QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00180

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vernon, A. E. Articles by Philpott, A. Search for Related Content PubMed PubMed Citation Articles by Vernon, A. E. Articles by Philpott, A.

A single cdk inhibitor, p27^Xic1, functions beyond cell cycle regulation to promote muscle differentiation in Xenopus

Department of Oncology, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK

View larger version (55K): [in a new window]   Fig. 1. Cell cycle exit is directly related to myogenic differentiation. (A) Longitudinal section of a stage 15 embryo stained with an antibody against MyoD (dark purple, anterior to left). (B) Longitudinal section of a stage 15 embryo was analyzed for the expression of BrdU (green, arrows) and muscle actin (MA) (red).

View larger version (76K): [in a new window]   Fig. 2. p27Xic1 is expressed in cells destined to a somitic fate. Embryos were analyzed by whole-mount in situ hybridization at the indicated stages for the expression of p27Xic1 (A-D,I,L) and MyoD (E-H,J). (A) p27Xic1 expression in the animal pole at stage 10. Lateral view with dorsal towards the left, asterisk marks the involuting dorsal lip. (B) p27Xic1 localizes to the presomitic mesoderm at stage 11 (arrow). Dorsal view, anterior is downwards. (C) Stage 15 embryo with p27Xic1 in the myotome, notochord, primary neurons and anterior placodes (arrows). Dorsal view with anterior downwards. (D) By stage 22, p27Xic1 is downregulated in the more mature, anterior somites. Lateral view, anterior left. (E) Lateral view of an embryo stained for MyoD at stage 10 with dorsal leftwards and animal pole upwards. V, ventral marginal zone; L, lateral marginal zone; asterisk, involuting dorsal lip. (F) MyoD is expressed in a horseshoe around the blastopore at stage 11. Dorsal view, asterisk indicates the notochord. (G) MyoD expression at stage 15. Dorsal view with anterior downwards. (H) MyoD expression at stage 22. (I) At stage 26, p27Xic1 is virtually absent from the anterior myotome. (J) MyoD is still highly expressed posteriorly at stage 26. (K) Double in situ hybridization demonstrating that p27Xic1 (light blue) is only expressed in the subset of MyoD (purple)-expressing cells that are destined to become skeletal muscle. Parallel horizontal lines indicate the extent of the region of overlap (purple/black). (L) Lateral view of a stage 13 embryo showing epidermal p27Xic1 expression. Dorsal left, anterior down. (M) p27Xic1 protein expression at stage 15. (N) Bisected embryo showing nuclear p27Xic1 expression at stage 15. (O) Hoechst staining of DNA in bisected embryo shown in N.

View larger version (59K): [in a new window]   Fig. 3. MyoD is unable to upregulate expression of p27Xic1 in vivo. (A) Quantitative RT-PCR for MA, p27Xic1 and ODC on cDNA from animal caps of embryos injected with 100 pg MyoD. (B-F') Embryos were injected with 100 pg MyoD (B,C,F,F') or 50 pg DNMyoD (D,E) into one cell of two-cell stage embryos, along with ß-gal as a tracer (light blue, injected side towards the left in B-E or upwards in F,F'). Dorsal views of embryos analyzed for (B,D) MA or (C,E) p27Xic1 expression by whole-mount in situ hybridization at stage 15 (F,F'). (F,F') MyoD-injected embryos allowed to develop to stage 21 were stained for phospho-histone-H3 then longitudinally sectioned and stained for MA (red) and Hoechst (blue, DNA specific). MyoD overexpression enlarges the area staining for MA (F) and also causes extra proliferation (F', arrows).

View larger version (64K): [in a new window]   Fig. 4. p27Xic1 can enlarge the myotome independently of its ability to arrest the cell cycle. One cell of two-cell stage embryos were injected with (A,E) 45 pg p27Xic1, (B,F) 15 pg p27Xic1 NT, (C,G) 50 pg p27Xic1 CT or (D,H) 50 pg p27Xic1 35-96 and ß-gal as a tracer (light blue, injected side to left) and allowed to develop until stage 15 (A-D) or 22 (E-H). Dorsal views with anterior downwards (midline indicated by broken white line) demonstrate that phosphohistone-H3 staining (purple) is reduced after injection of all four p27Xic1 constructs (A-D). Transverse sections stained with an antibody against muscle actin (dark blue) indicate that full-length p27Xic1 (A) and p27Xic1 NT (B) can increase the size of the myotome (arrows), while p27Xic1 CT (C) and p27Xic1 35-96 (D) have no effect.

View larger version (49K): [in a new window]   Fig. 5. p27Xic1 synergizes with MyoD to promote muscle differentiation. (A) Embryos were injected in both cells of two-cell stage embryos with MyoD (100 pg), p27Xic1 (45 pg), p27Xic1 NT (15 pg), p27Xic1 CT (50 pg), p27Xic1 35-96 (50 pg) or a combination thereof. Ventral marginal zones dissected from injected embryos at stage 10 were allowed to develop until parallel embryos reached stage 19 and analyzed by RT-PCR for expression of muscle actin (MA), myosin heavy chain (MHC) and ornithine decarboxylase (ODC) as an internal control. (B) Quantification of the synergistic upregulation of MA by MyoD and p27Xic1 mutant overexpression in ventral marginal zones. The relative induction of MA was determined by measuring the incorporated [32P]dATP and normalizing to ODC. The standard error of the mean was obtained by performing this typical experiment in triplicate.

View larger version (56K): [in a new window]   Fig. 6. p27Xic1 is required for muscle differentiation. Western blot for endogenous p27Xic1 protein in uninjected embryos and embryos injected with 20 ng Con Mo or 20 ng p27Xic1 Mo harvested at stage 22. Cytoskeletal ß-tubulin is used as a loading control. Embryos were injected with 20 ng p27Xic1 Mo (B-D,F,H,K), 20 ng Con Mo (E,J) or 20 ng p27Xic1 Mo + 20 pg p21Cip1 (G) along with ß-gal (light blue, injected side towards the left) and analyzed at stage 15 for expression of MyoD (B), Myf5 (C), MA (E) and MHC (E-G) by whole-mount in situ hybridization. Embryos injected with 20 ng p27Xic1 Mo were incubated in HUA from gastrulation until stage 22 and analyzed by whole-mount antibody staining for 12/101 expression (H,I). Embryos injected with 20 ng Con Mo (J) or p27Xic1 Mo (K) were analyzed for apoptotic cells at stage 15 by whole-mount TUNEL staining.

View larger version (92K): [in a new window]   Fig. 7. Ablation of p27Xic1 causes excess proliferation and loss of differentiated muscle. Embryos were injected in one cell of two-cell stage embryos with (A,A') 10 ng Con Mo or (B-C') 10 ng p27Xic1 Mo and ß-gal (light blue in A',B') (injected side upwards) and allowed to develop until stage 21 (A-B') or stage 26 (C,C'). Embryos were stained for phospho-histone-H3 (purple in A',B'), longitudinally sectioned and stained with an antibody against muscle actin (MA) (red in A,B,C'). DNA is stained with Hoechst (blue, A,B,C).

View larger version (13K): [in a new window]   Fig. 8. p27Xic1 function during myogenic differentiation. During myogenesis, p27Xic1 is required in parallel with, or downstream of, the determination factors MyoD and Myf5. p27Xic1 both arrests the cell cycle and, independent of its cell cycle role, promotes differentiation.