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.     

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.     

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.     

N-ethylmaleimide sensitive factor is required for fusion of the C. elegans uterine anchor cell

The fusion of the Caenorhabditis elegans uterine anchor cell (AC) with the uterine-seam cell (utse) is an excellent model system for studying cell-cell fusion, which is essential to animal development. We obtained an egg-laying defective (Egl) mutant in which the AC fails to fuse with the utse. This defect is highly specific: other aspects of utse development and other cell fusions appear to occur normally. We find that defect is due to a missense mutation in the nsf-1 gene, which encodes N-ethylmaleimide-sensitive factor (NSF), an intracellular membrane fusion factor. There are two NSF-1 isoforms, which are expressed in distinct tissues through two separate promoters. NSF-1L is expressed in the uterus, including the AC. We find that nsf-1 is required cell-autonomously in the AC for its fusion with the utse. Our results establish AC fusion as a paradigm for studying cell fusion at single cell resolution and demonstrate that the NSF ATPase is a key player in this process.     

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.     

Cell autonomy of lin-12 function in a cell fate decision in C. elegans

The lin-12 gene of C. elegans encodes a predicted transmembrane protein that controls a decision by two cells, Z1.ppp and Z4.aaa, between the anchor cell (AC) and ventral uterine precursor cell (VU) fates. We performed laser ablation experiments to demonstrate that specification of the VU fate of Z1.ppp or Z4.aaa depends on an "AC-to-VU" signal from the presumptive AC. We generated genetic mosaics in which defined cells lacked lin-12 activity. By correlating the fates of Z1.ppp and Z4.aaa with the lin-12 genotype of nearly every cell in these mosaics, we conclude that lin-12 function is VU cell autonomous. We present a model in which lin-12 functions in the receiving mechanism for the "AC-to-VU" signal leading to the specification of the AC and VU fates of Z1.ppp and Z4.aaa.     

Connection of vulval and uterine epithelia in Caenorhabditis elegans

In the Caenorhabditis elegans hermaphrodite, the establishment of the egg-laying system requires the connection of two epithelial tubes: the uterus of the gonad and the vulva in the underlying ectoderm. A specialized uterine cell, the anchor cell (AC), plays a central role in specifying the fates of the uterine and vulval precursor cells via the EGF-Ras-MAP kinase and the Notch/Delta signaling pathways. This central and common inducing source ensures that the two sets of cells are in register and it specifies the cell types that build the T-shaped connection between uterus and vulva. On either side, progeny of the induced cells form lumen structures and undergo stereotyped cell-to-cell fusion, thereby building epithelial tubes. Finally, the anchor cell fuses with a uterine syncytium and thus leaves only a thin cellular process between the lumen of the uterus and the vulva. In the adult, the fertilized eggs exit the lumen of the uterus through the vulva. This relatively simple developmental process serves as a model to study the biology of cells during organogenesis, such as intercellular signaling, cell polarity, invasion of basal laminae and epithelia, cell recognition and cell fusion. The anchor cell is a particularly interesting cell as it coordinates the development of its neighboring cells by using different signaling pathways at different times.     

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.     

mig-5/Dsh controls cell fate determination and cell migration in C. elegans

Cell fate determination and cell migration are two essential events in the development of an organism. We identify mig-5, a Dishevelled family member, as a gene that regulates several cell fate decisions and cell migrations that are important during C. elegans embryonic and larval development. In offspring from mig-5 mutants, cell migrations are defective during hypodermal morphogenesis, QL neuroblast migration, and the gonad arm migration led by the distal tip cells (DTCs). In addition to abnormal migration, DTC fate is affected, resulting in either an absent or an extra DTC. The cell fates of the anchor cell in hermaphrodites and the linker cells in the male gonad are also defective, often resulting in the cells adopting the fates of their sister lineage. Moreover, 2 degrees vulval precursor cells occasionally adopt the 3 degrees vulval cell fate, resulting in a deformed vulva, and the P12 hypodermal precursor often differentiates into a second P11 cell. These defects demonstrate that MIG-5 is essential in determining proper cell fate and cell migration throughout C. elegans development.     

The Let-60 Locus Controls the Switch between Vulval and Nonvulval Cell Fates in Caenorhabditis Elegans

During induction of the Caenorhabditis elegans hermaphrodite vulva by the anchor cell of the gonad, six multipotent vulval precursor cells (VPCs) have two distinct fates: three VPCs generate the vulva and the other three VPCs generate nonspecialized hypodermis. Genes that control the fates of the VPCs in response to the anchor cell signal are defined by mutations that cause all six VPCs to generate vulval tissue (Multivulva or Muv) or that cause all six VPCs to generate hypodermis (Vulvaless or Vul). Seven dominant Vul mutations were isolated as dominant suppressors of a lin-15 Muv mutation. These mutations are dominant alleles of the gene let-60, previously identified only by recessive lethal mutations. Our genetic studies of these dominant Vul recessive lethal mutations, recessive lethal mutations, intragenic revertants of the dominant Vul mutations, and the closely mapping semi-dominant multivulva lin-34 mutations suggest that: (1) loss-of-function mutations of let-60 are recessive lethal at a larval stage, but they also cause a Vul phenotype if the lethality is rescued maternally by a lin-34 gain-of-function mutation. (2) The dominant Vul alleles of let-60 are dominant negative mutations whose gene products compete with wild-type activity. (3) lin-34 semidominant Muv alleles are either gain-of-function mutations of let-60 or gain-of-function mutations of an intimately related gene that elevates let-60 activity. We propose that let-60 activity controls VPC fates. In a wild-type animal, reception by a VPC of inductive signal activates let-60, and it generates into a vulval cell type; in absence of inductive signal, let-60 activity is low and the VPC generates hypodermal cells. Our genetic interaction studies suggest that let-60 acts downstream of let-23 and lin-15 and upstream of lin-1 and lin-12 in the genetic pathway specifying the switch between vulval and nonvulval cell types.     

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.