The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase

The Caenorhabditis elegans gene laf-1 is critical for both embryonic development and sex determination. Laf-1 is thought to promote male cell fates by negatively regulating expression of tra-2 in both hermaphrodites and males. We cloned laf-1 and established that it encodes a putative DEAD-box RNA helicase related to Saccharomyces cerevisiae Ded1p and Drosophila Vasa. Three sequenced laf-1 mutations are missense alleles affecting a small region of the protein in or near helicase motif III. We demonstrate that the phenotypes resulting from laf-1 mutations are due to loss or reduction of laf-1 function, and that both laf-1 and a related helicase vbh-1 function in germline sex determination. Laf-1 mRNA is expressed in both males and hermaphrodites and in both the germline and soma of hermaphrodites. It is expressed at all developmental stages and is most abundant in embryos. LAF-1 is predominantly, if not exclusively, cytoplasmic and colocalizes with PGL-1 in P granules of germline precursor cells. Previous results suggest that laf-1 functions to negatively regulate expression of the sex determination protein TRA-2, and we find that the abundance of TRA-2 is modestly elevated in laf-1/+ females. We discuss potential functions of LAF-1 as a helicase and its roles in sex determination.     

EGF Signal Propagation during C. elegans Vulval Development Mediated by ROM-1 Rhomboid

During Caenorhabditis elegans vulval development, the anchor cell (AC) in the somatic gonad secretes an epidermal growth factor (EGF) to activate the EGF receptor (EGFR) signaling pathway in the adjacent vulval precursor cells (VPCs). The inductive AC signal specifies the vulval fates of the three proximal VPCs P5.p, P6.p, and P7.p. The C. elegans Rhomboid homolog ROM-1 increases the range of EGF, allowing the inductive signal to reach the distal VPCs P3.p, P4.p and P8.p, which are further away from the AC. Surprisingly, ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. This observation led to the discovery of an AC–independent activity of EGF in the VPCs that promotes vulval cell fate specification and depends on ROM-1. Of the two previously reported EGF splice variants, the longer one requires ROM-1 for its activity, while the shorter form acts independently of ROM-1. We present a model in which ROM-1 relays the inductive AC signal from the proximal to the distal VPCs by allowing the secretion of the LIN-3L splice variant. These results indicate that, in spite of their structural diversity, Rhomboid proteins play a conserved role in activating EGFR signaling in C. elegans, Drosophila, and possibly also in mammals.     

EGF signal propagation during C. elegans vulval development mediated by ROM-1 rhomboid

During Caenorhabditis elegans vulval development, the anchor cell (AC) in the somatic gonad secretes an epidermal growth factor (EGF) to activate the EGF receptor (EGFR) signaling pathway in the adjacent vulval precursor cells (VPCs). The inductive AC signal specifies the vulval fates of the three proximal VPCs P5.p, P6.p, and P7.p. The C. elegans Rhomboid homolog ROM-1 increases the range of EGF, allowing the inductive signal to reach the distal VPCs P3.p, P4.p and P8.p, which are further away from the AC. Surprisingly, ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. This observation led to the discovery of an AC–independent activity of EGF in the VPCs that promotes vulval cell fate specification and depends on ROM-1. Of the two previously reported EGF splice variants, the longer one requires ROM-1 for its activity, while the shorter form acts independently of ROM-1. We present a model in which ROM-1 relays the inductive AC signal from the proximal to the distal VPCs by allowing the secretion of the LIN-3L splice variant. These results indicate that, in spite of their structural diversity, Rhomboid proteins play a conserved role in activating EGFR signaling in C. elegans, Drosophila, and possibly also in mammals.     

Predictive modeling of signaling crosstalk during C. elegans vulval development

Caenorhabditis elegans vulval development provides an important paradigm for studying the process of cell fate determination and pattern formation during animal development. Although many genes controlling vulval cell fate specification have been identified, how they orchestrate themselves to generate a robust and invariant pattern of cell fates is not yet completely understood. Here, we have developed a dynamic computational model incorporating the current mechanistic understanding of gene interactions during this patterning process. A key feature of our model is the inclusion of multiple modes of crosstalk between the epidermal growth factor receptor (EGFR) and LIN-12/Notch signaling pathways, which together determine the fates of the six vulval precursor cells (VPCs). Computational analysis, using the model-checking technique, provides new biological insights into the regulatory network governing VPC fate specification and predicts novel negative feedback loops. In addition, our analysis shows that most mutations affecting vulval development lead to stable fate patterns in spite of variations in synchronicity between VPCs. Computational searches for the basis of this robustness show that a sequential activation of the EGFR-mediated inductive signaling and LIN-12 / Notch-mediated lateral signaling pathways is key to achieve a stable cell fate pattern. We demonstrate experimentally a time-delay between the activation of the inductive and lateral signaling pathways in wild-type animals and the loss of sequential signaling in mutants showing unstable fate patterns; thus, validating two key predictions provided by our modeling work. The insights gained by our modeling study further substantiate the usefulness of executing and analyzing mechanistic models to investigate complex biological behaviors.     

High sensitivity of C. elegans vulval precursor cells to the dose of posterior Wnts

Cell competence is a key developmental property. The Caenorhabditis elegans vulval competence group consists of P(3–8).p, six cells aligned along the antero-posterior axis in a wide central body region. The six cells are not equal in their competence: 1) P3.p quits the competence group in half of the individuals; 2) the posterior cells P7.p and P8.p are less competent than central vulval precursor cells. Competence to adopt a vulval fate is controlled by expression of the HOM-C gene lin-39, and maintained through Wnt signals that are secreted from the tail in a long-range gradient. Here we quantify the LIN-39 protein profile in vulval precursor cells of early L2 stage larvae, prior to P3.p fusion and inductive signaling. We show that LIN-39 levels are very low in P3.p and P4.p, peak in P5.p and progressively decrease until P8.p. This unexpectedly centered profile arises independently from the gonad. Posterior Wnt signaling reduces LIN-39 level in the posterior cells by activating the next-posterior HOM-C gene, mab-5. On the anterior side, P3.p and P4.p competence and division are sensitive to the already low LIN-39 and Wnt doses; most dramatically, each of the cwn-1/Wnt and egl-20/Wnt genes show haplo-insufficience for P3.p fate. In contrast to previous results, we find that these Wnts maintain P3.p and P4.p competence without affecting their LIN-39 level. The centered vulval competence profile is thus under the control of the posterior Wnts and of cross-regulation of three HOM-C genes and prepatterns the later induction of vulval fates.     

C. elegans PlexinA PLX-1 mediates a cell contact-dependent stop signal in vulval precursor cells

PLX-1 is a PlexinA transmembrane protein in Caenorhabditis elegans, and the transmembrane-type semaphorin, SMP-1, is a ligand for PLX-1. The SMP-1/PLX-1 system has been shown to be necessary for proper epidermal morphogenesis in the male tail and seam cells. Here, we show that the SMP-1/PLX-1 system also regulates vulval morphogenesis. In plx-1 and smp-1 mutants, hermaphrodites sometimes exhibit a protruding vulva or multiple vulva-like protrusions. Throughout the vulval development of plx-1 and smp-1 mutants, the arrangement of vulval cells is often disrupted. In the initial step of vulval morphogenesis, vulval precursor cells (VPCs) are generated normally but are subsequently arranged abnormally in mutants. Continuous observation revealed that plx-1 VPC fails to terminate longitudinal extension after making contact with neighbor VPCs. The arrangement defects of VPCs in plx-1 and smp-1 mutants are rescued by expressing the respective cDNA in VPCs. plx-1::egfp and smp-1::egfp transgenes are both expressed in all vulval cells, including VPCs, throughout vulval development. We propose that the SMP-1/PLX-1 system is responsible for a cell contact-mediated stop signal for VPC extension. Analyses using cell fate-specific markers showed that the arrangement defects of VPCs also affect cell fate specification and cell lineages, but in a relatively small fraction of plx-1 mutants.     

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.     

Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development

The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TORC1 proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. We found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. We found that ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, our genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from our data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development.     

Wnt and EGF pathways act together to induce C. elegans male hook development

Comparative studies of vulva development between Caenorhabditis elegans and other nematode species have provided some insight into the evolution of patterning networks. However, molecular genetic details are available only in C. elegans and Pristionchus pacificus. To extend our knowledge on the evolution of patterning networks, we studied the C. elegans male hook competence group (HCG), an equivalence group that has similar developmental origins to the vulval precursor cells (VPCs), which generate the vulva in the hermaphrodite. Similar to VPC fate specification, each HCG cell adopts one of three fates (1°, 2°, 3°), and 2° HCG fate specification is mediated by LIN-12/Notch. We show that 2° HCG specification depends on the presence of a cell with the 1° fate. We also provide evidence that Wnt signaling via the Frizzled-like Wnt receptor LIN-17 acts to specify the 1° and 2° HCG fate. A requirement for EGF signaling during 1° fate specification is seen only when LIN-17 activity is compromised. In addition, activation of the EGF pathway decreases dependence on LIN-17 and causes ectopic hook development. Our results suggest that WNT plays a more significant role than EGF signaling in specifying HCG fates, whereas in VPC specification EGF signaling is the major inductive signal. Nonetheless, the overall logic is similar in the VPCs and the HCG: EGF and/or WNT induce a 1° lineage, and LIN-12/NOTCH induces a 2° lineage. Wnt signaling is also required for execution of the 1° and 2° HCG lineages. lin-17 and bar-1/β-catenin are preferentially expressed in the presumptive 1° cell P11.p. The dynamic subcellular localization of BAR-1-GFP in P11.p is concordant with the timing of HCG fate determination.     

HPL-2/HP1 Prevents Inappropriate Vulval Induction in Caenorhabditis elegans by Acting in Both HYP7 and Vulval Precursor Cells

A current model for Caenorhabditis elegans vulval cell fate specification is that SynMuv genes act redundantly in the hyp7 hypodermal syncytium to repress the LIN-3/EGF inducer and prevent ectopic vulval induction of vulva precursor cells (VPCs). Here we show that the SynMuv gene hpl-2/HP1 has an additional function in VPCs, where it may act through target genes including LIN-39/Hox.     

PI3K/Akt pathway regulates retinoic acid‐induced Hox gene expression in F9 cells

Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA‐induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA‐induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA‐induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time‐course gene expression profiles for all 39 Hox genes located in four different clusters—Hoxa, Hoxb, Hoxc, and Hoxd—were analyzed. Collinear expression of Hoxa and ‐b cluster genes was initiated earlier than that of the ‐c and ‐d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA‐induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.