Loss-of-function (lj) mutations in any of the signaling proteins in the LET-23 RTK pathway result in fewer than three VPCs undergoing. The let-23(sy97) mutant animals are defective in all let-23-mediated phenotypes except the hermaphrodite ovulation [ 62 ]. One is to regulate the basal activity of signaling in the absence of the ligand.
Waterfield (1993).: Assembly of signaling complexes by receptor tyrosine kinases. 1993).: SH2 domains recognize specific phosphopeptide sequences. Sternberg (1994).: The lin-3/let-23 pathway mediates inductive signaling during male spicule development in Caenorhabditis elegans. Sternberg (1991).: Multiple intercellular signaling systems control the development of C. 1990).: The let-23 gene required for vulval induction in Caenorhabditis elegans encodes a tyrosine kinase of the EGF receptor subfamily.
Horvitz (1990): The Caenorhabditis elegans ras let-60 gene acts as a switch in the vulva induction pathway. Horvitz (1995): The Caenorhabditis elegans mek-2 gene is required for vulva induction and encodes a MEK protein kinase-like protein. Han (1998).: SUR-8, a conserved Ras-binding protein with leucine-rich repeats, positively regulates Ras-mediated signaling in C. 1998). signal transduction during oogenesis and vulvar development.
Horvitz (1989).: The combined action of two intercellular signaling pathways specifies the fate of three cells during vulval induction in C. Alternatively, gonad-ablated let-60(gf) animals may be subject to a form of negative regulation in the vulva, namely independent of SOS-1 and is abolished in a let-60(gf); let-341(null) background perhaps due to the presence of the ligand. Reduction-of-function (rf) mutations in any of the signaling proteins in the LET-23 RTK pathway result in fewer than three VPCs undergoing vulval differentiation.
In the most severe cases, disruption of LET-23-mediated signaling results in a complete failure to generate vulval tissue (non-vulva or Vul phenotype). The let-23 gene required for vulval induction in Caenorhabditis elegans encodes a tyrosine kinase of the EGF receptor subfamily.
The extent of vulval induction was determined by examining vulvar anatomy at the early to mid-L4 stage of development under Nomarski optics using a Plan 100 objective 15 . Ablations of the AC were performed with a laser microbeam as described by Sulston and White (1980). . The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis.
Basolateral localization of the Caenorhabditis elegans epidermal growth factor receptor in epithelial cells by the PDZ protein LIN-10. Changes in cell lineage after laser ablation of cells in the somatic gonad of Caenorhabditis elegans. Here, we use the development of uterine-vulval connection in Caenorhabditis elegans as a model system to study reciprocal signaling.
LIN-3, an EGF family protein, is first produced by the anchor cell of the gonad to induce vulvar precursor cells to generate vulvar tissue. Using genetic and cell biological analyses, we found that uv1 uterine cell specification is dependent on EGF signaling from vulval lineage 1 o cells to a subset of ventral uterine gonadal cells. We also find that EGL-38, a member of the PAX protein family, is required for lin-3 transcription in the vulva, but not anchor cell.
A let-23 mutation conferring ligand-independent activity bypasses the requirement for EGL-38 for uv1 cell fate specification.
Background
Vulva cells form the invagination, with 1 o of the vulvar cells at the tip of the invagination (vulF) attached to the ventral most uterine cells (n cell progeny). All then cell progeny (except four that make direct contact with vulF and become uv1 cells) fuse with the AC to form a thin laminar. This fusion and differentiation of the utse moves the bulky AC and additional n-cell progeny out of the way, allowing the junction to form.
In the meantime, the 1° vulval cells separate anterio-posteriorly and left-right to create a hole for eggs to pass through. Two series of observations led us to conclude that proper uterine morphogenesis might depend on signals from the vulva to the uterus.
Results
To determine whether this expression might have a functional significance, we examined the expression pattern of let-23::GFP during the period corresponding to vulval expression of lin-3::lacZ. We detected the expression of let-23::GFP in the uv1 cells, the most ventral uterine cells, suggesting a possible signaling from the vulva to the uterus (Figure 1B,C). Because let-23::GFP expression in uv1 cells peaks later than the proposed induction, its expression could be upregulated by lin-3.
In vulvalless let-23(sy97) hermaphrodites, in which all six VPCs adopt the non-vulval 3° cell fate, the putative uv1 cells adopt the utse cell fate instead (Table 1). As discussed above, although vulvaless let-23 hermaphrodites fail to specify uv1 cell fate, this result is not sufficient to implicate the LIN-3/LET-23 pathway in vulva-to-uv1 signaling, as laser ablation of the VPCs also results in UV1-defective animals. We therefore examined animals harboring partial loss-of-function mutations in genes that function in the LIN-3/LET-23 signaling pathway that mediates vulva induction.
Another way to investigate LET-23 pathway function in uv1 specification is to alleviate the vulval induction defect by mutating negative regulators. SLI-1, a homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation in C. The high penetrance of uvl defect observed in let-23; gap-1 suggests that LET-23-mediated signaling is less regulated by GAP-1 in the presumptive uv1 cells than in the VPCs.
Let-23(sa62) thus circumvents the requirement for lin-3 expression in the vulva to mediate UVL specification.
Discussion
We also find that lin-3 is expressed in vulF cells at the right time and place to be the ligand for this induction; Furthermore, uv1 cell fate specification is impaired in animals carrying mutations in let-23 or in the downstream components of LET-23-. A lin-3::lacZ transgene is expressed in vulval cells of the 1 ° lineage at the early L4 stage, as well as in AC as previously reported [ 8 ]. We cannot use the same experiments to address the question of whether LIN-3 protein in the AC or indin 1 o vulval tissue is used for vulval 1 o morphogenesis as suggested by observations from egl-38 mutants due to physical invasion of the AC.
Proper vulval morphogenesis would then allow lin-3 to be expressed in the vulva to regulate uv1 specification. In addition to being necessary for lin-3 expression in vulF cells, EGL-38 also regulates the expression of other genes in the same cells (M. Wang, C. Chang, and P. Sternberg, unpublished observations). EGL-38 acts upstream of lin-3 as a positive regulator of its expression in the vulval lineage.
We propose that this signaling event triggers the expression of LIN-3 in the vulval cells, which in turn specifies the UVL cell fate involved in the intimate cells. It is possible that LIN-3 expression in the vulva also plays an autocrine amplification role on which proper vulva development depends. We show that lin-3 expression in the 10 vulval cells, which is positively regulated by EGL-38, is required for UVL cell specification.
A BamHI-Saci fragment containing the last 5 kb of a 3'-lin-3 genomic sequence in pMob KS with a lacZ cassette fused into the first cytoplasmic exon of lin-3 was excised from plasmid pRH56 [8] and end-filled at the sad side.
Acknowledgements
A point mutation in the extracellular domain activates LET-23, the C. Beitel G, ClarkS, Horvitz HR: The Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction. Jacobs D, Beitel GJ, Clark SG, Horvitz HR, Kornfeld K: Gain-of-function mutations in the Caenorhabditis elegans lin-1 ETS gene identify a C-terminal regulatory domain phosphorylated by ERK MAP kinase. Nomarski photomicrographs illustrating the cellular defects in the uterine-vulval junctions of mid-L4let-23; sli-1 mutants.
Expression of a lin-3::lacZ reporter construct is abrogated in the vulF cells of the egl-38 animal. B) ~-galactosidase expression cannot be detected from the lin-3::lacZ reporter construct in the vulF descendants (arrows) of P6.p from a mid-14th instar egl-38 animal. Staining directly above the vulF cells represents expression in the partially defocused anchor cell.
Fl cross progeny express ~-galactosidase from the lin-3::lacZ reporter construct in the vulF progeny (arrows), whereas their homozygous parents did not. EGL-38 acts directly or indirectly upstream of lin-3 as a positive regulator in the vulF cells. Expression of lin-3 in the vulF cells will then specify the uvl cell fate through a RAS-RAF-dependent signaling pathway in a cell non-autonomous manner.
Only seven out of twenty animals have been followed for expression of egl-17::GFP in the 1° and 2° vulval lineages.
The RING finger/B-Box factor TAM-1 and a retinoblastoma-like protein LIN-35
In the first model, a weak cis -silencing element may be present in each member of the tandem repeat. The size of the tam-1 (cc567) effect on the myo-3:: gfp transgene was estimated by optical quantitation of fluorescence to be ∼18-fold ( Fig. 1C ). The change in expression of the endogenous myo-3 locus in a tam-1 background was first tested genetically (Table 1).
Sequence analysis of this fragment predicted a single coding region (C. elegans Sequencing Consortium 1998). Class B synMuv family genes have been shown to play a role in modulating the activity of the EGFR-RAS pathway during vulvar specification. At some level, the effects of class B synMuv genes are likely to involve changes in the acetylation state of histones in chromatin.
The directionality of the class B synMuv effect should be considered when evaluating the following: transgene expression is reduced in the absence of LIN-35 or T AM-1 function. The multicopy tandem array nature of the ccls4251 transgene was confirmed by Southern blot analysis (showing ∼100 copies of pSAK2 and pSAK4 each; data not shown). Isolation and genetic characterization of cell line mutants of the nematode Caenorhabditis elegans.
Mutations with dominant effects in the behavior and morphology of the nematode Caenorhabditis elegans.
