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Figure 2


Fig. 2. Dorsal neural cell identity is not affected if either BMPRIA or BMPRIB are constitutively activated in the chick spinal cord after HH stage 14/15. (A-H) Following electroporation of either CMV::caBMPRIA (A,B,E,F,I,J) or CMV::caBMPRIB (C,D,G,H,K,L) constructs in combination with a CMV::GFP vector, changes in cellular identity were examined in stage 22/23 embryos using antibodies against (A-D) pLh2 and (E-H) pIsl. There was no significant difference in the number of dI1 and dI3 neurons on the electroporated and non-electroporated sides of the spinal cord (electroporated side marked with +). (I-L) The activation status of the BMP-specific Smad (Smad1/Smad5/Smad8) second messenger intermediates was assessed using antibodies against the phosphorylated (phos) forms of Smad1/Smad5/Smad8, which endogenously labels the progenitor domain of dI1 neurons (open arrowheads, I,K). Both constructs can activate Smad1/Smad5/Smad8 to equally high levels. (M) Quantification of the activity levels of the CMV::caBMPRIA-HA or CMV::caBMPRIB-HA constructs, by assessing the percentage of HA+ cells that were simultaneously positive for phosSmad1/Smad5/Smad8. There was no significant difference (P>0.17) between the percentage of cells activated by BMPRIA (97.0%±0.6 s.e.m., n=97 sections from 8 embryos) and that activated by BMPRIB (96.0%±0.8 s.e.m., n=101 sections, 11 embryos). (N) Summary of spinal cell types labeled. (O) Quantification of the number of dI1 and dI3 neurons on the electroporated side verses the non-electroporated side following electroporation of either CMV::caBMPRIA or CMV::caBMPRIB at stage 11/12 or 14/15. For both receptors, there was a significant increase in the number of dI1 neurons (BMPRIA: P<0.0004, n=32 sections from 3 embryos; BMPRIB: P<3.8x10-5, n=32 sections, 2 embryos) and dI3 neurons (BMPRIA: P<0.0032, n=25 sections from 2 embryos; BMPRIB: P<0.019, n=24 sections, 2 embryos) on the electroporated side, following electroporation at stage 11/12. However, there was no increase in dI1 (BMPRIA: P>0.29, n=56 sections, 5 embryos; BMPRIB: P>0.48, n=36 sections, 4 embryos) or dI3 (BMPRIA: P>0.36, n=53 sections, 5 embryos; BMPRIB: P>0.22, n=35 sections, 6 embryos) cell numbers, following electroporation at stage 14/15. Scale bar in L: 100 µm for A-L.