C. elegans mutants have been identified in which certain of the six vulval precursor cells adopt fates normally expressed by other vulval precursor cells. These mutants define genes that appear to function in the response to an intercellular signal that induces vulval development. The multivulva (Muv) phenotype of one such mutant, CB1322, results from an interaction between two unlinked mutations, lin-8 and lin-9. In this paper, eighteen new mutations are identified that are alleles of eight genes, that interact with either lin-8 or lin-9 to generate a Muv phenotype. None of these 20 mutations alone causes any vulval cell lineage defects. The 'silent Muv' mutations fall into two classes: hermaphrodites carrying a mutation of each class are Muv, while hermaphrodites carrying two mutations of the same class have a wild-type vulval phenotype. These results indicate that the Muv phenotype of these mutants results from defects in two functionally-redundant pathways, thereby demonstrating that redundancy can occur at the level of gene pathways as well as at the level of gene families (Ferguson, 1989).
The C. elegans gene lin-9 functions in an Rb-related pathway that acts antagonistically to a receptor tyrosine kinase/Ras signal transduction pathway controlling vulval induction. lin-9 is also required for the development of the sheath cells in the hermaphrodite gonad and for the development of the male spicule, rays and gonad. lin-9 is transcribed as two alternatively spliced 2.4kb messages, which use two distinct polyadenylation sites and are SL1 trans-spliced. The conceptual translation of lin-9 cDNA sequences predicts proteins of 642 and 644 amino acids with a significant similarity to predicted Drosophila and vertebrate proteins. It is suggested that lin-9 is the founding member of a new protein family that functions in Rb-related pathways in many species (Beitel, 2000).
In C. elegans, the formation of the hermaphrodite vulva is induced by an RTK/Ras signaling pathway. The vulva is generated from six multipotent ventral ectodermal blast cells, P3.p-P8.p. Each of these six P(3-8).p cells can potentially adopt either the 1° vulval cell fate, the 2° vulval cell fate, or the 3° nonvulval cell fate. During wild-type development, a signal from the gonadal anchor cell induces the nearest P(3-8).p cell, P6.p, to adopt the 1° fate and the adjacent P5.p and P7.p cells to adopt the 2° fate. The cells furthest from the anchor cell, P3.p, P4.p, and P8.p, adopt the uninduced 3° fate. Vulval induction acts through a signaling pathway, which includes an EGF-like ligand; a receptor tyrosine kinase, Ras and MAP kinase, to regulate the activities of the ETS transcription factor LIN-1 and the winged-helix transcription factor LIN-31 (Lu, 1998 and references).
Vulval induction is negatively regulated by the synthetic multivulva (synMuv) genes. Loss-of-function mutations in these genes result in a multivulva (Muv) phenotype as a consequence of the expression of vulval cell fates by the P3.p, P4.p, and P8.p cells. The Muv phenotype of these mutants requires mutations in two genes. Specifically, these synMuv mutations fall into two classes, referred to as A and B. Animals carrying a class A and a class B mutation have a Muv phenotype, while animals carrying one or more mutations of the same class have a wild-type vulval phenotype. These mutations appear to define two functionally redundant pathways that negatively regulate the expression of vulval cell fates (Lu, 1998).
Four class A genes (lin-8, lin-15A, lin-38, and lin-56) and ten class B genes (lin-9, lin-15B, lin-35, lin-36, lin-37, lin-51, lin-52, lin-53, lin-54, and lin-55) have been identified. lin-15 encodes both A and B activities in two nonoverlapping transcripts. lin-15A, lin-15B, lin-9, and lin-36 encode novel proteins. Two genes, an Rb related protein and its binding partner, have been characterized in one of these pathways. lin-35 encodes a protein similar to the tumor suppressor Rb and the closely related proteins p107 and p130. lin-53 encodes a protein similar to RbAp48, a mammalian protein that binds Rb. In mammals, Rb and related proteins act as regulators of E2F transcription factors, and RbAp48 may act with such proteins as a transcriptional corepressor. It is proposed that LIN-35 and LIN-53 antagonize the Ras signaling pathway in C. elegans by repressing transcription in the vulval precursor cells of genes required for the expression of vulval cell fates (Lu, 1998).
It is proposed that the class B synMuv genes inhibit vulval induction by a conserved mechanism: LIN-35 Rb forms a complex with a sequence-specific transcription factor, presumably an E2F-like protein, and recruits a corepressor complex containing HDA-1, LIN-53 p48, and other proteins to turn off the transcription of vulval specification genes via E2F-binding sites. In the wild type, in the P3.p, P4.p, and P8.p cells, synMuv gene activity antagonizes the basal activity of the RTK/Ras pathway by repressing transcription of vulval genes. As a result, those cells adopt the nonvulval 3° fate. However, in P5.p, P6.p, and P7.p the antagonistic effect of synMuv gene activity is inactivated or can be overcome by the activated RTK/Ras pathway, thereby releasing transcriptional repression and permitting the expression of vulval fates. In the P(3-8).p cells of a synMuv mutant, repression cannot occur and all six P(3-8).p cells express vulval fates, resulting in a Muv phenotype. The synMuv genes do not appear to exert their effects by regulating cell cycle progression of the P(3-8).p cells, since all six of these cells have very similar cell cycle profiles. The synMuv genes must act genetically upstream of or in parallel to the Ras pathway. Action in parallel would be consistent with recent findings from studies of mammalian cells: dominant-negative Ras and Ras neutralizing antibodies induce an Rb-dependent block in DNA synthesis and G1 arrest, suggesting that Rb functions to inhibit mitogenesis downstream of or in parallel to Ras (Lu, 1998 and references).
The single C. elegans member of the retinoblastoma gene family, lin-35 Rb, was originally identified as a synthetic Multivulva (synMuv) gene. These genes form two redundant classes, A and B, that repress ectopic vulval cell fate induction. lin-35 Rb also acts as a negative regulator of G(1) progression and likely is the major target of cyd-1 Cyclin D and cdk-4 CDK4/6. G(1) control functions are described for several other class B synMuv genes. efl-1 E2F negatively regulates cell cycle entry, while dpl-1 DP appeared to act both as a positive and negative regulator. A negative G(1) regulatory function has been identified for lin-9 ALY, as well as lin-15B and lin-36, which encode novel proteins. Inactivation of lin-35 Rb, efl-1, or lin-36 allowed S phase entry in the absence of cyd-1/cdk-4 and increased ectopic cell division when combined with cki-1 Cip/Kip RNAi. These data are consistent with lin-35 Rb, efl-1, and lin-36 acting in a common pathway or complex that negatively regulates G(1) progression. In contrast, lin-15B appears to act in parallel to lin-35. These results demonstrate the potential for genetic identification of novel G(1) regulators in C. elegans (Boxem, 2002).
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