A plethora of intercellular signals during Caenorhabditis elegans development.

Reproducible cell-cell interactions contribute to the invariance of Caenorhabditis elegans development and allow high resolution study of molecular mechanisms of intercellular signaling. A number of new cell interactions have been discovered in the past year. The power of nematode molecular genetics has been increased through several technical advances and the genome project, and these new approaches are now being successfully applied both to familiar and new signaling mechanisms.

A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae.

The relative uniformity of microtubule ultrastructure in almost all eukaryotic cells is thought to be a consequence of the conserved elements of tubulin sequence. In support of this idea, a mutation in a beta-tubulin gene of Drosophila melanogaster, occurring at a highly conserved position, produces U-shaped microtubules, suggesting a defect in either nucleation or packing during assembly (M. T. Fuller, J. H. Caulton, J. A. Hutchens, T. C. Kaufman, and E. C. Raff, J. Cell Biol. 104:385-394, 1987, and J. E. Rudolph, M. Kimble, H. D. Hoyle, M. A. Subler, and E. C. Raff, Mol. Cell. Biol. 7:2231-2242, 1987). Surprisingly, we find that introducing the same mutation into the sole beta-tubulin gene of Saccharomyces cerevisiae has virtually no consequences for microtubule structure or function in that organism.

Diversity among beta-tubulins: a carboxy-terminal domain of yeast beta-tubulin is not essential in vivo.

Sequences of genes for beta-tubulins from many different organisms demonstrate that they encode highly conserved proteins but that these proteins diverge considerably at their carboxyl termini. The patterns of interspecies conservation of this diversity suggest that it may have functional significance. We have taken advantage of the properties of Saccharomyces cerevisiae to test this hypothesis in vivo. The sole beta-tubulin gene of this species is one of the most divergent of all beta-tubulins and encodes 12 amino acids which extend past the end of most other beta-tubulin molecules. We have constructed strains in which the only beta-tubulin gene is an allele lacking these 12 codons. We show here that this carboxy-terminal extension is not essential. The absence of these 12 amino acids had no effect on a number of microtubule-dependent functions, such as mitotic and meiotic division and mating. It did confer dominant supersensitivity to a microtubule-depolymerizing drug.

A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

Caenorhabditis elegans HOM-C genes regulate the response of vulval precursor cells to inductive signal.

Factors that determine the competence of cells to respond to extracellular cues are not well understood. We demonstrate that two HOM-C transcription factors have antagonistic roles in determining the ability of Caenorhabditis elegans vulval precursor cells (VPCs) to respond to the inductive signal from the anchor cell of the somatic gonad. The vulva develops from a subset of ectodermal vulval precursor cells distributed along the anteroposterior axis. Vulval patterning depends on both a localized inductive signal, the LIN-3 growth factor, and lateral signaling between induced VPCs. One HOM-C gene, the Antp homolog mab-5, is expressed in the posterior two VPCs. By examining the response of single VPCs to controlled doses of inductive signal in wild-type and in mab-5 mutant animals, we demonstrate that mab-5 reduces the competence of these two cells. Moreover, a gain-of-function allele of mab-5 that causes ectopic expression of MAB-5 in all VPCs reduces the sensitivity of all VPCs to inductive signal. Additional experiments suggest that another HOM-C gene, the Scr homolog lin-39, is required for VPCs in wild-type animals to respond to activation of inductive signal. Genetic epistasis tests are consistent with models in which lin-39 acts downstream of the RAS pathway to regulate response to inductive signal. We propose that the spatial pattern of HOM-C gene expression may enhance the precision of vulval fate patterning.

Different levels of the C. elegans growth factor LIN-3 promote distinct vulval precursor fates.

An invariant spatial pattern of three cell fates (3 degrees-3 degrees-2 degrees-1 degree-2 degrees-3 degrees) is generated from a field of multipotent precursor cells during C. elegans vulval development. We demonstrate that the epidermal growth factor-like domain of the LIN-3 protein can induce either of two distinct vulval cell fates: a high dose of LIN-3 induces a 1 degree fate; a lower dose of LIN-3 induces a 2 degrees fate. A high dose of LIN-3 can also induce adjacent vulval precursor cells to assume 1 degree fates; thus, high levels of LIN-3 can override the lateral signaling that normally inhibits formation of adjacent 1 degree fates. We propose that the invariant pattern of vulval cell fates is generated by a graded distribution of LIN-3 that promotes different vulval fates according to local concentration and by a lateral signal that reinforces this initial bias.

LET-23-mediated signal transduction during Caenorhabditis elegans development.

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming alpha-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the "vulvaless" phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the "multivulva" phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development--larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways.