Roles of the Wnt effector POP-1/TCF in the C. elegans endomesoderm specification gene network

In C. elegans the 4-cell stage blastomere EMS is an endomesodermal precursor. Its anterior daughter, MS, makes primarily mesodermal cells, while its posterior daughter E generates the entire intestine. The gene regulatory network underlying specification of MS and E has been the subject of study for more than 15 years. A key component of the specification of the two cells is the involvement of the Wnt/β-catenin asymmetry pathway, which through its nuclear effector POP-1, specifies MS and E as different from each other. Loss of pop-1 function results in the mis-specification of MS as an E-like cell, because POP-1 directly represses the end-1 and end-3 genes in MS, which would otherwise promote an endoderm fate. A long-standing question has been whether POP-1 plays a role in specifying MS fate beyond repression of endoderm fate. This question has been difficult to ask because the only chromosomal lesions that remove both end-1 and end-3 are large deletions removing hundreds of genes. Here, we report the construction of bona fide end-1 end-3 double mutants. In embryos lacking activity of end-1, end-3 and pop-1 together, we find that MS fate is partially restored, while E expresses early markers of MS fate and adopts characteristics of both MS and C. Our results suggest that POP-1 is not critical for MS specification beyond repression of endoderm specification, and reveal that Wnt-modified POP-1 and END-1/3 further reinforce E specification by repressing MS fate in E. By comparison, a previous work suggested that in the related nematode C. briggsae, Cb-POP-1 is not required to repress endoderm specification in MS, in direct contrast with Ce-POP-1, but is critical for repression of MS fate in E. The findings reported here shed new light on the flexibility of combinatorial control mechanisms in endomesoderm specification in Caenorhabditis.     

Wnt signaling controls the stem cell-like asymmetric division of the epithelial seam cells during C. elegans larval development

Metazoan stem cells repopulate tissues during adult life by dividing asymmetrically to generate another stem cell and a cell that terminally differentiates. Wnt signaling regulates the division pattern of stem cells in flies and vertebrates. While the short-lived nematode C. elegans has no adult somatic stem cells, the lateral epithelial seam cells divide in a stem cell-like manner in each larval stage, usually generating a posterior daughter that retains the seam cell fate and an anterior daughter that terminally differentiates. We show that while wild-type adult animals have 16 seam cells per side, animals with reduced function of the TCF homolog POP-1 have as many as 67 seam cells, and animals with reduced function of the β-catenins SYS-1 and WRM-1 have as few as three. Analysis of seam cell division patterns showed alterations in their stem cell-like divisions in the L2-L4 stages: reduced Wnt signaling caused both daughters to adopt non-seam fates, while activated Wnt signaling caused both daughters to adopt the seam fate. Therefore, our results indicate that Wnt signaling globally regulates the asymmetric, stem cell-like division of most or all somatic seam cells during C. elegans larval development, and that Wnt pathway regulation of stem cell-like behavior is conserved in nematodes.     

C. elegans fat storage and metabolic regulation

C. elegans has long been used as an experimentally tractable organism for discovery of fundamental mechanisms that underlie metazoan cellular function, development, neurobiology, and behavior. C. elegans has more recently been exploited to study the interplay of environment and genetics on lipid storage pathways. As an experimental platform, C. elegans is amenable to an extensive array of forward and reverse genetic, a variety of “omics” and anatomical approaches that together allow dissection of complex physiological pathways. This is particularly relevant to the study of fat biology, as energy balance is ultimately an organismal process that involves behavior, nutrient digestion, uptake and transport, as well as a variety of cellular activities that determine the balance between lipid storage and utilization. C. elegans offers the opportunity to dissect these pathways and various cellular and organismal homeostatic mechanisms in the context of a genetically tractable, intact organism.     

Wnt signaling controls temporal identities of seam cells in Caenorhabditis elegans

The Wnt signaling pathway regulates multiple aspects of the development of stem cell-like epithelial seam cells in Caenorhabditis elegans, including cell fate specification and symmetric/asymmetric division. In this study, we demonstrate that lit-1, encoding the Nemo-like kinase in the Wnt/β-catenin asymmetry pathway, plays a role in specifying temporal identities of seam cells. Loss of function of lit-1 suppresses defects in retarded heterochronic mutants and enhances defects in precocious heterochronic mutants. Overexpressing lit-1 causes heterochronic defects opposite to those in lit-1(lf) mutants. LIT-1 exhibits a periodic expression pattern in seam cells within each larval stage. The kinase activity of LIT-1 is essential for its role in the heterochronic pathway. lit-1 specifies the temporal fate of seam cells likely by modulating miRNA-mediated silencing of target heterochronic genes. We further show that loss of function of other components of Wnt signaling, including mom-4, wrm-1, apr-1, and pop-1, also causes heterochronic defects in sensitized genetic backgrounds. Our study reveals a novel function of Wnt signaling in controlling the timing of seam cell development in C. elegans.     

The PAM-1 aminopeptidase regulates centrosome positioning to ensure anterior-posterior axis specification in one-cell C. elegans embryos

In the one-cell Caenorhabditis elegans embryo, the anterior-posterior (A-P) axis is established when the sperm donated centrosome contacts the posterior cortex. While this contact appears to be essential for axis polarization, little is known about the mechanisms governing centrosome positioning during this process. pam-1 encodes a puromycin sensitive aminopeptidase that regulates centrosome positioning in the early embryo. Previously we showed that pam-1 mutants fail to polarize the A-P axis. Here we show that PAM-1 can be found in mature sperm and in cytoplasm throughout early embryogenesis where it concentrates around mitotic centrosomes and chromosomes. We provide further evidence that PAM-1 acts early in the polarization process by showing that PAR-1 and PAR-6 do not localize appropriately in pam-1 mutants. Additionally, we tested the hypothesis that PAM-1's role in polarity establishment is to ensure centrosome contact with the posterior cortex. We inactivated the microtubule motor dynein, DHC-1, in pam-1 mutants, in an attempt to prevent centrosome movement from the cortex and restore anterior-posterior polarity. When this was done, the aberrant centrosome movements of pam-1 mutants were not observed and anterior-posterior polarity was properly established, with proper localization of cortical and cytoplasmic determinants. We conclude that PAM-1's role in axis polarization is to prevent premature movement of the centrosome from the posterior cortex, ensuring proper axis establishment in the embryo.     

The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis

Tubulogenesis and lumen formation are critical to the development of most organs. We study Caenorhabditis elegans vulval and uterine development to probe the complex mechanisms that mediate these events. Development of the vulva and the ventral uterus is coordinated by the inductive cell-signaling activity of a gonadal cell called the anchor cell (AC). We demonstrate that in addition to its function in specifying fate, the AC directly promotes dorsal vulval tubulogenesis. Two types of mutants with defective anchor cell behavior reveal that anchor cell invasion of the vulva is important for forming the toroidal shape of the dorsal vulval cell, vulF. In fos-1 mutants, where the AC cannot breakdown the basement membranes between the gonad and the vulva, and in mutants in unc-6 netrin or its receptor unc-40, which cause AC migration defects, the AC fails to invade the vulva and no lumen is formed in vulF. By examining GFP markers of dorsal vulval cell fate, we demonstrate that fate specification defects do not account for the aberrant vulF shape. We propose that the presence of the AC in the center of the developing vulF toroid is required for dorsal vulval lumen formation to complete vulval tubulogenesis.     

Different endocytic functions of AGEF-1 in C. elegans coelomocytes

Background

ADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that play roles in membrane dynamics and vesicle trafficking. AGEF-1, which is thought to act as a guanine nucleotide exchange factor of class I ARFs, is required for caveolin-1 body formation and receptor-mediated endocytosis in oocytes of Caenorhabditis elegans. This study explores additional roles of AGEF-1 in endocytic transport.

Methods

agef-1 expression was knocked down by using RNAi in C. elegans. Markers that allow analysis of endocytic transport in scavenger cells were investigated for studying the effect of AGEF-1 on different steps of membrane transport.

Results

Knockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of C. elegans. First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes, even though lysosomal acidification is normal and degradation still occurs.

Conclusion

Our results suggest that AGEF-1 regulates endosome/lysosome fusion or fission events, in addition to earlier steps in endocytic transport.

General significance

AGEF-1 is the first identified GTPase regulator that functions at the lysosome fusion or fission stage of the endocytic pathway. Our study provides insight into lysosome dynamics in C. elegans.     

SDN-1/syndecan regulates growth factor signaling in distal tip cell migrations in C. elegans

Mutations in the sdn-1/syndecan gene act as genetic enhancers of the ventral-to-dorsal distal tip cell (DTC) migration defects caused by a weak allele of the netrin receptor gene unc-5. The sdn-1(ev697) allele was identified in a genetic screen for enhancers of unc-5 DTC migration defects, and carried a nonsense mutation predicted to truncate the SDN-1 protein prior to the transmembrane domain. The enhancement of unc-5 caused by an sdn-1 mutation was rescued by expression of wild-type sdn-1 in the hypodermis or nervous system rather than the DTCs, indicating a cell non-autonomous function of sdn-1. The enhancement was also partially reversed by mutations in the egl-17/FGF or egl-20/Wnt genes, suggesting that sdn-1 affects UNC-5 function through a mis-regulation of signaling in growth factor pathways. egl-20 reporter constructs exhibited increased and mis-localized EGL-20 distribution in sdn-1 mutants compared to wild-type animals. Finally, using loss of function mutations, we show that egl-17/Fgf and egl-20/Wnt are partially redundant in regulating the migration pattern of the posterior DTC, as double mutants exhibit significant frequencies of defects in migration phases along both the anteroposterior and dorsoventral axes. Together these results suggest that SDN-1 affects UNC-5 function by regulating the proper extracellular distribution of growth factors.     

Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.     

Identification of genes driving lineage divergence from single-cell gene expression data in C. elegans

The nematode Caenorhabditis elegans (C. elegans) is an ideal model organism to study the cell fate specification mechanisms during embryogenesis. It is generally believed that cell fate specification in C. elegans is mainly mediated by lineage-based mechanisms, where the specification paths are driven forward by a succession of asymmetric cell divisions. However, little is known about how each binary decision is made by gene regulatory programs. In this study, we endeavor to obtain a global understanding of cell lineage/fate divergence processes during the early embryogenesis of C. elegans. We reanalyzed the EPIC data set, which traced the expression level of reporter genes at single-cell resolution on a nearly continuous time scale up to the 350-cell stage in C. elegans embryos. We examined the expression patterns for a total of 131 genes from 287 embryos with high quality image recordings, among which 86 genes have replicate embryos. Our results reveal that during early embryogenesis, divergence between sister lineages could be largely explained by a few genes. We predicted genes driving lineage divergence and explored their expression patterns in sister lineages. Moreover, we found that divisions leading to fate divergence are associated with a large number of genes being differentially expressed between sister lineages. Interestingly, we found that the developmental paths of lineages could be differentiated by a small set of genes. Therefore, our results support the notion that the cell fate patterns in C. elegans are achieved through stepwise binary decisions punctuated by cell divisions. Our predicted genes driving lineage divergence provide good starting points for future detailed characterization of their roles in the embryogenesis in this important model organism.     

Spatial and molecular cues for cell outgrowth during C. elegans uterine development

The Caenorhabditis elegans uterine seam cell (utse) is an H-shaped syncytium that connects the uterus to the body wall. Comprising nine nuclei that move outward in a bidirectional manner, this synctium undergoes remarkable shape change during development. Using cell ablation experiments, we show that three surrounding cell types affect utse development: the uterine toroids, the anchor cell and the sex myoblasts. The presence of the anchor cell (AC) nucleus within the utse is necessary for proper utse development and AC invasion genes fos-1, cdh-3, him-4, egl-43, zmp-1 and mig-10 promote utse cell outgrowth. Two types of uterine lumen epithelial cells, uterine toroid 1 (ut1) and uterine toroid 2 (ut2), mediate proper utse outgrowth and we show roles in utse development for two genes expressed in the uterine toroids: the RASEF ortholog rsef-1 and Trio/unc-73. The SM expressed gene unc-53/NAV regulates utse cell shape; ablation of sex myoblasts (SMs), which generate uterine and vulval muscles, cause defects in utse morphology. Our results clarify the nature of the interactions that exist between utse and surrounding tissue, identify new roles for genes involved in cell outgrowth, and present the utse as a new model system for understanding cell shape change and, putatively, diseases associated with cell shape change.     

Biophysical and biological meanings of healthspan from C. elegans cohort

Lifespan among individuals ranges widely in organisms from yeast to mammals, even in an isogenic cohort born in a nearly uniform environment. Needless to say, genetic and environmental factors are essential for aging and lifespan, but in addition, a third factor or the existence of a stochastic element must be reflected in aging and lifespan. An essential point is that lifespan or aging is an unpredictable phenomenon. The present study focuses on elucidating the biophysical and biological meanings of healthspan that latently indwells a stochastic nature. To perform this purpose, the nematode Caenorhabditis elegans served as a model animal. C. elegans fed a healthy food had an extended healthspan as compared to those fed a conventional diet. Then, utilizing this phenomenon, we clarified a mechanism of healthspan extension by measuring the single-worm ATP and estimating the ATP noise (or the variability of the ATP content) among individual worms and by quantitatively analyzing biodemographic data with the lifespan equation that was derived from a fluctuation theory.