Wang, Lin et al. (2017) International Worm Meeting "Dissecting the roles of the ADAM10 metalloprotease SUP-17 in the BMP signaling pathway in Caenorhabditis elegans."

Liu, Jun, Liu, Zhiyu, Shi, Herong, Wang, Lin

[International Worm Meeting, 2017]

BMPs (Bone Morphogenetic Proteins) belong to the TGFbeta superfamily of ligands, and mediate a highly conserved signal transduction cascade. Upon ligand binding, type II receptors phosphorylate type I receptors, which in turns phosphorylate R-Smads (receptor regulated Smads). Phosphorylated R-Smads then complex with co-Smad (common mediator smad) and enter the nucleus to regulate gene transcription with different co-factors. BMPs play important roles in developmental and physiological processes. Malfunction of the pathway in humans can cause various disorders, such as skeleton diseases, heart diseases and cancers. So it is critical to strictly regulate the level of BMP signaling spatiotemporally. Using a highly specific genetic screen, we have identified several modulators of the BMP pathway in C. elegans. These include the Neogenin homolog UNC-40 [1], two paralogous tetraspanin proteins, TSP-12 and TSP-14, and an ADAM10 metalloproteinase (A Disintegrin and Metalloproteinase 10) SUP-17 [2]. We use the CRISPR/Cas9 system and tagged the endogenous TSP-12, SUP-17 and UNC-40 proteins with different fluorescence tags. We found that TSP-12 is localized to the cell surface and intracellular vesicles, and that TSP-12 can bind to SUP-17 and promote its cell surface localization. We have genetic evidence and preliminary biochemical evidence showing that UNC-40 is one, but not the only, substrate of SUP-17 in BMP signaling. We are currently identifying additional substrate(s) of SUP-17 in BMP signaling. Our work highlights the importance of intracellular signaling and protease processing in the regulation of BMP signaling. [1] Tian C, Shi H, Xiong S, Hu F, Xiong WC, Liu J. The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans. Development. 2013;140(19):4070-80. doi: 10.1242/dev.099838. PubMed PMID: 24004951; PubMed Central PMCID: PMCPMC3775419. [2] Wang L, Liu Z, Shi H, Liu J. Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans. PLoS Genet. 2017;13(1):e1006568. doi: 10.1371/journal.pgen.1006568. PubMed PMID: 28068334; PubMed Central PMCID: PMCPMC5261805.

Amin, Nirav et al. (2009) International Worm Meeting "The forkhead transcription factor LET-381 functions to pattern the C. elegans postembryonic mesoderm."

Shi, Herong, Amin, Nirav, Liu, Jun

[International Worm Meeting, 2009]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful system in which to study mesodermal development at single cell resolution. This lineage produces 14 striated bodywall muscles (BWMs), 2 non-muscle coelomocytes (CCs), and 2 multipotent progenitor cells of non-striated muscles called the sex myoblasts (SMs). We have previously found that the MyoD homolog HLH-1, the Hox factor MAB-5 and a C2H2 zinc finger protein FOZI-1 function within the M lineage to specify both M-derived BWMs and CCs, and that the absence of all three factors cause almost all M lineage cells to become SMs. The distinction between the myogenic BWM and the non-myogenic CC fates requires the Six homeodomain protein CEH-34 and its cofactor EYA-1 to act as CC-promoting factors. However, additional factors must also exist in this process as CEH-34 and EYA-1 alone are not sufficient to convert all myogenic BWM precursors to CCs. Here, we have identified a role for the conserved forkhead transcription factor LET-381 in patterning the M lineage. let-381 is expressed in progenitors of the M-derived CCs and their sister BWM cells. Knockdown of let-381 caused fate transformation of CCs to SMs. Additionally, let-381 is required for ceh-34 expression. Our studies lead to a model for how LET-381 functions as a competence factor to pattern the C. elegans postembryonic mesoderm in order to produce both myogenic and non-myogenic cells.

Liu, Zhiyu et al. (2015) International Worm Meeting "Three conserved tetraspanin proteins promote bone morphogenetic protein (BMP) signaling ."

Shi, Herong, Liu, Jun, Wang, Lin, Liu, Zhiyu

[International Worm Meeting, 2015]

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta (TGFbeta) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. In C. elegans, mutations in the BMP-like Sma/Mab pathway exhibit altered body sizes and the suppression of the dorsoventral patterning defects of sma-9(0) mutants in the postembryonic M lineage, among a number of phenotypes. Our lab has been using the sma-9(0) suppression assay to identify new players in the Sma/Mab pathway. We recently identified three conserved tetraspanin proteins, TSP-12, TSP-14 and TSP-21, as positive modulators of the Sma/Mab pathway. In addition to suppressing the sma-9(0) M lineage defect, tsp-21(0) single mutants or tsp-12(0); tsp-14(0) double mutants exhibit other defects in Sma/Mab signaling, including a small body size and reduced RAD-SMAD reporter expression. Genetic epistasis and cell type-specific rescue experiments showed that TSP-21 functions in the signal-receiving cells and genetically at the level of the ligand-receptor complex in the Sma/Mab pathway. A functional TSP-21::GFP generated via CRISPR/Cas9-mediated homologous recombination is plasma membrane-localized in a number of tissues, including the hypodermal, intestinal and M lineage cells. Using the split-ubiquitin yeast two-hybrid system, we found that TSP-21 can associate with itself and with TSP-12 and TSP-14. Furthermore, TSP-12 and TSP-14 can also associate with, and promote the cell surface localization of, SUP-17, an ortholog of the ADAM protease (a disintegrin and metalloprotease) ADAM10, which we have found to function in the Sma/Mab pathway as well. Because TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway, in the yeast two-hybrid system, we are currently determining whether TSP-12 and TSP-14 may be required for the proper localization of SMA-6. Altered BMP signaling is known to play a role in cancer progression. In recent years, tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development. We therefore plan to determine if the roles of TSP-12, TSP-14 and TSP-21 in promoting BMP signaling are conserved in mammals.

Tian, Chenxi et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The RGMb/DRAGON homolog DRAG-1 positively regulates a BMP-like signaling pathway in Caenorhabditis elegans"

Liu, Jun, Tian, Chenxi, Shi, Herong, Plavskin, Yevgeniy, Sen, Debjeet, Foehr, Marisa

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The BMP (bone morphogenetic protein) signaling pathway regulates multiple developmental and homeostatic processes. Mutations in the pathway can cause a variety of somatic and hereditary disorders in humans. Multiple levels of regulation, including extracellular regulation, ensure proper spatiotemporal control of BMP signaling in the right cellular context. We have identified a modulator of the BMP-like Sma/Mab pathway in C. elegans called DRAG-1. DRAG-1 is the sole member of the RGM (repulsive guidance molecule) family of proteins in C. elegans , and is critical in regulating body size and mesoderm development. Using a combination of molecular genetic and biochemical studies we demonstrate that DRAG-1 is a membrane-associated protein that functions at the ligand-receptor level to modulate the Sma/Mab pathway in a cell type-specific manner. We further show that DRAG-1 positively modulates this BMP-like pathway by using a novel Sma/Mab responsive reporter. Our work provides a direct link between RGM proteins and BMP signaling in vivo and a simple and genetically tractable system for mechanistic studies of RGM protein regulation of BMP pathways.

Tian, Chenxi et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The HMG-box protein SEM-2 is required for specifying the fate of the sex muscle precursors and is a direct target of Hox/PBC factors"

Waterston, Robert, Colledge, Clark, Liu, Jun, Stern, Micheal, Shi, Herong, Tian, Chenxi

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In C.elegans , the egg-laying vulval and uterine muscles (collectively called sex muscles) are derived from two sex myoblasts (SMs), which are descendants of the postembryonic mesodermal lineage, the M lineage. Hermaphrodites carrying the sem-2(n1343) mutation are egg-laying defective due to a fate transformation of the SMs to their sister cells, the bodywall muscles (BWMs). We have established that sem-2(n1343) is a mutation in sox-1 (C32E12.5) and that the n1343 allele contains a TC1 transposon insertion in the first 4.5kb intron of sem-2 . A functional GFP::SEM-2 fusion is localized in the nuclei of the SM mother cells (M. vlpa and M. vrpa), SM cells and their descendants as well as a variety of other cell types, including hypodermal cells and gut cells. The M lineage expression of sem-2 is under the control of both dorsal-ventral and anterior-posterior patterning mechanisms that include the LIN-12/Notch pathway, the SMA-9/TGF-beta pathway and the Wnt/beta-catenin asymmetry pathway. Furthermore, sem-2 is directly activated by Hox (MAB-5 and LIN-39) and PBC (CEH-20) factors in the M lineage via a conserved Hox/PBC binding site in its first intron that is disrupted by TC1 insertion in n1343 mutants. In addition to the M lineage, sem-2 is also required for embryonic elongation and proper vulval development.

Tian, Chenxi et al. (2013) International Worm Meeting "UNC-40 positively modulates BMP signaling independent of netrin signaling."

Fu, Fenghua, Liu, Jun, Shi, Herong, Xiong, Wen-Cheng, Tian, Chenxi, Xiong, Shan

[International Worm Meeting, 2013]

UNC-40 is the sole C. elegans homolog of DCC and neogenin, and is well known for its role as a netrin receptor important in axon and cell migration. We have found that UNC-40 is a positive modulator of the BMP-like Sma/Mab pathway. We have previously shown that loss-of-function mutations in the zinc finger-containing protein SMA-9 cause a dorsal to ventral fate transformation in the postembryonic mesodermal M lineage. This phenotype can be specifically suppressed by mutations in the Sma/Mab pathway. In a sma-9 suppressor screen, we found that mutations in unc-40 can suppress the sma-9 M lineage defect. Phenotypic and genetic epistasis analyses showed that unc-40 mutants are small and UNC-40 functions in the signal-receiving cells at the ligand/receptor level in the Sma/Mab pathway to regulate body size. This function of UNC-40 is independent of the axon guidance molecule UNC-6/netrin and the receptor UNC-5, as unc-6 and unc-5 mutants do not display Sma/Mab pathway mutant phenotypes. Instead, UNC-40 functions together with the RGM (repulsive guidance molecule) protein DRAG-1, a putative co-receptor of the Sma/Mab pathway. UNC-40 physically interacts with DRAG-1, and we have identified the key residues in the extracellular domain of UNC-40 that are critical for UNC-40-DRAG-1 interaction in vitro and UNC-40 function in vivo. Surprisingly, the extracellular domain of UNC-40 is sufficient to mediate Sma/Mab signaling, in clear contrast to the requirement of the intracellular domain of UNC-40 in mediating axon and cell migration. We showed that the mammalian homologs of DRAG-1 and UNC-40 can respectively rescue the Sma/Mab pathway phenotypes of drag-1 and unc-40 mutants. Moreover, an intracellular domain-deleted version of the UNC-40 homolog neogenin is capable of mediating BMP signaling in mammalian cell culture. Thus, in addition to mediating axon and cell migration, UNC-40 has an independent but evolutionarily conserved function in modulating BMP signaling.

Martin Victor et al. (2001) International C. elegans Meeting "The HAT activity of CBP-1 is indispensable for its biological function during C. elegans embryogenesis"

Craig C Mello, Martin Victor, Yang Shi

[International C. elegans Meeting, 2001]

CBP/p300 represent a conserved family of transcriptional cofactors possessing histone acetyltransferase (HAT) activity. This group of proteins has been shown to play critical roles in differentiation and cell proliferation in C. elegans , Drosophila , mouse and human. Despite their importance, the mechanisms by which they regulate transcription and thus exert their biological functions are poorly understood. A central question is whether the HAT activity is essential for the biological functions of these proteins. In this study, we investigated the importance of the HAT activity for CBP-1 in C. elegans . We show that a truncated CBP-1 protein lacking the HAT domain fails to promote differentiation, consistent with the notion that the HAT activity of CBP-1 is essential for its biological functions. To further address this question, we used a chemical that has been shown to be a specific inhibitor of the enzymatic activity of CBP/p300 HAT. Injection of this chemical into the gonads of the hermaphrodite mothers resulted in dead embryos arrested at different stages of development. Significantly, some of these embryos appear identical to cbp-1(RNAi) embryos in every aspect, as judged by the excess cell number, lack of endoderm and mesoderm tissues but excess neuronal differentiation (Shi and Mello, 1998). Taken together, our findings provide in vivo evidence that the HAT activity is not only critical, but also may be the only biochemical activity that is necessary for the biological functions of CBP-1. Shi, Y. and Mello,C. (1998), 'A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans .' Genes Dev. 12(7), 943-55.

Howard Chang et al. (2003) International Worm Meeting "Functional analysis of AMPA-type glutamate receptor sequences and their contribution to receptor localization at synapses"

Christopher Rongo, Howard Chang

[International Worm Meeting, 2003]

The modulation of AMPA-type glutamate receptor localization to central nervous system synapses is an important component of synaptic plasticity, and can be triggered by LTP (Long Term Potentiation) and CaMKII (Type II calcium-calmodulin-dependent protein kinase) activity. (1, 2). In mammalian hippocampal neurons, the different AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4 are proposed to form heteromultimers, and individual subunits can confer localization specificity to the multimeric channels that they comprise (2,3). One likely explanation for such subunit specificity is that the targeting of AMPA receptors to the synapses is probably due to the interaction of PDZ domain-containing proteins and the receptor subunit cytosolic tail sequences (4). In C. elegans, there are four AMPA receptor subunits that are expressed in the command interneurons, including, GLR-1, GLR-2, GLR-4 and GLR-5 (5,6). GLR-1 has C-terminal sequence that contributes to channel turnover (7). We have identified a glutamate receptor subunit domain that appears to be instructive for channel localization. This new finding may shed a new light on the localization and regulation of AMPA-type glutamate receptors in response to plasticity at synapses. (1) Rongo et al. Nature (1999), vol. 402, p. 195 (2) Shi et al. Science (1999), vol. 284, p.1811 (3) Shi et al. Cell (2001), vol. 105, p. 331 (4) Rongo et al. Cell (1998), vol. 94, p. 751 (5) Brockie et al. J. Neuroscience (2001), vol. 21(5), p. 1510 (6) Mellem et al. Neuron 2002 Dec 5; 36:933-944 (7) Burbea M et al. Neuron 2002 Jul 3;35(1):107-20

David Katz et al. (2006) Development & Evolution Meeting "Histone H3 Lysine 4 Guards The Immortality Of The Germline"

William Kelly, David Katz, Thomas Edwards

[Development & Evolution Meeting, 2006]

In the C. elegans early embryo, the chromatin in all blastomeres initially contain the euchromatic mark, di-methylation of histone H3 on lysine 4 (H3K4me2). However, upon their birth, the primordial germ cells Z2 and Z3 uniquely lose this mark. This appears to be a conserved characteristic of primordial germ cells as Drosophila pole cells also initially lack this modification. The loss of H3K4me2 has been proposed to be necessary for protecting the totipotency of the germline through chromatin-based transcriptional repression, but the mechanism of its removal is not understood. Recently, Shi et al. demonstrated that the mammalian amine oxidase LSD1 can specifically demethylate H3K4me2 (Shi et al. 2004). C. elegans has three lsd1 homologs : spr-5, amx-1 and T08D10.2. In order to ask if these genes play a role in the chromatin remodeling of Z2 and Z3, we inactivated them individually and in combination. RNAi or genetic mutation of any of these genes individually has no affect on H3K4me2 levels in Z2 and Z3. To test for redundant roles in the chromatin remodeling of Z2 and Z3, we generated a spr-5;amx-1 double mutant from two existing putative null mutations. Although the double mutants display multiple pleiotropic phenotypes, we observed no immediate effects on H3meK4 levels in Z2 and Z3. However, after multiple generations the double mutant exhibits a germline mortality phenotype, in which an increasing fraction of progeny in the population grow up sterile(black sterile phenotype). Strikingly, in these later generations, the double mutants exhibit retention of H3K4me2 in Z2 and Z3, further correlating repressive chromatin with maintenance of the germ cell lineage. In addition, the multiple generations that are required for this phenotype suggest that there can be stable inheritance of epigenetic defects through the germline and that the H3K4 demethylases may play a role in resetting H3K4 methylation at each generation.

Walstrom KM et al. (1998) West Coast Worm Meeting "AN RNA HELICASE IN C. ELEGANS IMPORTANT FOR DEVELOPMENT"

Walstrom KM, Swan KA

[West Coast Worm Meeting, 1998]

We have identified a putative DExH RNA helicase in C. elegans homologous to the Drosophila maleless protein and to human RNA helicase A. This protein, called RHA-1 for C. elegans RNA helicase A, contains an N-terminal double-stranded RNA binding domain, a central DExH ATPase domain, and a C-terminal RGG domain, which is also found in other RNA binding proteins. The human homologue of RHA-1 is capable of mediating an association between CREB binding protein (CBP) and RNA polymerase II and is suggested to be required for the activation of transcription by CBP (Nakajima et al. (1997) Cell 90, 1107-1112). To determine if RHA-1 is required during C. elegans development, we used RNA interference (RNAi) to deplete the embryo of RHA-1. We injected double-stranded RNA transcribed from a 1 kb fragment from the N-terminus of rha-1 and found a range of phenotypes culminating in dead embryos. Dead embryos exhibited muscle development but lacked gut granules suggesting endodermal development was compromised. Less severely affected progeny reached adulthood but were sterile or laid mainly oocytes. Recently, RNAi was performed with RNA transcribed fromC. elegans cbp-1 and the resulting embryos lacked mesoderm, endoderm, and hypodermal cells (Shi & Mello (1998) Genes and Develop. 12, 943-955). This suggests that one explanation of our rha-1 RNAi results is a partial block of CBP function. Other possibilities are that RHA-1 may be required for the function of other transcription factors or that it may be generally required for transcription or translation. This is the first evidence that RNA helicase A is required for development.