Hypodermal Expression Research Articles

O-GlcNAc transferase plays a non-catalytic role in C. elegans male fertility.

Animal behavior is influenced by the competing drives to maintain energy and to reproduce. The balance between these evolutionary pressures and how nutrient signaling pathways intersect with mating remains unclear. The nutrient sensor O-GlcNAc transferase, which post-translationally modifies intracellular proteins with a single monosaccharide, is responsive to cellular nutrient status and regulates diverse biological processes. Though essential in most metazoans, O-GlcNAc transferase (ogt-1) is dispensable in Caenorhabditis elegans, allowing genetic analysis of its physiological roles. Compared to control, ogt-1 males had a four-fold reduction in mean offspring, with nearly two thirds producing zero progeny. Interestingly, we found that ogt-1 males transferred sperm less often, and virgin males had reduced sperm count. ogt-1 males were also less likely to engage in mate-searching and mate-response behaviors. Surprisingly, we found normal fertility for males with hypodermal expression of ogt-1 and for ogt-1 strains with catalytic-dead mutations. This suggests OGT-1 serves a non-catalytic function in the hypodermis impacting male fertility and mating behavior. This study builds upon research on the nutrient sensor O-GlcNAc transferase and demonstrates a role it plays in the interplay between the evolutionary drives for reproduction and survival.

A Caenorhabditis elegans ortholog of human selenium-binding protein 1 is a pro-aging factor protecting against selenite toxicity

Human selenium-binding protein 1 (SELENBP1) was originally identified as a protein binding selenium, most likely as selenite. SELENBP1 is associated with cellular redox and thiol homeostasis in several respects, including its established role as a methanethiol oxidase that is involved in degradation of methanethiol, a methionine catabolite, generating hydrogen sulfide (H2S) and hydrogen peroxide (H2O2). As both H2S and reactive oxygen species (such as H2O2) are major regulators of Caenorhabditis elegans lifespan and stress resistance, we hypothesized that a SELENBP1 ortholog in C. elegans would likely be involved in regulating these aspects.Here we characterize Y37A1B.5, a putative selenium-binding protein 1 ortholog in C. elegans with 52% primary structure identity to human SELENBP1. While conferring resistance to toxic concentrations of selenite, Y37A1B.5 also attenuates resistance to oxidative stress and lowers C. elegans lifespan: knockdown of Y37A1B.5 using RNA interference resulted in an approx. 10% increase of C. elegans lifespan and an enhanced resistance against the redox cycler paraquat, as well as enhanced motility. Analyses of transgenic reporter strains suggest hypodermal expression and cytoplasmic localization of Y37A1B.5, whose expression decreases with worm age. We identify the transcriptional coregulator MDT-15 and transcription factor EGL-27 as regulators of Y37A1B.5 levels and show that the lifespan extending effect elicited by downregulation of Y37A1B.5 is independent of known MDT-15 interacting factors, such as DAF-16 and NHR-49. In summary, Y37A1B.5 is an ortholog of SELENBP1 that shortens C. elegans lifespan and lowers resistance against oxidative stress, while allowing for a better survival under toxic selenite concentrations.

The C. elegans heterochronic gene lin-28 coordinates the timing of hypodermal and somatic gonadal programs for hermaphrodite reproductive system morphogenesis.

C. elegans heterochronic genes determine the timing of expression of specific cell fates in particular stages of developing larvae. However, their broader roles in coordinating developmental events across diverse tissues have been less well investigated. Here, we show that loss of lin-28, a central heterochronic regulator of hypodermal development, causes reduced fertility associated with abnormal somatic gonadal morphology. In particular, the abnormal spermatheca-uterine valve morphology of lin-28(lf) hermaphrodites traps embryos in the spermatheca, which disrupts ovulation and causes embryonic lethality. The same genes that act downstream of lin-28 in the regulation of hypodermal developmental timing also act downstream of lin-28 in somatic gonadal morphogenesis and fertility. Importantly, we find that hypodermal expression, but not somatic gonadal expression, of lin-28 is sufficient for restoring normal somatic gonadal morphology in lin-28(lf) mutants. We propose that the abnormal somatic gonadal morphogenesis of lin-28(lf) hermaphrodites results from temporal discoordination between the accelerated hypodermal development and normally timed somatic gonadal development. Thus, our findings exemplify how a cell-intrinsic developmental timing program can also control proper development of other interacting tissues, presumably by cell non-autonomous signal(s). This article has an associated 'The people behind the papers' interview.

C. elegans patched-3 is an essential gene implicated in osmoregulation and requiring an intact permease transporter domain

The nematode Caenorhabditis elegans has retained a rudimentary Hedgehog (Hh) signalling pathway; Hh and Smoothened (Smo) homologs are absent, but two highly related Patched gene homologs, ptc-1 and ptc-3, and 24 ptc-related (ptr) genes are present. We previously showed that ptc-1 is essential for germ line cytokinesis. Here, we report that ptc-3 is also an essential gene; the absence of ptc-3 results in a late embryonic lethality due to an apparent defect in osmoregulation. Rescue of a ptc-3 mutant with a ptc-3::gfp translational reporter reveals that ptc-3 is dynamically expressed in multiple tissues across development. Consistent with this pattern of expression, ptc-3(RNAi) reveals an additional postembryonic requirement for ptc-3 activity. Tissue-specific promoter studies indicate that hypodermal expression of ptc-3 is required for normal development. Missense changes in key residues of the sterol sensing domain (SSD) and the permease transporter domain GxxxD/E motif reveal that the transporter domain is essential for PTC-3 activity, whereas an intact SSD is dispensable. Taken together, our studies indicate that PTC proteins have retained essential roles in C. elegans that are independent of Smoothened (Smo). These observations reveal novel, and perhaps ancestral, roles for PTC proteins.

Extracellular Sugar Modifications Provide Instructive and Cell-Specific Information for Axon-Guidance Choices

Heparan sulfates (HSs) are extraordinarily complex extracellular sugar molecules that are critical components of multiple signaling systems controlling neuronal development. The molecular complexity of HSs arises through a series of specific modifications, including sulfations of sugar residues and epimerizations of their glucuronic acid moieties. The modifications are introduced nonuniformly along protein-attached HS polysaccharide chains by specific enzymes. Genetic analysis has demonstrated the importance of specific HS-modification patterns for correct neuronal development. However, it remains unclear whether HS modifications provide a merely permissive substrate or whether they provide instructive patterning information during development. We show here with single-cell resolution that highly stereotyped motor axon projections in C. elegans depend on specific HS-modification patterns. By manipulating extracellular HS-modification patterns, we can cell specifically reroute axons, indicating that HS modifications are instructive. This axonal rerouting is dependent on the HS core protein lon-2/glypican and both the axon guidance cue slt-1/Slit and its receptor eva-1. These observations suggest that a changed sugar environment instructs slt-1/Slit-dependent signaling via eva-1 to redirect axons. Our experiments provide genetic in vivo evidence for the "HS code" hypothesis which posits that specific combinations of HS modifications provide specific and instructive information to mediate the specificity of ligand/receptor interactions.

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.

DAF-5 is a Ski oncoprotein homolog that functions in a neuronal TGF beta pathway to regulate C. elegans dauer development.

An unconventional TGF beta superfamily pathway plays a crucial role in the decision between dauer diapause and reproductive growth. We have studied the daf-5 gene, which, along with the daf-3 Smad gene, is antagonized by upstream receptors and receptor-regulated Smads. We show that DAF-5 is a novel member of the Sno/Ski superfamily that binds to DAF-3 Smad, suggesting that DAF-5, like Sno/Ski, is a regulator of transcription in a TGF beta superfamily signaling pathway. However, we present evidence that DAF-5 is an unconventional Sno/Ski protein, because DAF-5 acts as a co-factor, rather than an antagonist, of a Smad protein. We show that expressing DAF-5 in the nervous system rescues a daf-5 mutant, whereas muscle or hypodermal expression does not. Previous work suggested that DAF-5 and DAF-3 function in pharyngeal muscle to regulate gene expression, but our analysis of regulation of a pharynx specific promoter suggests otherwise. We present a model in which DAF-5 and DAF-3 control the production or release of a hormone from the nervous system by either regulating the expression of biosynthetic genes or by altering the connectivity or the differentiated state of neurons.

An Essential Role in Molting and Morphogenesis of Caenorhabditis elegans for ACN-1, a Novel Member of the Angiotensin-converting Enzyme Family That Lacks a Metallopeptidase Active Site

Genome sequence analyses predict many proteins that are structurally related to proteases but lack catalytic residues, thus making functional assignment difficult. We show that one of these proteins (ACN-1), a unique multi-domain angiotensin-converting enzyme (ACE)-like protein from Caenorhabditis elegans, is essential for larval development and adult morphogenesis. Green fluorescent protein-tagged ACN-1 is expressed in hypodermal cells, the developing vulva, and the ray papillae of the male tail. The hypodermal expression of acn-1 appears to be controlled by nhr-23 and nhr-25, two nuclear hormone receptors known to regulate molting in C. elegans. acn-1(RNAi) causes arrest of larval development because of a molting defect, a protruding vulva in adult hermaphrodites, severely disrupted alae, and an incomplete seam syncytium. Adult males also have multiple tail defects. The failure of the larval seam cells to undergo normal cell fusion is the likely reason for the severe disruption of the adult alae. We propose that alteration of the ancestral ACE during evolution, by loss of the metallopeptidase active site and the addition of new protein modules, has provided opportunities for novel molecular interactions important for post-embryonic development in nematodes.

Hypodermal Expression of Caenorhabditis elegans TGF-β Type I Receptor SMA-6 Is Essential for the Growth and Maintenance of Body Length

There are several transforming growth factor-β (TGF-β) 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-β 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.

Graded expression of ceh-14 reporters in the hypodermis is induced by a gonadal signal.

ceh-14, a LIM class homeobox gene from Caenorhabditis elegans, is the orthologue of the vertebrate Lhx3/Lhx4 genes. ceh-14 reporter constructs are expressed in several different cell types: head and tail neurons, spermatheca and hypodermis. An intriguing aspect of the hypodermal expression pattern is that it takes the form of a gradient which is strongest in the central body region in L4 to young adult hermaphrodites. Promoter deletion analyses revealed that important regulatory elements for hypodermal expression are located within the transcribed region of ceh-14. Since a large part of the hypodermis is a syncytium, we hypothesized that this expression is triggered in a non-cell-autonomous fashion, a possible source being the underlying gonad. In males, which have a different gonadal organisation, the ceh-14 reporter constructs are expressed in a gradient that is strongest in the tail. By laser ablation of the gonadal precursor cells we found that ceh-14 reporter construct expression is eliminated in the hermaphrodite hypodermis, suggesting that the gonad plays a role in the generation of the gradient. Several signaling pathways are known in the gonad and the vulva, thus we crossed the mutations lin-3, egl-17 and lin-12 with the ceh-14 reporter lines. However, the expression of the reporter constructs is not affected in these mutant backgrounds. This suggests that another, presently unknown, signal triggers the graded hypodermal expression.

Identification of heterochronic mutants in Caenorhabditis elegans. Temporal misexpression of a collagen::green fluorescent protein fusion gene.

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 specify the timing of lateral hypodermal seam cell terminal differentiation in Caenorhabditis elegans. We devised a screen to identify additional genes involved in this developmental timing mechanism based on identification of mutants that exhibit temporal misexpression from the col-19 promoter, a downstream target of the heterochronic gene pathway. We fused the col-19 promoter to the green fluorescent protein gene (gfp) and demonstrated that hypodermal expression of the fusion gene is adult-specific in wild-type animals and temporally regulated by the heterochronic gene pathway. We generated a transgenic strain in which the col-19::gfp fusion construct is not expressed because of mutation of lin-4, which prevents seam cell terminal differentiation. We have identified and characterized 26 mutations that restore col-19::gfp expression in the lin-4 mutant background. Most of the mutations also restore other aspects of the seam cell terminal differentiation program that are defective in lin-4 mutant animals. Twelve mutations are alleles of three previously identified genes known to be required for proper timing of hypodermal terminal differentiation. Among these are four new alleles of lin-42, a heterochronic gene for which a single allele had been described previously. Two mutations define a new gene, lin-58. When separated from lin-4, the lin-58 mutations cause precocious seam cell terminal differentiation and thus define a new member of the heterochronic gene pathway.