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doi: 10.1242/10.1242/dev.00193

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The cdk inhibitor p27^Xic1 is required for differentiation of primary neurones in Xenopus

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

View larger version (99K): [in a new window]   Fig. 1. p27Xic1 is expressed in cells destined to become primary neurones. Whole-mount in situ hybridisation at indicated stages show expression of p27Xic1 (A-C,G,I) or XMyT-1 (D-F,H). Except for G, figures show a dorsal view with anterior towards the bottom. Both p27Xic1 and XMyT-1 are found in lateral, intermediate and medial stripes of primary neurones, placodes (arrows) and the trigeminal ganglia (arrowheads). (G) Stage 13 embryos show p27Xic1 staining in the epidermis. (H) Double in situ hybridisation demonstrates overlap between p27Xic1 (purple) and XMyT-1 (light blue). (I) Vibratome section of a stage 15 embryo shows p27Xic1 expression in the myotome (My), notochord (Not) and primary neurone precursors (PNP) in the sensorial layer of the neural plate (arrow).

View larger version (97K): [in a new window]   Fig. 2. Embryos depleted of p27Xic1 protein fail to produce differentiated primary neurones. (A) Western blot for endogenous p27Xic1 protein levels on stage 18 embryos that were injected with 20 ng Con Mo or p27Xic1 Mo. Cytoskeletal-ß-tubulin demonstrates equal loading. Con Mo injection has no effect on any of the neural markers examined (B, data not shown). (C-F) p27Xic1 Mo-injected embryos were analysed for expression of X-NGNR-1, XMyT-1, NeuroD and Nßtub (purple) by whole-mount in situ hybridisation, injected side towards the left (ß-gal, light blue). Injection of 20 ng p27Xic1 Mo has no effect on X-NGNR-1 (C) but ablates XMyT-1 (D), NeuroD (E) and Nßtub (F) expression.

View larger version (65K): [in a new window]   Fig. 3. p27Xic1 promotes primary neurone differentiation independently of its cell cycle role. Nßtub expression abolished by p27Xic1 Mo (A) is rescued by co-injection of p21Cip1 (B). Embryos were co-injected with p27Xic1 Mo and p21Cip1 NT (C), p21Cip1 CT (D), p21Cip1 20-82 (E) or p21Cip1 N50S (F). Co-injection with p21Cip1 NT (C) and or p21Cip1 N50S (F) rescued Nßtub, while p21Cip1 CT (D) and p21Cip1 20-82 (E) had no effect.

View larger version (62K): [in a new window]   Fig. 4. p27Xic1 acts at an early stage of neurogenesis. Embryos co-injected with 50 pg X-NGNR-1 and 20 ng Con Mo (A) or p27Xic1 Mo (B) were analysed for Nßtub expression (purple) by whole-mount in situ hybridisation, injected side to the left (ß-gal, light blue). Co-injection of X-NGNR-1 with Con Mo produces extensive ectopic neurones (A), while p27Xic1 Mo prevents both endogenous and ectopic primary neurogenesis (B). (C) Co-injection of p21Cip1 with X-NGNR-1 rescues downregulation of Nßtub expression by p27Xic1 Mo. Embryos were co-injected with 400 pg NeuroD and 20 ng Con Mo (D) or p27Xic1 Mo (E). NeuroD is able to induce extensive ectopic Nßtub even in the absence of p27Xic1 protein (E).

View larger version (65K): [in a new window]   Fig. 5. Ectopic p27Xic1 promotes primary neurogenesis. Embryos were injected with (A) 45 pg p27Xic1 and ßgal (light blue, injected side towards the left) and analysed at stage 15 for BrdU incorporation. Embryos were injected with (B) 60 pg p27Xic1, (D) 30 pg p27Xic1 NT, (E) 50 pg p27Xic1 CT or (F) 50 pg p27Xic1 35-96 with ßgal and analysed at stage 15 for Nßtub expression (purple) by in situ hybridisation. Dorsal views show that p27Xic1 (B) and p27Xic1 NT (D) induce ectopic neurones within the neural plate (arrows), whereas p27Xic1 CT (E) and p27Xic1 35-96 (F) have no effect. (C) A section of a stage 15 embryo injected with 45 pg p27Xic1 indicating cell-autonomous Nßtub upregulation by p27Xic1. (G) Western blot for p27Xic1 levels on stage 10.5 embryos injected with increasing doses (30-150 pg) of p27Xic1 RNA.

View larger version (59K): [in a new window]   Fig. 6. p27Xic1 stabilises X-NGNR-1 protein. Western blot for X-NGNR-1 levels on stage 20 embryos injected with X-NGNR-1 MT alone or in combination with p27Xic1, p27Xic1 NT, p27Xic1 CT or p27Xic1 35-96. Cytoskeletal-ß-tubulin demonstrates equal loading.

View larger version (25K): [in a new window]   Fig. 7. Model demonstrating that p27Xic1 functions upstream of NeuroD, in parallel with X-NGNR-1, to stabilise X-NGNR-1 protein and promote primary neurone differentiation.