Vernon AE and Philpott A (2003), A single cdk inhibitor, p27Xic1, functions beyo...

XB-ART-6174

Development 2003 Jan 01;1301:71-83. doi: 10.1242/dev.00180.

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A single cdk inhibitor, p27Xic1, functions beyond cell cycle regulation to promote muscle differentiation in Xenopus.

Vernon AE , Philpott A .

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The molecular basis of the antagonism between cellular proliferation and differentiation is poorly understood. We have investigated the role of the cyclin-dependent kinase inhibitor p27(Xic1) in the co-ordination of cell cycle exit and differentiation during early myogenesis in vivo using Xenopus embryos. In this report, we demonstrate that p27(Xic1) is highly expressed in the developing myotome, that ablation of p27(Xic1) protein prevents muscle differentiation and that p27(Xic1) synergizes with the transcription factor MyoD to promote muscle differentiation. Furthermore, the ability of p27(Xic1) to promote myogenesis resides in an N-terminal domain and is separable from its cell cycle regulation function. This data demonstrates that a single cyclin-dependent kinase inhibitor, p27(Xic1), controls in vivo muscle differentiation in Xenopus and that regulation of this process by p27(Xic1) requires activities beyond cell cycle inhibition.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actl6a cdknx elavl1 gal.2 myf5 myh4 myh6 myod1 odc1 znrd2
???displayArticle.antibodies??? Act3 Ab1 Cdknx Ab1 Myod1 Ab1
???displayArticle.morpholinos??? cdknx MO1

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	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).
	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.
	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 β-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).
	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 β-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 β -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.
	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 β-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 phospho- histone-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.
	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,Aʹ′) 10 ng Con Mo or (B-Cʹ′) 10 ng p27Xic1 Mo and β-gal (light blue in Aʹ′,Bʹ′) (injected side upwards) and allowed to develop until stage 21 (A-Bʹ′) or stage 26 (C,Cʹ′). Embryos were stained for phospho-histone-H3 (purple in Aʹ′,Bʹ′), longitudinally sectioned and stained with an antibody against muscle actin (MA) (red in A,B,Cʹ′). DNA is stained with Hoechst (blue, A,B,C).