergo morphogenetic movements. The adherens junction, a key cell-cell adhesion structure, contains a highly conserved cadherin-catenin complex (CCC). a-catenin is an important regulator that connects the CCC to F-actin at adherens junctions. C. elegans provides a good model to study the function of a-catenin in vivo, since hmp-1 is the sole a-catenin homolog. Given the potential role of a-catenin as a physical linker between CCC and the actin cytoskeleton, we hypothesized that recruiting a-catenin to junctions irrespective of its normal binding partners is sufficient for its function. To test this, we fused full length HMP-1 with the junctional transmembrane protein VAB-9 (VAB-9::HMP-1::GFP). This fusion protein localizes correctly to junctions in embryos, and is able to rescue the hmp-1 strong loss-of-function mutant allele zu242. HMP-1 is normally recruited to junctions via HMP-2/b-catenin. The presumed HMP-2/b-catenin binding site of HMP-1 is located in the N-terminus of HMP-1. To test if this binding site has additional functional roles, we fused a truncated form of HMP-1 lacking the HMP-2/b-catenin binding site with VAB-9 (VAB-9::HMP-1(315-927)::GFP). This fusion protein also localizes correctly to junctions; however, it only partially rescues zu242 embryos. Our results suggest that hmp-1 is a stable physical linker between the cadherin-catenin complex and F-actin, while its N-terminal interaction with hmp-2 may indeed be required for robust linkage. The N terminus of HMP-1 may do so by recruiting additional binding partners that strengthen the connection to F-actin at the junction. In order to find potential binding partners for hmp-1, we performed co-immunoprecipitation followed by mass spectrometry and are currently testing the candidates to determine functional roles for these proteins.

Praitis, Vida et al. (2011) International Worm Meeting "The C. elegans Hailey-Hailey disease homolog pmr-1 is essential for embryonic development."

Feddersen, Charlotte, Yohannes, Lensa, Praitis, Vida, Kniss, Sarah, Yilma, Zelealem, Liszewski, Walter, Schacht, Angela, Simske, Jeffrey, Ha, Dae Gon, Mushi, Juliet, Imlay, Leah, Chakravorty, Adityarup, Sullivan-Wilson, Alexander, Miller, Michael, Stock, Tyson

[International Worm Meeting, 2011]

ATP2C1/PMR1 is a Ca2+/Mn2+ ATPase that localizes to the golgi where it acts in protein processing, metal homeostasis, and Ca2+ signaling. In humans, lesions in the gene cause Hailey-Hailey disease or benign familial pemphigus (MIM# 169600), which shows a semi-dominant inheritance pattern and is marked by severe skin lesions that typically occur in the second decade of life. Defects in the gene have also been associated with squamous cell carcinomas. In two independent screens designed to identify temperature-sensitive alleles of genes essential for embryonic enclosure, we isolated jc10 and ru5. Complementation analysis demonstrated these mutations were allelic and several independent lines of evidence indicate that it is lesions in the C. elegans ATP2C1 homolog pmr-1 that are responsible for the phenotypes observed in these mutants. Whole-genome and directed sequencing revealed mutations in pmr-1 in both jc10 and ru5, the phenotypes are rescued by fosmids carrying the pmr-1 gene, and the ru5 allele fails to complement pmr-1(tm1840), which contains a deletion in the first exon of the gene. Embryos homozygous for mutations in pmr-1 have defects in epidermal cell migration and adhesion during enclosure, as well as later pharynx unattached (PUN) and body morphogenesis phenotypes at 25C. Temperature shift experiments indicate that the temperature sensitive period occurs earlier in development, just after ventral cleft formation and during a period of extensive cell birth. Using lineage analysis and GFP markers that are expressed in a subset of cells during and just after the temperature-sensitive period, we determined that the timing of divisions and cell fate are normal in pmr-1(ru5) embryos, but that the position of some cells is defective. This analysis demonstrates that the C. elegans PMR-1 Ca2+/Mn2+ ATPase plays an important role in cell positioning during embryonic development.

MF Maduro et al. (1999) International C. elegans Meeting "An RNAi screen for embryonic patterning genes"

MF Maduro, JH Rothman, J Sumerel

[International C. elegans Meeting, 1999]

We are taking advantage of RNAi as a tool for identifying genes with potentially important roles in the developing embryo. This approach circumvents the extensive effort required to molecularly identify the relevant genes identified from mutagenesis or deficiency screening and facilitates identification of genes that might be required both maternally and zygotically. In our screen, we obtain RNA synthesis templates from an extant cDNA library. The templates are pooled in groups of eight and used to synthesize RNA. Although the library we are using has not been normalized, this has not been a major limitation; in preliminary experiments, we found that approximately 35% of pools cause penetrant embryonic arrest. For any phenotype recovered from a pool, we inject or soak individual RNAs and identify the gene from a small amount of cDNA sequence. To expedite analysis of mutant phenotypes, we are using a set of GFP markers, including a translational fusion of the gene encoding the MH27 antigen (a gift from Jeff Simske), which marks the adherens junctions of epithelia and allows us to recover embryos with abnormal epidermal patterning. For example, one pool produced viable L1 animals with a penetrant hypodermal protrusion near the posterior pharyngeal bulb; the MH27::GFP marker revealed a bilateral alteration in the local pattern of hypodermal seam cells. The gene responsible for the phenotype, which has previously been named nmy-1 , encodes a non-muscle myosin; we are currently investigating the role of this gene in seam cell development. We will report our progress with nmy-1 and other genes identified in the screen.