Gleason, Ryan et al. (2009) International Worm Meeting "Genetic mapping and characterization of a C. elegans Sma mutant."

Byrne, Rich, Padgett, Richard, Gleason, Ryan

[International Worm Meeting, 2009]

The transforming growth factor-b (TGFb) superfamily controls various processes of development and homeostasis of most tissues in metazoan organisms by exploiting the multifunctional TGFb signal transduction pathway. The TGFb superfamily consists of 42 structurally related members including members of the BMP family, TGFb family, activin family, glial derived neurotrophic factor (GDNF) and Mullerian Inhibitory Factor (MIF). The members of this superfamily act most commonly as local mediators to regulate a wide range of biological processes in all organisms. They have been functionally identified as essential regulators of developmental processes including; pattern formation, proliferation, cell division (positive and negative regulators), differentiation, extracellular matrix production, cell migration, cell death, tissue repair, and immune regulation. Aberrant expression of members of the TGFb superfamily has been detected in many diseases and cancer. The TGFb signaling pathways are conserved, and genetic screens in both C. elegans and D. melanogaster have been essential in delineating the transduction pathway. To identify novel members of this signal transduction pathway including receptors, intracellular components, extracellular regulators, and transcription factors we have characterized Sma mutants identified in genetic screens within the lab. wk94, a new allele identified as a novel Sma/Mab mutant has been mapped to chromosome I. wk94, regulates a gene reporter wkEx52 (spp-9::GFP), a reporter for Dbl-1 TGFb signaling, further characterizing its role in TGFb signaling. Efforts are underway to map and clone wk94. Current status will be presented at the meeting.

Gleason, Ryan et al. (2021) International Worm Meeting "Developmentally programmed H3 expression changes embryonic plasticity and reinforces cell fate specification"

Gleason, Ryan, Chen, Xin

[International Worm Meeting, 2021]

Early embryonic cells are pluripotent, possessing the transient capacity to generate all the cells of an organism. A fundamental question in developmental biology concerns identifying the epigenetic factors that underlie this temporary developmental plasticity, as well as understanding how commitment to a specific cell lineage is achieved and maintained. Using C. elegans as a model to dissect the role of distinct histone incorporation during gametogenesis and embryogenesis, we have uncovered an unappreciated developmental regulation of the incorporation of key molecular carriers of epigenetic information, the replication-coupled histone H3, and histone variant, H3.3, during gametogenesis that influence the epigenetic organization in the early embryo, with a lasting effect on pluripotency and lineage commitment. To characterize the dynamics of endogenous histones throughout the C. elegans lineage, I have generated knock-in strains inserting protein translational tags at the endogenous histone H3 loci (15 copies of H3 are encoded in the C. elegans genome), as well as generating knock-out and point-mutations to study the developmental impact of the loss of tissue-specific histone incorporation. In doing so, I have uncovered a surprising difference in the epigenome established in the germline, and maintained during early embryogenesis, which incorporates low levels of canonical H3 in favor of the histone variant H3.3. Unlike somatic cells where all 15 copies of H3 are expressed, germ cells uniquely restrict canonical H3 expression to 4 loci. Furthermore, upon cellular differentiation, I have identified a 400-fold increase in canonical H3 incorporation in the somatic cell lineage. This onset of canonical H3 incorporation in late-stage embryos correlates with a window of developmental plasticity which has been characterized in C. elegans embryonic blastomeres (Yuzyuk et al., 2009). To address the contribution of this increased H3 incorporation during gastrulation, I generated an endogenously encoded, mutated histone H3-H113D, to genetically alter chromatin assembly by destabilizing the H3-H4 histone tetramer at the onset of gastrulation. Utilizing a cell fate challenge assay to measure the degree of embryonic plasticity, we find embryonic plasticity is prolonged in H3-H113D mutants. Taken together, the data presented demonstrate H3 incorporation is developmentally programmed to restrict plasticity during embryogenesis and reinforce cell fate acquisition.

Gleason, Ryan et al. (2011) International Worm Meeting "Intracellular trafficking of the type I and type II TGFb receptors, SMA-6 and DAF-4, in C. elegans."

Grant, Barth, Akintobi, Adenrele, Padgett, Richard, Gleason, Ryan

[International Worm Meeting, 2011]

Receptor-mediated endocytosis regulates the internalization, degradation, and recycling of a diverse group of membrane bound, cell-signaling receptors. This regulation of cell signaling receptors modulates receptor availability, duration of signaling, and signaling strength. We have been developing C. elegans as a model to study the trafficking of TGFb receptors SMA-6 and DAF-4. Interestingly, using endocytic mutations that block recycling and transport to the trans-Golgi network (TGN), we find that trafficking of the type I and type II receptors are handled differently in the animal. Mutations in vps-35, a component of the retromer, block C. elegans TGFb signaling, indicating that retromer recycling of one or both receptors is necessary for full signaling strength. This work was prompted by the identification of sma-10, which was discovered through a genetic screen in the Padgett Lab, and which we show also affects the trafficking of the type I and type II receptors, sma-6 and daf-4. Experiments were done to distinguish between a role in receptor secretion or in internalization of receptors. Using double mutants for the AP-2 complex (required for entry into the cell) and sma-10, we show that sma-10 does not act in secretion of the receptors but acts after receptor internalization. In sma-10 mutants, both receptors accumulate in the cytoplasm, indicating that recycling and/or degradation are altered. Further data will be presented to show how TGFb traffics in C. elegans and what role sma-10 plays in this process.

Gleason, Ryan et al. (2013) International Worm Meeting "Disparate endocytic recycling of the TGFb signaling receptors, Sma-6 and Daf-4, regulates signaling of the Sma/Mab pathway."

Li, Ying, Padgett, Richard, Grant, Barth, Gleason, Ryan, Kane, Nanci, Akintobi, Adenrele (Dee)

[International Worm Meeting, 2013]

Our study aims to delineate the intracellular trafficking pathways that regulate the type I and type II TGFb receptors, Sma-6 and Daf-4 respectively. The TGFb superfamily of ligands regulate a diverse array of developmental processes across metazoan biology. Genetic and genomic analysis in C. elegans has contributed to the delineation of the highly conserved TGFb signal transduction pathway. An unanswered and critical question is how receptor-mediated endocytosis orchestrates the availability and intracellular trafficking (degradation versus recycling) of TGFb signaling receptors. By utilizing C. elegans we have identified a novel role of two distinct endocytic trafficking pathways in regulating both the recycling and signaling of the two TGFb receptors, Sma-6 and Daf-4. To test the receptor trafficking of the Sma/Mab pathway, functional translational fusions of both Sma-6 and Daf-4 were developed to various fluorescent proteins to assess the in vivo localization and availability of the receptors in genetically tractable endocytic mutants. Here we show that the protein sorting complex, the Retromer, is an essential linchpin in the decision to recycle or degrade Sma-6. In the absence of the Retromer complex Sma-6 is not recycled back to the Plasma membrane and is mis-sorted to the Lysosome for degradation. In contrast, Daf-4 recycles through the recycling endosome and is not dependent on the Retromer complex. Co-IP experiments demonstrate a direct interaction between the Retromer complex and Sma-6. Together, this work establishes a novel role for disparate intracellular trafficking in the regulation of TGFb receptor availability and signaling.

Musset-Bilal F et al. (1994) Worm Breeder's Gazette "Characterization of the C. elegans Basement Membrane-Associated Protein SPARC"

Musset-Bilal F, Schwarzbauer JE, Ryan CS

[Worm Breeder's Gazette, 1994]

Characterization of the C. elegans Basement Membrane- Associated Frederique Musset-Bilal, Carol S. Ryan, and Jean E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544

Vora, Mehul et al. (2015) International Worm Meeting "LRIGs: Novel Regulators of TGF-beta / BMP Signaling."

Padgett, Richard, Kane, Nanci, Gleason, Ryan, Vora, Mehul

[International Worm Meeting, 2015]

Transforming Growth Factor - beta (TGFbeta) / Bone Morphogenetic Protein (BMP) signaling is a conserved signaling pathway that governs fundamental developmental processes across metazoans. In the nematode Caenorhabditis elegans, the pathway controls body size and male tail morphology, but also has functions in innate immunity and aging. In a screen to identify regulators of pathway signaling, we identified the gene sma-10 that appears to interfere with the trafficking of the TGFbeta receptors, after internalization, leading to a decreased signaling output. The Leucine-Rich Immunoglobulin-containing (LRIG1-3) genes are the mammalian orthologs of sma-10. A central question is if sma-10 trafficking effects are conserved in vertebrates. To address these questions, we are utilizing fly and mammalian tissue culture models. We show that overexpression of LRIGs in HeLa cells leads to decreased abundance of co-transfected TGFbeta and BMP receptors. Experiments are underway, using RNAi, to assess the effect of knockdowns of LRIGs on receptor trafficking.

Liao, Kelvin et al. (2017) International Worm Meeting "C. elegans SMA-10 regulates BMP Receptor Trafficking."

Liao, Kelvin, Padgett, Richard, Vora, Mehul, Gleason, Ryan, Kane, Nanci, Li, Ying

[International Worm Meeting, 2017]

Signal transduction of the conserved BMP signaling pathway functions through two distinct serine/threonine transmembrane receptors, the type I and type II receptors. Endocytosis orchestrates the assembly of signaling complexes by coordinating the entry of receptors with their downstream signaling mediators. Recently, we showed that the C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6, part of the transforming growth factor- beta (TGF beta ) superfamily, is recycled through the retromer complex unlike the type II receptor, DAF-4. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, we reported on SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. Here we show that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, MVB, and the late endosome with a reduction in signaling strength. Our studies show that trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, we find that the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling.

Liu, Z. et al. (2019) International Worm Meeting "Tetraspanins TSP-12 and TSP-14 function redundantly to promote the trafficking of the type II BMP receptor in Caenorhabditis elegans."

Liu, Z., Nzessi, A., Grant, B., Liu, J., Shi, H.

[International Worm Meeting, 2019]

Tetraspanins are a unique family of four-pass transmembrane proteins that play important roles in a variety of cell biological processes. One of their roles is to regulate the trafficking of their associated proteins. We have previously shown that two paralogous tetraspanins in C. elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling [1]. To dissect the functions of TSP-12 and TSP-14 in BMP signaling, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple tissues, including the hypodermis, where the BMP receptors function to regulate body size, one of the phenotypic outputs of the BMP pathway. TSP-12 and TSP-14 also share overlapping localization patterns on the cell surface and partial co-localization in intracellular vesicles. Using markers for various intracellular vesicles in hypodermal cells, we found that TSP-12 and TSP-14 are primarily localized to the early endosomes, late endosomes and recycling endosomes. In tsp-12(0); tsp-14(0)double mutants, the morphology of the endosomes is impaired and an intestinally expressed DAF-4::GFP is mis-localized to lysosomes or lysosome-related organelles, indicating that TSP-12 and TSP-14 are required for the intracellular trafficking of the type II BMP receptor DAF-4. Together with previous findings by Gleason et al. showing that the type I receptor SMA-6 is recycled via the retromer complex [2], our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors, and highlights the importance of intracellular trafficking in the regulation of BMP signaling in vivo. 1. Wang, L. et al. (2017) Two paralogous tetraspanins TSP-12 and TSP-14 function with the ADAM10 metalloprotease SUP-17 to promote BMP signaling in C. elegans.PLoS GeneticsDOI:10.1371/journal.pgen.1006568. 2. Gleason, R. J. et al. (2014) BMP signaling requires retromer-dependent recycling of the type I receptor. PNASDOI:10.1073/pnas.1319947111

Toshia Myers et al. (2005) International Worm Meeting "Towards an understanding of how Wnt signals regulate VPC patterning"

Iva Greenwald, Toshia Myers

[International Worm Meeting, 2005]

In addition to the well characterized inductive and lateral signals (from the gonad and P6.p, respectively), other signaling events also affect vulval precursor cell (VPC) patterning. We are interested in identifying additional signals that influence VPC patterning and their cellular sources. We recently confirmed that hyp7 communicates with the VPCs to regulate their fates, and hope to determine the nature of this communication and whether it involves an "inhibitory signal" (see Abstract by Berkovits et al.). In considering potential non-autonomous regulators of VPC patterning, we wondered whether Wnt signaling could be related to 'inhibitory signaling', as both affect VPC fusion and vulval induction (see Eisenmann et al., 1998; Chen and Han, 2001; Gleason et al., 2002). However, the identity of the Wnt(s) and its cellular source is not known.Available single wnt mutants do not display highly penetrant VPC defects, suggesting that more than one wnt gene may be involved. In order to identify potential Wnt ligands involved in VPC fate specification, we made transcriptional reporters for the three wnts for which expression data had not been reported, cwn-1, cwn-2 and mom-2. We found that the cwn-1 and cwn-2 reporters are expressed in neurons near the VPCs, raising the possibility that they are involved in regulating VPC specification and potentially implicating neurons in VPC specification. We are coordinating our efforts with David Eisenmann and colleagues (see Abstract by Gleason et al.), who also have expression data and have seen numerous synergistic effects among mutations in wnt genes. In particular, the cwn-1(ok546); egl-20(n585) double mutant displays a highly penetrant defect in VPC fate specification. We have analyzed the cell fates in this double mutant using arIs99, an integrated dpy-7::2NLS::yfp reporter that is continuously visible in hyp7 nuclei, and have seen varying defects in VPC induction, ranging from underinduction for P3.p-P8.p, as well as overinduction for P8.p. In addition, we saw anatomical abnormalities of Pn.p cells and their decendants and aberrant expression of arIs99 in ventral cord neurons. We will use the highly penetrant fused (F) fate of P4.p in the double mutant to conduct mosaic analysis to gain insight into the cellular source of cwn-1. We hope to have data on this point at the meeting.

Zocher E et al. (2018) MicroPubl Biol "Dominant-negative VPS-4 disrupts ODR-10::GFP distribution but has limited effects on chemotaxis"

Ruth N, Dahlberg C, Zocher E

[MicroPubl Biol, 2018]

Accumulation of ODR-10 depends on the sex and fed state of C. elegans (Ryan et al. 2014), though the precise mechanism of its regulation is not known. We tested whether the ESCRT pathway is required to maintain normal accumulation and distribution of ODR-10::GFP in AWA neurons under well-fed and starved conditions. The conserved AAA ATPase VPS-4 is required as the final step of the ESCRT pathway, to dissociate the machinery as cargo is moved from endosomes to multivesicular bodies (Babst et al. 2011, Kim et al. 2011). Mutation of Glutamate 219 to Glutamine (E219Q) in VPS-4 results in a dominant negative enzyme (VPS-4 DN). VPS-4 DN disrupts normal accumulation of the glutamate receptor, GLR-1, in a cell autonomous manner (Chun et al. 2008). We investigated a potential role for VPS-4 in ODR-10 regulation using fluorescence microscopy and an attraction chemotaxis assay. We expressed VPS-4 DN under the odr-10 promoter in animals expressing ODR-10::GFP (Figure 1A). Using live-imaging (200X (total) magnification), ODR-10::GFP was not visible in over half of the wild type, well-fed animals. Using this baseline, we measured changes in ODR-10::GFP (abundance and localization) and found that VPS-4 DN increased the frequency with which we observed animals with aberrant ODR-10 accumulation (Figure 1B). In 8 animals (out of 58) there was observable fluorescence in the cell bodies of AWA neurons (1 with fluorescence exclusively in the cell body). Starvation exacerbated this effect: 26 out of 39 animals had fluorescence in the cell bodies of AWA neurons (7 with fluorescence exclusively in the cell body). In contrast to previously published data that relied on different imaging parameters (Ryan, et al., 2014) we also observed that starvation of animals led to increased frequency of visible fluorescence in the cilia of AWA in WT animals (Figure 1B). Expression of VPS-4 DN had no effect on the ability of animals to react to the chemoattractant diacetyl under well-fed conditions (Figure 1C). However, VPS-4 DN expression in the AWA neuron resulted in significantly lower chemotaxis indices, compared to wild type, fed animals (Figure 1C, asterisk). Starvation of animals has a subtle effect on the abundance of ODR-10 that accumulates in the cilia of AWA neurons, but that accumulation does not affect the efficiency of chemotaxis in wild type animals (Figure 1B and C). However, when food stress was paired with a disruption of the ESCRT pathway the aberrant accumulation of ODR-10::GFP correlated with a modest but significant decrease in chemotaxis index (Figure 1C). It is still unknown whether ODR-10 is a direct target of the ESCRT machinery or other ubiquitin-related pathways. These data could also represent indirect effects due to changes in availability or localization of cofactors that are required for normal receptor processing and/or localization within the AWA neuron.