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First published online 20 February 2008
doi: 10.1242/dev.014969

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CDC-25.1 stability is regulated by distinct domains to restrict cell division during embryogenesis in C. elegans

Department of Biology, McGill University, Montreal, Quebec, H3A 1B1, Canada.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Two cdc-25.1(gf) mutations affect a putative β-TrCP-like phosphodegron, while rr36 corresponds to a new temperature-sensitive lf mutation. Schematic representation of the CDC-25.1 phosphatase highlighting the corresponding amino acid changes: the two gain-of-function mutations within the β-TrCP motif (blue) in the N-terminal regulatory domain and the intragenic rr36 mutation adjacent (N-terminal) to the catalytic phosphatase domains (black and red) (see Materials and methods for description). The amino acid changes are in red. The consensus β-TrCP phosphodegron motif is boxed, with its phosphorylable serines in green.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Stabilization of CDC-25.1 triggers intestinal hyperplasia during a specific developmental window. The wild-type intestinal lineage is shown on the left with its typical division timing in minutes after pronuclear meeting at 20°C. The end-3 gene is transcribed during the E1 and E2 stages (black bar) (Maduro et al., 2007), while GFP alone expressed from the end-3 promoter is visible from E2 to threefold stage (550 minutes) (green bar). DIC/GFP overlays of wild-type transgenic embryos expressing the indicated GFP::CDC-25.1 fusion protein variants under the control of the end-3 promoter. The average time in minutes at which the degradation was complete is shown beneath each column. Degradation of the GFP::CDC-25.1[WT] fusion protein occurs 20 minutes after the E4-8 cell division. GFP::CDC-25.1[G47D] resists degradation for more than 120 minutes and triggers a supernumerary division 40-45 minutes into the E8-cell stage, resulting in a precocious E16-like stage and an abnormal hyperplasic E32-cell stage. The intragenic mutation restores proper degradation of GFP::CDC-25.1[G47D;L273F] with kinetics similar to the wild type, while GFP::CDC-25.1[L273F] is destabilized prematurely. Stabilization and hyperplasia typical of GFP::CDC-25.1[G47D] protein are detected in lin-23(RNAi)-treated animals expressing GFP::CDC-25.1[WT], while the [L273F] substitution suppresses these phenotypes. These lin-23(RNAi) embryos do not undergo cell fate transformation (see text). Exposure time was increased to similar levels in all 220 and 260 minute embryos. All embryos were assayed at 20°C. In the last two columns, these embryos were classified based on their intestinal cell stage, owing to the effect of lin-23(RNAi) treatment on the E division timing (Z. Bao, personal communication).

View larger version (66K): [in this window] [in a new window]   Fig. 3. Loss-of-function in lin-23 triggers cell fate transformations and cdc-25.1-dependent hyperplasia. (A) DIC/GFP overlays of representative embryos expressing the intestinal-specific elt-2::gfp marker at 20°C. A wild-type embryo with 16 intestinal cells and a cdc-25.1(rr31) mutant with 30 intestinal cells are shown before morphogenesis. lin-23(e1883) null, lin-23(RNAi) and cdc-25.1(rr31); lin-23(RNAi) embryos at their terminal stages display obvious hyperplasia and lack morphogenetic movements. A cdc-25.1(rr31rr36) mutant treated with lin-23(RNAi) showing typical suppression of the lin-23-dependent intestinal hyperplasia. (B) Time lapse imaging of an embryo dissected from a lin-23(RNAi)-treated hermaphrodite undergoing Cp and MSp to E transformations. This embryo carries an integrated end-3::end-3(P202L)::gfp transgene that marks descendants of intestinal cells. Cells in the E, C- and MS-like lineages are highlighted. Double-headed arrows indicate the division axis of Ca- and Cp-like. Daughters of the normal E blastomere and those of two ectopic E-like cells are indicated by arrowheads as they begin to express the GFP marker. (C) Quantification of intestinal cell number in terminal stage lin-23(RNAi) and control embryos. The lin-23(RNAi)-dependent hyperplasia is not additive to cdc-25.1(gf), while penetrant and significant suppression (*) of the cell cycle defects is seen when CDC-25.1 levels are reduced by rr36. E blastomeres (n=10) from lin-23(RNAi)-treated embryos (rightmost bar) were isolated by laser ablation as described by Zhu et al. (Zhu et al., 1997) and the number of intestinal cells, based on the expression of elt-2::gfp marker, was quantified 24 hours thereafter.

View larger version (37K): [in this window] [in a new window]   Fig. 4. lin-23(ot1) mutants display a mild cdc-25.1-dependent intestinal-specific hyperplasia during embryogenesis. (A) DIC/GFP overlays of wild-type and lin-23(ot1) L1 hatchlings expressing the intestinal-specific elt-2::gfp marker. The wild-type larvae hatch with 20 intestinal cells, whereas the lin-23(ot1) mutants typically display mild intestinal hyperplasia (here, 28 intestinal cells). (B) Quantification of intestinal cell number in L1 hatchlings at 20°C (n=40). The cdc-25.1(gf) and lin-23(ot1) hyperplasia are not additive, whereas the loss-of-function cdc-25.1(rr36) intragenic mutation significantly suppresses both the cdc-25.1(rr31) and the lin-23(ot1)-dependent cell cycle defects. Similar results were observed after hypomorphic cdc-25.1(RNAi) treatment of cdc-25.1(rr31) (data not shown) and lin-23(ot1).

View larger version (80K): [in this window] [in a new window]   Fig. 5. The β-TrCP orthologue lin-23 is differentially expressed during embryogenesis. (A) The progeny of transgenic hermaphrodites expressing GFP::LIN-23 exclusively in their germ line (pie-1 promoter) display ubiquitous and mainly cytoplasmic localization of maternal GFP::LIN-23 until the beginning of the E8-cell stage (180 minutes). Asterisks indicate the intestinal cells. (B) Zygotic LIN-23::GFP is detected 120 minutes after fertilization (E4-stage), and shows differential expression levels between the E lineage and the rest of the embryo. The differential expression of lin-23 continues throughout embryogenesis, with the highest levels in the head region and the lowest in the intestine. Intestinal cells were recognized by morphology, localization and the presence of birefringent gut granules (pseudo-red). The corresponding developmental time in minutes following pronuclear meeting is shown on the left.