(A–C) Summary of dorsal progenitors (dP1-6) and post-mitotic neurons (dI1-6) in the developing spinal cord. The combinatorial use of antibodies against Lhx2/9 (green), Lhx1/5 (blue), Isl1 (blue), Pax2 (green) and Tlx3 (red) permit the unambiguous identification of dI1-dI5. (D–U) Chicken spinal cords were electroporated at HH stage 15 with Gfp (D, G, J, M, P, S), Bmp4 (E, H, K, N, Q, T) or Bmp7 (F, I, L, O, R, U), under control of the CAG enhancer (Miyazaki et al., 1989), and incubated until HH stage 25. Thoracic transverse sections were labeled with antibodies against Tlx3 (red, S–U), Lhx2/9 (red, J–L), Lhx1/5 (red,M–O), Isl1 (red P-R; green, S–U) or Pax2 (green M-O), pSmad1/5/8 (red, D–F) and Mafb (red, G–I). (D–F) Ectopic expression of Bmp4 (n = 52 sections from 5 embryos, p<0.0001) more effectively activates the R-Smads (Smad1/5/8) than Bmp7 (n = 30 sections 4 embryos, p<0.005), while the expression of Gfp has no effect (n = 37 sections from 4 embryos, p>0.72). (G–I) Mis-expression of Bmp4 or Bmp7 has dramatic, but distinct, effects on dorsal cell differentiation. Specifically, the ectopic expression of Bmp7 (I, n = 28 sections from 4 embryos, p<7.01×10−5) resulted in consistently more Mafb+ RP cells than Bmp4 (H, n = 37 sections from 5 embryos, p<0.12), and the Gfp control (G, n=36 sections from 2 embryos, p>0.67). (J–L, P–R) Mis-expression of Bmp4 however, most effectively directs cells towards the Lhx2/9+ dI1 (K, n = 28 sections from 3 embryos, p<0.0001) and Isl1+ dI3 fates (Q, 91 sections from 5 embryos, p<0.0001) compared to Bmp7 (dI1, L, n = 26 sections from 3 embryos, p<0.0001; dI3, R, n = 59 sections from 5 embryos, p<5.58×10−6). Mis-expression of Gfp has no effect (dI1, J, n = 46 sections from 3 embryos, p>0.47; dI3, P, n = 45 sections from 3 embryos, p>0.46). (P–R) Bmp4 (N, n = 47 sections from 3 embryos, p<0.0001) is the only BMP sufficient to direct cells toward an Lhx1/5+ dI2 identity, while Bmp7 can suppress Pax2+ dI4 fate (O, n = 24 sections from 3 embryos, p<0.01). The ectopic expression of Gfp has no effect (M, n = 35 sections from 3 embryos, p>0.77). Note that the presence of Bhlhlb5 (Skaggs et al., 2011), permitted the Pax2+ Lhx1/5- Bhlhb5- dI4s to be unambiguously distinguished from the Pax2- Lhx1/5+ Bhlhb5- dI2s and the Pax2+ Lhx1/5+ Bhlhb5+ dI6s (data not shown). (S–U) The mis-expression of the BMPs has no effect on the Tlx3+ dI5 fate (S, GFP: n = 38 sections from 3 embryos p>0.16; BMP4: T, n = 50 sections from 4 embryos, p>0.39; BMP7: U, n = 29 sections from 3 embryos, p>0.08) fates. (V) Quantification of the fold change in cell number normalized to Gfp control. The probability Bmp4 and Bmp7 misexpression result in the same distribution of cellular activities is p<0.0002 (Fisher test). (W–Y) Summary of the cellular changes directed by BMP4 and BMP7. We found that there are there are spatial organizational changes for Bmp4, but not Bmp7, misexpression. While the RP, dI1 and dI3 populations remain in the correct spatial order with respect to each other, the dI2s both expand and change location, such that they are intermingled with/ventral to the dI3 population. Probability of similarity between control and experimental groups, *=p<0.05, **p<0.005, ***p<0.0005. Student’s t-test or Mann-Whitney test. Scale bar: 65 µm.