Richard-Parpaillon L et al. (2004), G1/S phase cyclin-dependent kinase overexpressi...

XB-ART-3621

Development 2004 Jun 01;13111:2577-86. 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.

Richard-Parpaillon L , Cosgrove RA , Devine C , Vernon AE , Philpott A .

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Cell division and differentiation are largely incompatible but the molecular links between the two processes are poorly understood. Here, we overexpress G1/S phase cyclins and cyclin-dependent kinases in Xenopus embryos to determine their effect on early development and differentiation. Overexpression of cyclin E prior to the midblastula transition (MBT), with or without cdk2, results in a loss of nuclear DNA and subsequent apoptosis at early gastrula stages. By contrast, overexpressed cyclin A2 protein does not affect early development and, when stabilised by binding to cdk2, persists to tailbud stages. Overexpression of cyclin A2/cdk2 in post-MBT embryos results in increased proliferation specifically in the epidermis with concomitant disruption of skin architecture and delay in differentiation. Moreover, ectopic cyclin A2/cdk2 also inhibits differentiation of primary neurons but does not affect muscle. Thus, overexpression of a single G1/S phase cyclin/cdk pair disrupts the balance between division and differentiation in the early vertebrate embryo in a tissue-specific manner.

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Species referenced: Xenopus laevis
Genes referenced: actl6a bcl2l1 cdk2 tubb
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	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 Î² 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).
	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).
	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).
	Fig. 6. Cyclin A2/cdk2 overexpression results in disruption of skin architecture. Embryos were injected with RNAs encoding cyclin A2 with cdk2 (A-C) orÎ² -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.
	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).
	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 Î²-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.
	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: Î²-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).