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doi: 10.1242/10.1242/dev.00500

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The C. elegans LIM homeobox gene lin-11 specifies multiple cell fates during vulval development

Bhagwati P. Gupta, Minqin Wang^* and Paul W. Sternberg

GHHMI and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA
^* Present address: University of Illinois College of Law, Champaign, IL 61820, USAG

View larger version (25K): [in a new window]   Fig. 1. Wild-type vulval development. During L2/L3 stages, the anchor cell (AC) induces P5.p, P6.p and P7.p VPCs to adopt 1° and 2° cell fates. The relative positions of the cell nuclei have been drawn. The terminal cell division axes are NTLL for the 2° lineage and TTTT for the 1° lineage (N, not divided; T, transverse; L, longitudinal). The Pn.pxxx cells invaginate during L4 stage to give rise to the future vulval opening. The seven differentiated cell types have been marked with colors (1° lineage vulE and vulF are red; 2° lineage vulA are white; vulB1 and vulB2 are yellow; vulC are light green; and vulD are dark green).

View larger version (74K): [in a new window]   Fig. 2. Cell fusion events in vulva revealed by the expression of ajm-1::GFP. (A,B) Wild-type. (C-G) lin-11(n389). The regions occupied by VPC progeny are marked with brackets. All images are the ventral views of the vulva. (A) Wild-type vulval rings (vulA to vulF) are visible at different focal planes. The outermost boundaries of the rings are marked with respective cell types. (B) A schematic representation of the vulval rings from lateral and ventral sides. For easier viewing, alternative rings have been coded with dark and light colors. The ventral view shows all seven rings together. (C) A n389 animal with abnormal vulval fusions. The outermost ring (big arrow) corresponds to the 2° lineage cells. The inside rings (small arrows) belong to the 1° lineage cells. Brackets marked with red star point to the unfused vulval cells (punctate GFP). (F) Same animal as in C, under Nomarski. (D) Another n389 animal. In this case, all 2° lineage cells have fused together (big arrow). The small arrows point to the 1° lineage rings. (G) Same animal as in D, under Nomarski. (E) P6.p lineage cells were ablated in this n389 animal. The outermost big ring is still visible (arrow). (H) n389 animal. P5.p and P7.p lineage cells were ablated during early L3 stage. Vulval rings corresponding to the 1° lineage cells (arrows) have abnormal morphology. Scale bars: in A, 12 µm; in G, 10 µm for C-H.

View larger version (97K): [in a new window]   Fig. 3. Expression pattern of the vulval markers in wild-type and lin-11(n389) vulva. (A,C,E,G,I,K) Nomarski images of the L4 stage vulval cells. (B,D,F,H,J,L) Corresponding images showing GFP fluorescence. Primary lineage cells have been marked with stars and 2° lineage cells with arrows. Anterior is towards the left. (A,B) Wild-type egl-17::GFP expression in the vulC and vulD cells. (C,D) In n389 background presumptive vulC and vulD do not reveal any egl-17::GFP. Instead, weak egl-17::GFP is expressed in all the 1° lineage cells. (E,F) Wild-type cdh-3::GFP expression is detected in all the 1° lineage cells and in the vulC, vulD of the 2° lineage cells. (G,H) n389; cdh-3::GFP animals have no detectable GFP fluorescence in the 2° lineage cells. In this animal, the presumptive vulF cells are expressing weak GFP, whereas vulE reveal no detectable expression. (I,J) Wild-type zmp-1::GFP expression is seen in the vulA and vulD of the 2° lineage. By contrast, n389; zmp-1::GFP animals (K,L) do not express GFP in any of the vulval cells. The bright fluorescence is seen in some uterine lineage cells. Scale bar: 8 µm.

View larger version (101K): [in a new window]   Fig. 4. Developmental expression of lin-11::GFP (syIs80) in vulval cells. (A,C,E) Nomarski photographs. (B,D,F) lin-11::GFP-expressing vulval cells marked with arrows. Few VC neurons are also visible (star). (A,B) During Pn.px stage, weak GFP fluorescence is detected in the P5.pp and P7.pa cells (arrows in B). (C,D) By Pn.pxx stage, vulval cells express high levels of GFP. The VPC lineage tree has been drawn. In this animal P5.ppx, P6.ppx and P7.pax cells are seen expressing GFP. (E,F) During L4 stage, Pn.pxxx cells express GFP in the P5.p and P7.p progeny. The region occupied by each VPC progeny has been marked. In this focal plane, only a subset of the cells is visible (arrows). Anterior is towards the left. Scale bar: 10 µm.

View larger version (19K): [in a new window]   Fig. 5. Penetrance of the vulval lin-11::GFP expression at different developmental stages in two transgenic lines: syIs80 (A-C) and syIs53 (D,E). x-axis is the vulval cells, whereas the y-axis is the percentage of animals expressing GFP (sample size for each set is 50). (A) GFP penetrance is higher in P5.pp and P7.pa cells compared with others. syIs53 animals do not reveal vulval expression at this stage. (B,D) By Pn.pxx stage, P5.p and P7.p N,T cells express high levels of GFP. The penetrance is complete in syIs80 animals but lower (60-70%) in syIs53 animals. (C,E) By Pn.pxxx stage, GFP expression is detected in all 2° lineage cells.

View larger version (80K): [in a new window]   Fig. 6. Effect of the heat shock induced lin-11 expression. (A) Pn.pxx stage heat pulse causes ectopic invagination in some of the P5.p and P7.p lineage cells. (B) The same animal as in A. The ectopic expression of egl-17::GFP can be seen in the presumptive vulA of the P7.p lineage. (C) In this animal (heat pulsed during Pn.px stage), all vulval cells have invaginated. The hs::lin-11 transgenic strains are syEx530 (A,B) and syEx500 (C). utse, uterine seam cell. In all images, anterior is towards the left. Scale bar: 8 µm.

View larger version (60K): [in a new window]   Fig. 7. Vulval morphogenesis defects caused by lin-11 RNAi. (A,B) Wild-type. (C-E) Early Pn.pxxx stage heat shocked hs-dslin-11i animals. (A) During mid-L4 stage, the vulD nuclei are located in the same plane of focus (arrows). (B) Same animal as in A when viewed at different focal plane. Thick arrows mark the 1° lineage cell nuclei, vulE and vulF–all seen together. (C) A hs-dslin-11i animal having defects in the positioning of vulval nuclei. In this focal plane, only the P5.p lineage presumptive vulD nucleus is visible. (D) The same animal as in C when viewed at different focal plane. The presumptive vulD nucleus of the P7.p lineage (thin arrow) is seen along with the vulF nuclei (thick arrows). vulE pair is not visible in this plane. (E) A weak Pvul phenotype seen in some hs-dslin-11i adults. Anterior is towards the left. Scale bars: in A, 8 µm for A-D; in E, 10 µm.

View larger version (78K): [in a new window]   Fig. 8. Expression pattern of ldb-1 during vulval development. Thick arrows indicate vulval opening. (A,C,E,G) Nomarski images, (B,D,F,H) GFP fluorescence photomicrographs. Number of animals examined at each stage are 28 (Pn.px), 25 (Pn.pxx), 34 (early Pn.pxxx), 26 (mid Pn.pxxx), 15 (late Pn.pxxx) and 13 (adult). (A,B) Pn.pxx stage animal expressing faint ldb-1::GFP in vulval progeny. Vulval lineages are drawn. (C,D) An early-L4 stage animal, arrows indicate some of the cells expressing GFP. (E,F) A mid-L4 stage animal. Arrowheads indicate all 1° lineage cells in this focal plane that are expressing GFP. (G,H) A young adult expressing GFP in all the 2° lineage cells. (I) Penetrance of ldb-1::GFP expression at different stages. Anterior is towards the left. Scale bar: 10 µm.

View larger version (122K): [in a new window]   Fig. 9. Effect of ldb-1 RNAi on vulval morphology and marker gene expression. Vulval cells are marked with thin arrows. Thick arrows indicate the position of the vulval opening. Nomarski images showing morphological defects in the vulva during mid (A,C) and late (E) L4 stages. (B,D,F) GFP fluorescence photomicrographs of the corresponding animals. (A,B) P7.p lineage presumptive vulC fails to express egl-17::GFP (ayIs4). (C,D) Only presumptive vulD, but not presumptive vulC, vulE and vulF, are expressing cdh-3::GFP (syIs50). (E,F) Presumptive vulC of P5.p and P7.p lineage, and presumptive vulB1 of the P5.p lineage fail to express ceh-2::GFP (syIs54). Anterior is towards the left. Scale bar: 10 µm.

View larger version (59K): [in a new window]   Fig. 10. Yeast two-hybrid interaction test of LIN-11 and LDB-1. Yeast cells were transformed with plasmids pGBKT7 and pGADT7 (1), pGBKT7-lin-11 and pGADT7 (2), pGBKT7 and pGADT7-ldb-1 (3), pVA3 and pTD1 (4), and pGBKT7-lin-11 and pGADT7-ldb-1 (5). In a transformation control (A), all cells can be seen growing on plate that lacks leucine and tryptophan (SD/–Leu/–Trp). However, only positive control (pVA3 and pTD1) and test (pGBKT7-lin-11 and pGADT7-ldb-1) can promote HIS3 expression (B), leading to growth on plate that lacks histidine, leucine and tryptophan (SD/–His/–Leu/–Trp).