An antagonistic role for the C. elegans Schnurri homolog SMA-9 in modulating TGFbeta signaling during mesodermal patterning

In C. elegans, the Sma/Mab TGFbeta signaling pathway regulates body size and male tail patterning. SMA-9, the C. elegans homolog of Schnurri, has been shown to function as a downstream component to mediate the Sma/Mab TGFbeta signaling pathway in these processes. We have discovered a new role for SMA-9 in dorsoventral patterning of the C. elegans post-embryonic mesoderm, the M lineage. In addition to a small body size, sma-9 mutant animals exhibit a dorsal-to-ventral fate transformation within the M lineage. This M lineage defect of sma-9 mutants is unique in that animals carrying mutations in all other known components of the TGFbeta pathway exhibit no M lineage defects. Surprisingly, mutations in the core components of the Sma/Mab TGFbeta signaling pathway suppressed the M lineage defects of sma-9 mutants without suppressing their body size defects. We show that this suppression specifically happens within the M lineage. Our studies have uncovered an unexpected role of SMA-9 in antagonizing the TGFbeta signaling pathway during mesodermal patterning, suggesting a novel mode of function for the SMA-9/Schnurri family of proteins.     

Genetic screen for small body size mutants in C. elegans reveals many TGFbeta pathway components

In the nematode Caenorhabditis elegans, a TGFbeta-related signaling pathway regulates body size and male tail morphogenesis. We sought to identify genes encoding components or modifiers of this pathway in a large-scale genetic screen. Remarkably, this screen was able to identify essentially all core components of the TGFbeta signaling pathway. Among 34 Small mutants, many mutations disrupt genes encoding recognizable components of the TGFbeta pathway: DBL-1 ligand, DAF-4 type II receptor, SMA-6 type I receptor, and SMA-2, SMA-3, and SMA-4 Smads. Moreover, we find that at least 11 additional complementation groups can mutate to the Small phenotype. Four of these 11 genes, sma-9, sma-14, sma-16, and sma-20 affect male tail morphogenesis as well as body size. Two genes, sma-11 and sma-20, also influence regulation of the developmentally arrested dauer larval stage, suggesting a role in a second characterized TGFbeta pathway in C. elegans. Other genes may represent tissue-specific factors or parallel pathways for body size control. Because of the conservation of TGFbeta signaling pathways, homologs of these genes may be involved in tissue specificity and/or crosstalk of TGFbeta pathways in other animals.     

Hypodermal expression of Caenorhabditis elegans TGF-beta type I receptor SMA-6 is essential for the growth and maintenance of body length.

There are several transforming growth factor-beta (TGF-beta) pathways in the nematode Caenorhabditis elegans. One of these pathways regulates body length and is composed of the ligand DBL-1, serine/threonine protein kinase receptors SMA-6 and DAF-4, and cytoplasmic signaling components SMA-2, SMA-3, and SMA-4. To further examine the molecular mechanisms of body-length regulation in the nematode by the TGF-beta pathway, we examined the regional requirement for the type-I receptor SMA-6. Using a SMA-6::GFP (green fluorescent protein) reporter gene, sma-6 was highly expressed in the hypodermis, unlike the type-II receptor DAF-4, which is reported to be ubiquitously expressed. We then examined the ability of SMA-6 expression in different regions of the C. elegans body to rescue the sma-6 phenotype (small) and found that hypodermal expression of SMA-6 is necessary and sufficient for the growth and maintenance of body length. We also demonstrate that GATA sequences in the sma-6 promoter contribute to the hypodermal expression of sma-6.     

The expression of TGFbeta signal transducers in the hypodermis regulates body size in C. elegans

In C. elegans, a TGFbeta-related signaling pathway regulates body size. Loss of function of the signaling ligand (dbl-1), receptors (daf-4 and sma-6) or Smads (sma-2, sma-3 and sma-4) results in viable, but smaller animals because of a reduction in postembryonic growth. We have investigated the tissue specificity of this pathway in body size regulation. We show that different tissues are reduced in size by different proportions, with hypodermal blast cell size most closely proportional to body size. We show that SMA-3 Smad is expressed in pharynx, intestine and hypodermis, as has been previously reported for the type I receptor SMA-6. Furthermore, we find that SMA-3::GFP is nuclear localized in all of these tissues, and that nuclear localization is enhanced by SMA-6 activity. Interestingly, SMA-3 protein accumulation was found to be negatively regulated by the level of Sma/Mab pathway activity. Using genetic mosaic analysis and directed expression of SMA-3, we find that SMA-3 activity in the hypodermis is necessary and sufficient for normal body size. As dbl-1 is expressed primarily in the nervous system, these results suggest a model in which postembryonic growth of hypodermal cells is regulated by TGFbeta-related signaling from the nervous system to the hypodermis.     

Glypican LON-2 is a conserved negative regulator of BMP-like signaling in Caenorhabditis elegans

Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.     

Regulation of Extracellular Matrix Organization by BMP Signaling in Caenorhabditis elegans

In mammals, Bone Morphogenetic Protein (BMP) pathway signaling is important for the growth and homeostasis of extracellular matrix, including basement membrane remodeling, scarring, and bone growth. A conserved BMP member in Caenorhabditis elegans, DBL-1, regulates body length in a dose-sensitive manner. Loss of DBL-1 pathway signaling also results in increased anesthetic sensitivity. However, the physiological basis of these pleiotropic phenotypes is largely unknown. We created a DBL-1 over-expressing strain and show that sensitivity to anesthetics is inversely related to the dose of DBL-1. Using pharmacological, genetic analyses, and a novel dye permeability assay for live, microwave-treated animals, we confirm that DBL-1 is required for the barrier function of the cuticle, a specialized extracellular matrix. We show that DBL-1 signaling is required to prevent animals from forming tail-entangled aggregates in liquid. Stripping lipids off the surface of wild-type animals recapitulates this phenotype. Finally, we find that DBL-1 signaling affects ultrastructure of the nematode cuticle in a dose-dependent manner, as surface lipid content and cuticular organization are disrupted in animals with genetically altered DBL-1 levels. We propose that the lipid layer coating the nematode cuticle normally prevents tail entanglement, and that reduction of this layer by loss of DBL-1 signaling promotes aggregation. This work provides a physiological mechanism that unites the DBL-1 signaling pathway roles of not only body size regulation and drug responsiveness, but also the novel Hoechst 33342 staining and aggregation phenotypes, through barrier function, content, and organization of the cuticle.     

SMA-3 smad has specific and critical functions in DBL-1/SMA-6 TGFbeta-related signaling

A TGFbeta signal transduction cascade controls body size and male tail morphogenesis in the nematode Caenorhabditis elegans. We have analyzed the function of the sma-3 Smad gene, one of three Smad genes that function in this pathway. Null mutations in sma-3 are at least as severe as null mutations in the ligand and type I receptor genes, dbl-1 and sma-6, indicating that the other Smads do not function in the absence of SMA-3. Furthermore, null mutations in sma-3 do not cause defects in egg laying or in regulation of the developmentally arrested dauer larva stage, indicating no overlapping function with another C. elegans TGFbeta signaling pathway. The sma-3 gene is widely expressed at all developmental stages in hermaphrodites and males. The molecular lesions associated with eight sma-3 alleles of varying severity have been determined. The missense mutations cluster in two previously identified regions important for Smad function.     

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.     

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.     

Patterning of dopaminergic neurotransmitter identity among Caenorhabditis elegans ray sensory neurons by a TGFbeta family signaling pathway and a Hox gene

We have investigated the mechanism that patterns dopamine expression among Caenorhabditis elegans male ray sensory neurons. Dopamine is expressed by the A-type sensory neurons in three out of the nine pairs of rays. We used expression of a tyrosine hydroxylase reporter transgene as well as direct assays for dopamine to study the genetic requirements for adoption of the dopaminergic cell fate. In loss-of-function mutants affecting a TGFbeta family signaling pathway, the DBL-1 pathway, dopaminergic identity is adopted irregularly by a wider subset of the rays. Ectopic expression of the pathway ligand, DBL-1, from a heat-shock-driven transgene results in adoption of dopaminergic identity by rays 3-9; rays 1 and 2 are refractory. The rays are therefore prepatterned with respect to their competence to be induced by a DBL-1 pathway signal. Temperature-shift experiments with a temperature-sensitive type II receptor mutant, as well as heat-shock induction experiments, show that the DBL-1 pathway acts during an interval that extends from two to one cell generation before ray neurons are born and begin to differentiate. In a mutant of the AbdominalB class Hox gene egl-5, rays that normally express EGL-5 do not adopt dopaminergic fate and cannot be induced to express DA when DBL-1 is provided by a heat-shock-driven dbl-1 transgene. Therefore, egl-5 is required for making a subset of rays capable of adopting dopaminergic identity, while the function of the DBL-1 pathway signal is to pattern the realization of this capability.     

Nervous wreck interacts with thickveins and the endocytic machinery to attenuate retrograde BMP signaling during synaptic growth

Regulation of synaptic growth is fundamental to the formation and plasticity of neural circuits. Here, we demonstrate that Nervous wreck (Nwk), a negative regulator of synaptic growth at Drosophila NMJs, interacts functionally and physically with components of the endocytic machinery, including dynamin and Dap160/intersectin, and negatively regulates retrograde BMP growth signaling through a direct interaction with the BMP receptor, thickveins. Synaptic overgrowth in nwk is sensitive to BMP signaling levels, and loss of Nwk facilitates BMP-induced overgrowth. Conversely, Nwk overexpression suppresses BMP-induced synaptic overgrowth. We observe analogous genetic interactions between dap160 and the BMP pathway, confirming that endocytosis regulates BMP signaling at NMJs. Finally, we demonstrate a correlation between synaptic growth and pMAD levels and show that Nwk regulates these levels. We propose that Nwk functions at the interface of endocytosis and BMP signaling to ensure proper synaptic growth by negatively regulating Tkv to set limits on this positive growth signal.     

Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family

We have identified a new member of the TGF-beta superfamily, CET-1, from Caenorhabditis elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants, suggesting cet-1, sma-2, sma-3 and sma-4 share a common pathway. Overexpression experiments demonstrated that cet-1 function requires wild-type sma genes. Interestingly, CET-1 appears to affect body length in a dose-dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild-type worms elongated body length close to lon mutants. In male sensory ray patterning, lack of cet-1 function results in ray fusions. Epistasis analysis revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway in the male tail. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.