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First published online 28 April 2004
doi: 10.1242/dev.01121

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G1/S phase cyclin-dependent kinase overexpression perturbs early development and delays tissue-specific differentiation in Xenopus

Laurent Richard-Parpaillon^*, Ruth A. Cosgrove, Christine Devine, Ann E. Vernon and Anna Philpott

Department of Oncology, Cambridge University, Hutchison/MRC Research Centre, Addenbrookes Hospital, Hills Road, Cambridge CB2 2XZ, UK

View larger version (82K): [in a new window]   Fig. 2. Cyclin E but not cyclin A2 overexpression leads to apoptosis in the gastrula embryo. RNAs encoding cyclin E alone (a,A) or cyclin E with cdk2 (d,D,G), cyclin A2 alone (b,B) or cyclin A2 with cdk2 (e,E), cdk2 alone (f,F), Bgal as a control (c,C) or cyclin E with cdk2 and BclXL (H) were injected into one cell of a 2-cell embryo and allowed to develop to stage 9 (a-f) or stage 11 (A-H). Areas of slowed cell division are indicated by black arrows (a,d,H). Areas of apoptosis are indicated by white arrows (A,D,G).

View larger version (80K): [in a new window]   Fig. 6. Cyclin A2/cdk2 overexpression results in disruption of skin architecture. Embryos were injected with RNAs encoding cyclin A2 with cdk2 (A-C) or ß-gal as a control (D-F), along with GFP as a lineage tracer. Embryos were fixed at stage 23, sectioned, then stained for expression of the skin marker, epi (A,D), confined to the epidermis in these sections. Region of epi expression deep within the thickened skin is indicated by a white arrow. GFP fluorescence (B,E). DNA is stained with Hoechst in blue (C,F). Alternatively, uninjected embryos or embryos injected with RNAs encoding cyclin A2/cdk2, as indicated, were photographed in whole-mount (G). A raised hyperpigmented lump, which often formed after cyclin A2/cdk2 injection, is indicated by the black arrow.

View larger version (39K): [in a new window]   Fig. 4. Cyclin A2/cdk2 overexpression is sustained throughout early development. (A) Embryos were injected with RNA encoding cdk2, cyclin A2, cyclin A2 with cdk2, or Bgal as a control. Embryos were allowed to develop to stage 23, then immunoprecipitated cdk2 from injected embryos was tested for its ability to phosphorylate histone H1. (B) Embryos were injected with RNA encoding cyclin A2, cdk2, cyclin A2 with cdk2 or GFP as control and allowed to develop to stage 9 (lanes 1-3, 7) or stage 23 (lanes 4-6, 8). Extracts from embryos, injected as labelled, were western blotted to detect cyclin A2 protein levels. (C,D) Cyclin A2/cdk2-injected embryos were grown until tailbud stages (injected side to the right). Tagged cyclin A2 is stable in all tissues on the injected side (D), including neural tube (nt), notochord (no), muscle (m) and epidermis (ep); see Hoechst staining of the same section (C).

View larger version (47K): [in a new window]   Fig. 1. Activation of H1 kinase activity by overexpression of cyclins and cdk2. RNA encoding cyclin E alone or with cdk2, cyclin A2 alone or with cdk2, or cdk2 alone were injected into fertilised eggs and allowed to develop until stage 8. Cdk2 was immunoprecipitated from injected embryos (lanes as labelled) and tested for its ability to phosphorylate histone H1.

View larger version (112K): [in a new window]   Fig. 3. Cyclin E overexpression results in nuclear loss. RNAs encoding cyclin E1 alone (A) or cyclin E1 with cdk2 (D), cyclin A2 alone (B) or cyclin A2 with cdk2 (E), cdk2 alone (C) or ß-gal as a control (F) were injected into one cell of a 2-cell embryo and allowed to develop to stage 8.5. Embryos were fixed, depigmented and the DNA stained with Hoechst. Animal caps were dissected and viewed by fluorescence microscopy. White arrows indicate regions in which nuclei are absent.

View larger version (78K): [in a new window]   Fig. 5. Cyclin A2/cdk2 overexpression promotes cell proliferation in the embryonic epidermis. Embryos were injected into one cell at the 2-cell stage with cyclin A2 and cdk2 RNA along with Bgal (light blue). (A) Whole-mount immunostaining with antiphosphohistone H3 antibodies shows an increase in cell proliferation on the injected side (left). (B-G) Cyclin A2/cdk2-injected embryos were grown until tailbud stages and then allowed to incorporate BrdU for 1 hour before fixing (B,C,E; BrdU uptake) red; nuclei stained in blue with Hoechst dye; bottom left of each panel: ß-gal auto-fluorescence. Epidermis overexpressing cyclin A2/cdk2 (C) is disorganised and shows increased BrdU incorporation compared with epidermis on the uninjected side (B). By contrast, cell proliferation does not differ in the neural tube and muscle between the injected side (left) and the uninjected side (right) (E). (D,F,G) Number of BrdU-positive cells expressed as a percentage of total number of nuclei (Hoechst-positive) were counted in regions of equal side on the injected and uninjected sides of the embryos. BrdU incorporation is increased in the epidermis on the injected side compared with the uninjected side of embryos (D). No significant difference in BrdU incorporation is seen for muscle (F) and neural tube (G).

View larger version (102K): [in a new window]   Fig. 7. Cyclin A2/cdk2 delays differentiation of epidermis and neurons. Whole-mount in-situ hybridisation analysis shows a delay in skin differentiation (A-C: epidermal keratin, stage 14, 16 and 18, respectively); and neural differentiation (D-F: neural ß tubulin, stage 14, 19 and 25, respectively) in embryos injected with cyclin A2 and cdk2 RNA (injected side to the left, indicated by asterisks). Differentiation of the muscle is not affected (G-I: muscle actin, stage 13, 16 and 21, respectively).