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Role of C. elegans lin-40 MTA in vulval fate specification and morphogenesis

Zhe Chen and Min Han*

Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309, USA



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  		Fig. 1. Major events and regulators in generation of the VPCs (A), vulval induction (B) and early stages of vulval morphogenesis (C). (A) During the L1/L2 stages, the fusion between P(3-8).p and hyp7 is blocked by the action of lin-39 Hox, which is expressed in these six cells (Clark et al., 1993; Wang et al., 1993). P3.p through P8.p (VPCs) have the potential to adopt the vulval fate, whereas other Pn.ps fuse with the hypodermis. (B) During vulval induction at the L3 stage, the RTK/Ras/MAPK pathway transduces a vulval inductive signal from the anchor cell and the synMuv genes prevent the vulval fate in the VPCs (Riddle et al., 1997). lin-39 is also required at this stage to maintain the competence of the VPCs and their progeny for vulval induction (Maloof and Kenyon, 1998; Clandinin et al., 1997). In wild-type animals, three VPCs are induced to become vulval cells and the other three divide once and then fuse with the hypodermis. (C) During the early stages of vulval morphogenesis, the induced VPCs first divide twice in a longitudinal orientation, each giving rise to four granddaughters. All but two of the granddaughter cells undergo one more round of division, and each has a specific division plane. L stands for longitudinal division, T for transverse division and N for no division. The dividing cells also migrate dorsally as well as towards the center of the vulva (where the inner cell P6.p is located).

 


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  		Fig. 2. (A) Two transcripts, lin-40a and lin-40b, are generated from the lin-40 locus by alternative splicing. cDNA clones that represent both transcripts have a polyA tail at their 3' ends and an in-frame stop codon in front of the start codon. Arrows on top of the genomic sequence and protein products in B indicate the positions of mutations in lin-40 alleles. (B) The protein structure of MTA1 proteins. Human MTA1 (hMTA-1) and its homologues in C. elegans, LIN-40A, LIN-40B and EGL-27, share some consensus peptide motifs, including a leucine zipper, a SANT domain and a zinc-finger motif. The SH3-binding domain is only present in mammalian MTA1. The hatched domain in LIN-40B represents the sequence that interacts with LIN-36 and LIN-53 in a two-hybrid screen. (C,D) The expression pattern of a lin-40a::gfp reporter gene. A high level of GFP fusion protein was detected predominantly in the nuclei of most, if not all, somatic cells. Shown here is the presence of LIN-40A::GFP in vulval cells, the anchor cell (AC) and many other gonadal cells. (E,F) The expression pattern of a lin-40b::gfp reporter. LIN-40B::GFP was found to be present at a lower level than LIN-40A::GFP in both the cytoplasm and the nucleus. Ventral is downwards and anterior is towards the left. Scale bars: 10 µm.

 


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  		Fig. 3. (A) The Muv phenotype in lin-40(s1675) animals. P(5-7).p have formed a normal vulval invagination, and P8.p was ectopically induced and its progeny formed a pseudovulva in this animal. Arrows point to vulval invaginations formed by induced Pn.ps. (B) The ‘multiprotrusion’ phenotype in a lin-37(n758); lin-40(ku285) double mutant. Note that each of the P(5-7).p cells formed a vulval invagination in this animal. (C,E) jam-1::gfp expression in wild-type animals during the L2 stage. Arrowheads point to the boundaries between neighboring Pn.ps and arrows indicate the nuclei of Pn.ps. JAM-1::GFP can be detected on the apical surface of Pn.ps where they adjoin the hypodermal syncytium. Note that in E, but not in C, P3.p is already fused to hyp7. (D,F) DIC Nomarski images of the worms shown in C,E. Numbers next to arrows represent the identity of Pn.ps. (G) lin-39::lacZ expression in wild-type animals. Arrows point to the MH27 staining on the surface of Pn.ps and arrowheads indicate the nuclei with positive anti-ß-galactosidase staining. Ventral is downwards and anterior is towards the left. Scale bars: 10 µm.

 


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  		Fig. 4. Mutations in lin-40 and egl-27 affect the fusion between Pn.ps and hyp7 during the L2 stage. y-axis represents the percentage of Pn.ps that remain unfused. (A) A partial loss-of-function mutation in lin-40 caused an elevated percentage of the unfused cell fate in P3.p. This effect was suppressed by a mutation in lin-39(n1760). The egl-27(n170) null allele also enhanced the unfused cell fate in P3.p. *P<0.001, **P<0.05. P values were derived from comparing data from the mutants to that from the wild-type animals. (B) Although lin-40(ku285) alone did not affect the fusion in P(9-11).p, it dramatically increased the abnormal unfused fate in these cells in an egl-27(n170) background. P values were derived from comparing the fusion frequencies of P9.p, P10.p and P11.p between egl-27 and egl-27; lin-40.

 


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  		Fig. 5. How lin-40 regulates vulval fate specification. lin-39 Hox is a pivotal regulator in maintaining the competence of the VPCs at both the L1/L2 and later stages and therefore allowing vulval induction to occur. lin-40 MTA represses vulval fate specification by inhibiting lin-39 expression during the L2 stage and thus reducing the potential of the VPCs to be induced. By contrast, the Ras/MAPK pathway positively regulates lin-39 expression during the L3 stage (Maloof and Kenyon, 1998). Thus, lin-40 antagonizes the Ras/MAPK pathway at least partly at the level of lin-39 expression. lin-40 might also function in a lin-39-independent manner to repress downstream targets required for vulval fate specification, as indicated by a broken line.

 

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