The SEL-12 presenilin mediates induction of the Caenorhabditis elegans uterine pi cell fate

During Caenorhabditis elegans hermaphrodite development, the anchor cell induces the vulva and the uterine pi cells whose daughters connect to the vulva, thereby organizing the uterine-vulval connection. Both the initial selection of a single anchor cell during the anchor cell vs. ventral uterine precursor cell decision and the subsequent induction of the pi cell fate by the anchor cell are mediated by the lin-12 gene. Members of the presenilin gene family can cause early onset Alzheimer's disease when mutated and are also required for LIN-12/Notch signaling during development. We have shown that, in C. elegans, mutation of the sel-12-encoded presenilin results in pi cell induction defects. By contrast, other lin-12-mediated cell fate decisions occur normally in sel-12 mutants due to the redundant function of a second C. elegans presenilin called HOP-1. We found that the sel-12 egg-laying defect was partially rescued by expression of the sel-12 gene in the pi cells. sel-12-mediated pi cell fate specification provides a useful system for the analysis of presenilin function at single cell resolution.     

C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.     

EGF signaling overcomes a uterine cell death associated with temporal mis-coordination of organogenesis within the C. elegans egg-laying apparatus

We isolated cog-3(ku212) as a C. elegans egg-laying defective mutant that is associated with a connection-of-gonad defective phenotype. cog-3(ku212) mutants appear to have no connection between the vulval and the uterine lumens at the appropriate stage because the uterine lumen develops with a temporal delay relative to the vulva and, thus, is not present when the connection normally forms. The lack of temporal synchronization between the vulva and the uterus is not due to precocious or accelerated vulval development. Instead, global gonadogenesis is mildly delayed relative to development of extra-gonadal tissue. cog-3(ku212) mutants also have a specific uterine fate defect. Normally, four cells of the uterine pi lineage respond via their LET-23 epidermal growth factor-like receptors to a vulval-derived LIN-3 EGF signal and adopt the uterine vulval 1 (uv1) fate. In cog-3(ku212) mutants, these four pi progeny cells are set aside as a pre-uv1 population but undergo necrosis prior to full differentiation. A gain-of-function mutation in LET-23 EGF receptor and ectopic expression of LIN-3 EGF within the proper temporal constraints can rescue the uv1 defect, suggesting that a signaling defect, perhaps due to the temporal delay, is at fault. In support of this model, we demonstrate that lack of vulval-uterine coordination due to precocious vulval development also leads to uv1 cell differentiation defects.     

Cell cycle-dependent sequencing of cell fate decisions in Caenorhabditis elegans vulva precursor cells

In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1 degrees ) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1 degrees cell each express secondary (2 degrees ) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non-vulval tertiary (3 degrees ) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two developmental decisions separable by cell cycle phase: lin-12 must act prior to the end of VPC S phase to influence a 1 degrees versus 2 degrees cell fate choice, but must act after VPC S phase to influence a 3 degrees versus 2 degrees cell fate choice. Coupling developmental decisions to cell cycle transitions may provide a mechanism for prioritizing or ordering choices of cell fates for multipotential cells.     

The C. elegans LIM homeobox gene lin-11 specifies multiple cell fates during vulval development

LIM homeobox family members regulate a variety of cell fate choices during animal development. In C. elegans, mutations in the LIM homeobox gene lin-11 have previously been shown to alter the cell division pattern of a subset of the 2 degrees lineage vulval cells. We demonstrate multiple functions of lin-11 during vulval development. We examined the fate of vulval cells in lin-11 mutant animals using five cellular markers and found that lin-11 is necessary for the patterning of both 1 degrees and 2 degrees lineage cells. In the absence of lin-11 function, vulval cells fail to acquire correct identity and inappropriately fuse with each other. The expression pattern of lin-11 reveals dynamic changes during development. Using a temporally controlled overexpression system, we show that lin-11 is initially required in vulval cells for establishing the correct invagination pattern. This process involves asymmetric expression of lin-11 in the 2 degrees lineage cells. Using a conditional RNAi approach, we show that lin-11 regulates vulval morphogenesis. Finally, we show that LDB-1, a NLI/Ldb1/CLIM2 family member, interacts physically with LIN-11, and is necessary for vulval morphogenesis. Together, these findings demonstrate that temporal regulation of lin-11 is crucial for the wild-type vulval patterning.     

Cryptic quantitative evolution of the vulva intercellular signaling network in Caenorhabditis

BACKGROUND: The Caenorhabditis vulva is formed from a row of Pn.p precursor cells, which adopt a spatial cell-fate pattern-3 degrees 3 degrees 2 degrees 1 degrees 2 degrees 3 degrees -centered on the gonadal anchor cell. This pattern is robustly specified by an intercellular signaling network including EGF/Ras induction from the anchor cell and Delta/Notch signaling between the precursor cells. It is unknown how the roles and quantitative contributions of these signaling pathways have evolved in closely related Caenorhabditis species. RESULTS: Cryptic evolution in the network is uncovered by quantification of cell-fate-pattern frequencies obtained after displacement of the system out of its normal range, either by anchor-cell ablations or through LIN-3/EGF overexpression. Silent evolution in the Caenorhabditis genus covers a large neutral space of cell-fate patterns. Direct induction of the 1 degrees fate as in C. elegans appeared within the genus. C. briggsae displays a graded induction of 1 degrees and 2 degrees fates, with 1 degrees fate induction requiring a longer time than in C. elegans, and a reduced lateral inhibition of adjacent 1 degrees fates. C. remanei displays a strong lateral induction of 2 degrees fates relative to vulval-fate activation in the central cell. This evolution in cell-fate pattern space can be experimentally reconstituted by mild variations of Ras, Wnt, and Notch pathway activities in C. elegans and C. briggsae. CONCLUSIONS: Quantitative evolution in the roles of graded induction by LIN-3/EGF and Notch signaling is demonstrated for the Caenorhabditis vulva signaling network. This evolutionary system biology approach provides a quantitative view of the variational properties of this biological system.     

Regulation of anchor cell invasion and uterine cell fates by the egl-43 Evi-1 proto-oncogene in Caenorhabditis elegans

Cell invasion is a tightly controlled process occurring during development and tumor progression. The nematode Caenorhabditis elegans serves as a genetic model to study cell invasion during normal development. In the third larval stage, the anchor cell in the somatic gonad first induces and then invades the adjacent epidermal vulval precursor cells. The homolog of the Evi-1 oncogene, egl-43, is necessary for basement membrane destruction and anchor cell invasion. egl-43 is part of a regulatory network mediating cell invasion downstream of the fos-1 proto-oncogene. In addition, EGL-43 is required to specify the cell fates of ventral uterus cells downstream of or in parallel with LIN-12 NOTCH. Comparison with mammalian Evi-1 suggests a conserved pathway controlling cell invasion and cell fate specification.     

lin-17/Frizzled and lin-18 regulate POP-1/TCF-1 localization and cell type specification during C. elegans vulval development

The Caenorhabditis elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. We show that most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. We demonstrate that lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva.     

Tissue-specific regulation of the LIM homeobox gene lin-11 during development of the Caenorhabditis elegans egg-laying system

The egg-laying system of Caenorhabditis elegans hermaphrodites requires development of the vulva and its precise connection with the uterus. This process is regulated by LET-23-mediated epidermal growth factor signaling and LIN-12-mediated lateral signaling pathways. Among the nuclear factors that act downstream of these pathways, the LIM homeobox gene lin-11 plays a major role. lin-11 mutant animals are egg-laying defective because of the abnormalities in vulval lineage and uterine seam-cell formation. However, the mechanisms providing specificity to lin-11 function are not understood. Here, we examine the regulation of lin-11 during development of the egg-laying system. Our results demonstrate that the tissue-specific expression of lin-11 is controlled by two distinct regulatory elements that function as independent modules and together specify a wild-type egg-laying system. A uterine pi lineage module depends on the LIN-12/Notch signaling, while a vulval module depends on the LIN-17-mediated Wnt signaling. These results provide a unique example of the tissue-specific regulation of a LIM homeobox gene by two evolutionarily conserved signaling pathways. Finally, we provide evidence that the regulation of lin-11 by LIN-12/Notch signaling is directly mediated by the Su(H)/CBF1 family member LAG-1.     

Protruding vulva mutants identify novel loci and Wnt signaling factors that function during Caenorhabditis elegans vulva development

The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.