Effect of mdka overexpression on proliferation in MFP and tailbud. In the shh expressing MFP, BrdU positive cells in S phase were counted in ten transverse trunk sections each of ten control embryos (a) and ten embryos injected with 50pg mdka RNA (b). No significant changes in cell numbers were observed (Mann-Whitney U-test, n = 10, p = 0.631, Z = -0.571). This was also confirmed in independent BrdU analyses using nkx2.2b as LFP marker (n = 5; c,d). Identical results were obtained when MFP cells in M phase were counted after immunostaining with a phospho-histone H3 (PhH3) antibody and col2a1 as MFP marker (n = 5; e,f). Also in the tailbud, the number of M phase cells was not significantly increased (Mann-Whitney U-test, n = 10, p = 0.912, Z = -0.153; g,h). For counting cells in the tailbud, one focal plane (focused on MFP) of a defined trunk area (with respect to somite positions) was used. Arrowheads indicate BrdU or PhH3 positive cells in the MFP. For BrdU labelling, embryos were incubated for 30 minutes in 10 mM BrdU (Sigma) at the 18s stage and fixed in 4% paraformaldehyde after washing for 15 minutes. Incorporated BrdU, as well as PhH3 protein were detected after shh, col2a1 or nkx2.2b in-situ hybridization by immunostaining with the respective antibodies (anti-BrdU G3G4, Developmental Studies Hybridoma Bank, University of Iowa; anti-PhH3, Upstate Biotechnology, Lake Placid, NY) and the Vecta Stain Elite Kit (Vector Laboratories, Burlingame, CA).
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