Wen C-h et al. (1996) East Coast Worm Meeting "Genetic study of sel-9"

Greenwald IS, Wen C-h

[East Coast Worm Meeting, 1996]

sel-9 mutations were first isolated in a screen for suppressors of lin-12(n676n930), a lin-12 hypomorphic mutation(1). Recently, in a complementation screen for new sel-9 mutations, we have obtained more alleles of sel-9 which show strong interactions with both lin-12 and glp-1 mutations. One class of sel-9 mutations, probably gain-of-function alleles, increase lin-12 and glp-1activity. The other class of mutation, represented by a probable loss-of-function allele, decreases lin-12 and glp-1activity. From genetic analysis, we propose that sel-9 defines a postive regulator or effector in the lin-12 and/or glp-1 pathways. Some of the sel-9 alleles also show new phenotypes in a wild type background, suggesting additional roles for sel-9 other than the involvement in the lin-12 and glp-1 pathways. We have also been trying to clone the sel-9 gene. sel-9 maps to LG V, between the left hand break points of two deficiencies, sDf47 and mDf3, a region covered by a few cosmids and YACs. Preliminary experiments indicate that sel-9 can be rescued by a single cosmid clone. We are trying to verify this result with the new sel-9 alleles. 1) Sundaram, M. and I. Greenwald 1993 Suppressors of a lin-12 hypomorph define genes that interact with both lin-12 and glp-1 in Caenorhabditis elegans. Genetics 135: 765-783.

Soulavie, F. et al. (2015) International Worm Meeting "EGF/Ras signaling and the AFF-1 fusogen control seamless tube auto-fusion and shaping in the C. elegans excretory system."

Sundaram, M., Soulavie, F.

[International Worm Meeting, 2015]

Unicellular tubes with an intracellular lumen are found in the mammalian microvasculature and in some invertebrate organs, including the C. elegans excretory (renal-like) system. Many unicellular tubes are seamless -they lack autocellular junctions along their length - and they adopt elongated or branched shapes. How cells become seamless tubes with such complex shapes is poorly understood. We've shown that the C. elegans excretory duct forms a seamless tube by cell wrapping to form an autocellular junction, followed by membrane fusion to remove that junction and become a seamless toroid. The duct tube subsequently elongates more than five-fold and adopts an unusual asymmetric shape. Seamlessness and tube shape depend on the EGF-Ras-ERK signaling cascade. Surprisingly, we found that signaling promotes both seamlessness and elongated tube shape via a single downstream target, the plasma membrane fusogen AFF-1.Using transcriptional reporters for aff-1, we demonstrated that EGF-Ras-ERK signaling promotes aff-1 expression in the duct cell through a combination of relief of LIN-1/Ets-dependent repression and stimulation of LIN-1/Ets + EOR-1/BTB-Zn finger-dependent activation. Although duct auto-fusion occurs at the 1.5-fold stage of embryogenesis, aff-1 expression in the duct persists throughout much of larval development. In the absence of aff-1, the duct retains an autocellular junction and has a dramatically shortened morphology, with the lumen only ~a third of its normal length. Furthermore, lumen markers accumulate in a diffuse pattern adjacent to the main lumen, suggesting possible accumulation of a vesicular intermediate. Continuous expression of aff-1 cDNA in the duct cell rescues auto-fusion and tube morphogenesis, while late expression can rescue a part of the duct shape and lumen phenotypes independently of auto-fusion. Our results suggest a continuous role for AFF-1 in seamless tube morphogenesis, and reveal an unexpected link between fusogen activity and intracellular lumen elongation.

Pu, P. et al. (2015) International Worm Meeting "The lipocalin LPR-1 cooperates with LIN-3/EGF signaling to promote excretory duct tube morphogenesis ."

Sundaram, M., Pu, P.

[International Worm Meeting, 2015]

LPR-1 belongs to the lipocalin superfamily, which is a group of structurally related secreted proteins that transport lipophilic cargos and in some cases participate in intercellular signaling. lpr-1 loss-of-function(lf) mutants have an incompletely penetrant L1 lethal phenotype due to failure to maintain a continuous lumen between the excretory duct and pore tubes. Although lpr-1 is normally expressed in both the duct and pore, it can function cell non-autonomously in tissue-specific rescue experiments, consistent with a transport- or signaling-related function.Epistasis experiments suggest that LPR-1 may facilitate LIN-3/EGF signaling from the excretory canal cell to the excretory duct and pore. The EGF-Ras-ERK signaling pathway plays multiple roles in the development of the excretory system, including specifying excretory duct versus pore cell fate and promoting duct morphogenesis. lpr-1 mutant lethality was significantly suppressed by overexpressing a lin-3/EGF genomic fragment or by global hyperactivation of the downstream Ras signaling pathway, suggesting that LPR-1 and LIN-3/EGF act in a common pathway. Temporal rescue experiments with a hsp::LIN-3(EGF) transgene demonstrated that signaling acts early, around the time of initial duct and pore tubulogenesis, to rescue lpr-1(lf) lethality. Tissue-specific rescue experiments with LET-60/Ras(gf) showed that hyperactivation of Ras was required in both the duct and pore cells.lpr-1 mutants do not display duct-to-pore cell fate transformations or other phenotypes expected for a general defect in EGF-Ras-ERK signaling; therefore we wondered if LPR-1 facilitates signaling by a specific LIN-3/EGF isoform. LIN-3 is the only EGF-related ligand in C. elegans, but alternative splicing generates at least four LIN-3 protein isoforms that differ in their extracellular domains and in their biological activities. In order to test which isoform(s) of LIN-3 could function in the excretory system and which might require LPR-1, single copy integration lines expressing different LIN-3 isoforms were generated. All three of the LIN-3 isoforms we tested could rescue lin-3(lf) and lpr-1(lf) mutant lethality, although they differ in rescue efficiency. These data do not support the idea that LPR-1 is a dedicated cofactor for a specific LIN-3 isoform. Rather, the data suggest that either LPR-1 plays a more general role in facilitating LIN-3/EGF signaling in the context of the excretory system, or that signaling upregulates other targets that in some way compensate for the absence of LPR-1.

Soulavie, F. et al. (2017) International Worm Meeting "An unexpected link between fusogen activity and intracellular lumen elongation."

Soulavie, F., Sundaram, M., Hall, D.

[International Worm Meeting, 2017]

Unicellular tubes with intracellular lumens are found in the mammalian microvasculature and in some invertebrate organs, including the C. elegans excretory (renal-like) system. Many unicellular tubes are seamless -they lack autocellular junctions along their length - and they adopt elongated or branched shapes. How cells become seamless tubes with such complex shapes is poorly understood. One proposed mechanism for seamless tube formation involves endocytosis of basal membrane followed by vesicle merging or exocytosis to form a lumen. An alternative mechanism involves cell wrapping to form a lumen lined by an autocellular junction, followed by membrane auto-fusion to remove that junction and become a seamless toroid. We provide evidence that the plasma membrane fusogen AFF-1 is involved in both mechanisms of intracellular lumen growth. The C. elegans excretory duct tube forms by wrapping and auto-fusion, and subsequently elongates more than five-fold and adopts an unusual asymmetric shape. Both auto-fusion and subsequent tube elongation depend on the EGF-Ras-ERK signaling cascade and its downstream target, aff-1. In the absence of aff-1, the duct retains an autocellular junction and has a dramatically shortened morphology, with the lumen only ~a third of its normal length. Super-resolution and electron microscopy showed that aff-1 mutants accumulate excess vesicles with apical cargoes adjacent to the main lumen, and large inclusions with highly convoluted membranes adjacent to and continuous with the basal membrane. Accumulation of the membrane-binding dye FM4-64 in aff-1 mutant duct cells suggests that the basal inclusions may correspond to a blocked endocytic intermediate. AFF-1 is an exoplasmic fusogen, and therefore could be suitably positioned to mediate dynamin-independent vesicle scission during endocytosis. Based on our observations, we propose that EGF signaling stimulates basal endocytosis in the growing duct tube, and that AFF-1 could be needed for scission of the internalized tubulovesicles, which ultimately contribute apical membrane to drive lumen growth. To further test this model, we are currently using the ZIF-1-dependent proteolysis system to remove AFF-1 from the duct cell after auto-fusion is complete, and following the subsequent progress of membrane trafficking. This work was supported by NIH grant GM58540 to M.S.

Ning Chen et al. (2005) International Worm Meeting "lin-1 positively regulates the transcription of the lateral signal gene apx-1"

Ning Chen, Iva Greenwald

[International Worm Meeting, 2005]

During vulval development, the Ras-mediated inductive signal induces P6.p to adopt the 1<sym05> fate, while LIN-12/Notch-mediated lateral signaling causes P5.p and P7.p to adopt the 2<sym05> fate. We have shown that three dsl genes, lag-2, apx-1, and dsl-1, are functionally redundant components of the lateral signal. Transcription of all three genes is initiated or upregulated in VPCs in response to the inductive signal (Chen and Greenwald, 2004).We performed a computational analysis of the 5<sym07> flanking regions of all three lateral signal genes and identified an <sym08>EBSX<sym09> motif, which is composed of Ets Binding Site (EBS) and a conserved flanking sequence (X). We have found that an 800 bp fragment from the apx-1 promoter that has a cluster of <sym08>EBSX<sym09> motifs appears to be necessary and sufficient for expression in the presumptive 1<sym05> VPC. Furthermore, we have found that the 1<sym05> cell specific expression of apx-1 is dependent on lin-1, an Ets gene. In addition, in lin-1 mutants, adjacent VPCs often adopt the 1<sym05> fate, indicating a failure of lateral signaling. Our results provide a possible molecular basis for a positive role of LIN-1 in VPC specification: phosphorylation of LIN-1 by MAPK may convert LIN-1 into a transcriptional activator (see also Howard and Sundaram, 2002). References:Chen, N. and Greenwald, I. (2004). The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins. Dev Cell 6, 183-92. Howard, R. M. and Sundaram, M. V. (2002). C. elegans EOR-1/PLZF and EOR-2 positively regulate Ras and Wnt signaling and function redundantly with LIN-25 and the SUR-2 Mediator component. Genes Dev 16, 1815-27.

Marisa Foehr et al. (2007) International Worm Meeting "Dorsoventral patterning of the postembryonic mesoderm requires both LIN-12/Notch and TGF-beta signaling."

Marisa Foehr, Jun Liu

[International Worm Meeting, 2007]

The C. elegans post-embryonic mesodermal lineage arises from a single precursor cell, the M mesoblast, which will diversify to generate distinct dorsal and ventral cell types. The dorsal daughter of M gives rise to a subset of body wall muscles and two non-muscle coelomocytes, whereas the ventral daughter of M gives rise to two sex myoblasts in addition to a subset of body wall muscles. Mutations in the C. elegans Schnurri homolog sma-9 cause ventralization of the M lineage. We have previously shown that SMA-9 antagonizes the Sma/Mab TGF-beta signaling pathway to promote dorsal M lineage fates (Foehr et al., 2006). Interestingly, loss-of-function mutations in the Notch receptor homolog lin-12 cause dorsalization of the M lineage (Greenwald et al., 1983), an exact opposite phenotype of sma-9 mutants. We have found that while LIN-12 protein is present in both the dorsal and ventral M lineage cells, the ligands for LIN-12, LAG-2 and APX-1, are asymmetrically localized in cells adjacent to ventral M-derived cells, and they function redundantly in promoting ventral M lineage fates. To investigate how LIN-12/Notch signaling interacts with SMA-9 and the Sma/Mab TGF-beta pathway in regulating M lineage patterning, we generated double and triple mutant combinations among lin-12, sma-9 and dbl-1 (the ligand for the Sma/Mab TGF-beta pathway) and examined their M lineage phenotypes. Our results suggest that the LIN-12/Notch pathway and the Sma/Mab TGF-beta pathway function independently in regulating dorsoventral patterning of the M lineage, with LIN-12/Notch required for ventral M lineage fates, and SMA-9 antagonism of TGF-beta signaling required for dorsal M lineage fates. Our work provides a model for how combined Notch and TGF-beta signaling regulates the developmental potential of two equipotent cells along the dorsoventral axis.

Grant BD et al. (1996) East Coast Worm Meeting "sel-1, Negative Regulator of lin-12 and glp-1."

Grant BD, Greenwald IS

[East Coast Worm Meeting, 1996]

The lin-12 gene of C. elegans encodes a founding member of the lin-12/Notch family of putative receptor proteins. Two members of this family in C. elegans, the lin-12 and glp-1 genes, mediate many cell fate decisions throughout development. Suppressor analysis has yielded a number of candidate genes, including the sel-1 gene, which may function with lin-12 and glp-1 in determining cell fates (Sundaram and Greenwald, 1993) . We will present here further genetic and molecular characterization of the sel-1 gene (see Grant and Greenwald, 1996). We have shown that the null phenotype of sel-1 is wild-type, except for an elevation in lin-12 and glp-1 activities when assayed in sensitized genetic backgrounds. We also found that by genetic criteria, sel-1 is a specific modifier of lin-12 and glp-1 activities. Cloning and sequencing of the sel-1 locus revealed that SEL-1 is a predicted extracellular protein, with a putative secretory signal sequence, potential GPI linkage signal, and a region of homology to predicted proteins from humans and yeast. Sequence analysis of sel-1 mutants combined with structure / function studies indicate that the homologous region is very important to sel-1 function. Immuno-localization experiments indicate that SEL-1 is a widely distributed protein found at the cell-surface and within the cell body (possibly within membrane bound vesicles). SEL-1 is found in cells undergoing lin-12 mediated cell fate decisions. Experiments addressing local versus diffusable models for SEL-1 activity will be discussed. Sundaram, M. and I. Greenwald 1993 Suppressors of a lin-12 hypomorph define genes that interact with both lin-12 and glp-1 in Caenorhabditis elegans. Genetics 135: 765-783. Grant, B. and I. Greenwald 1996 The Caenorhabditis elegans sel-1 gene, a negative regulator of lin-12 and glp-1, encodes a predicted extracellular protein. Genetics in press.

Pu, Pu et al. (2013) International Worm Meeting "LPR-1 facilitates LIN-3/EGF signaling during the development of the excretory system."

Lemmon, M., Pu, Pu, Sundaram, M., Freed, D.

[International Worm Meeting, 2013]

In the C.elegans excretory system, the EGF/Ras/Erk signaling pathway plays multiple roles in specifying the duct versus the pore cell fate and maintaining the duct organ architecture. LPR-1 belongs to the large lipocalin family which is a group of small secreted proteins functioning as carriers for lipophilic cargos. lpr-1 loss-of-function mutants showed a highly penetrant L1 lethal excretory phenotype resembling that of lin-3/EGF or let-60/Ras mutants, whereas this lethality was almost completely suppressed by overexpressing a lin-3/EGF genomic fragment or hyperactivating the downstream Ras signaling pathway. Furthermore, lpr-1 loss could suppress the excretory pore-to-duct fate transformation of lin-15 SynMuv mutants. These epistasis results suggest that during excretory system development, LPR-1 facilitates efficient signaling when LIN-3/EGF is at endogenous or slightly increased levels, whereas LPR-1 can be bypassed by high level expression of LIN-3/EGF. Based on our epistasis results and the lipocalin research findings of others in other species, we hypothesize that LPR-1 directly binds to LIN-3/EGF or LET-23/EGFR to facilitate signaling between the adjacent excretory canal and excretory duct cells. We are testing for the in vitro interactions between LPR-1 and LIN-3/EGF extracellular domain or LET-23/EGFR extracellular domain using surface plasmon resonance with purified proteins from S2 or Sf9 cells. Further, in order to determine which one or more isoforms of LIN-3/EGF are functionally dependent on LPR-1 in vivo, we will generate single copy transgenic lines by MOS-SCI technique and then check the rescue ability of each LIN-3 isoform for lin-3(n1417) in the absence of LPR-1 or in the presence of LPR-1.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Yuan Jiang et al. (2004) East Coast Worm Meeting "MLS-2, an HMX class homeodomain protein essential for mesodermal patterning and cell fate specification"

Jun Kelly Liu, Yuan Jiang

[East Coast Worm Meeting, 2004]

We are interested in understanding mesodermal patterning and fate specification by studying the C. elegans postembryonic mesodermal lineage, the M lineage. The M lineage is derived from a single precursor cell, the M mesoblast, and gives rise to six cell types: striated bodywall muscles (BWMs), nonmuscle coelomocytes (CCs), and four classes of non-striated sex muscles which are descendants of the sex myoblasts (SMs). We are studying the function of the mls-2 (mesodermal lineage specification) gene in M lineage patterning and fate specification. The mls-2(cc615) mutation causes randomization of division planes in the M lineage, and subsequent fate transformation of CCs and BWMs to SMs. In addition, cc615mutants have defects in SM migration and show some larval and adult lethality. We have cloned the wild type mls-2 gene (C39E6.4). mls-2 encodes a homeodomain protein that belongs to the HMX family of homeodomain proteins that are also present in sea urchin, Drosophila and vertebrates., We examined the expression pattern of mls-2 using both functional mls-2::gfp fusion construct and affinity purified anti-MLS-2 antibodies. We found that the MLS-2 protein is localized in nuclei of early M lineage cells and a subset of head neurons. Furthermore, mls-2 expression in the M lineage and the head neurons appears to require distinct cis-acting elements. Overexpression of mls-2 in the early M lineage where mls-2 is normally expressed caused a variety of defects in the M lineage. Forced expression of mls-2 in the later M lineage such as in SMs where mls-2 is not normally expressed resulted in extra rounds of divisions of SMs. This suggests that mls-2 may have multiple roles in the M lineage and that MLS-2 protein level is critical for the correct patterning of the M lineage. The M lineage defects of mls-2(cc615) are very similar to those of mab-5, hlh-1, egl-27 and egl-20 mutants. We are currently carrying out molecular and genetic epistasis experiments to investigate the relationship between mls-2 and those factors.