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Fig. 5. Bmp7 induces hypothalamic identity in postmitotic cells that are ventralised by Shh. (A-C) Immunofluorescent and in situ hybridisation analyses of stage 4-5 aLNP explants, incubated with Shh. After 2 days, Lim1+/Shh+/Six3+, but not Nkx2.1+/Shh+ progenitor cells are detected (A,B). After 6 days, Th+ (arrowheads), but not Nkx2.1+/Th+ neurons are detected (C). (D) RT-PCR analysis of Shh mRNA in HH stage 5 and HH stage 6 prechordal mesoderm. No significant difference in Shh mRNA levels is detected ({Delta}Ct=5.0±0.4, 4.8±0.5, respectively). (E,F) Recombinate explants of stage 4-5 pHyp and prechordal mesoderm, incubated alone (E) or with anti-Shh IgG (F). No Nkx2.1+ cells are detected in the presence of anti-Shh IgG. (G-I) Shh and Bmp7 cooperate to induce Nkx2.1+/Shh+progenitors and Nkx2.1+/Th+ neurons. Stage 4-5 aLNP explants, incubated with Shh and Bmp7. After 2 days, Nkx2.1+/Shh+ (G) and Nkx2.1+/Lim1+ (H) progenitors are detected. Msx+/Th+ neurons are detected on day 5 (I, inset) and Nkx2.1+/Th+ neurons detected on day 7 (I, white arrows). Th+ (blue arrowheads) neurons can also be detected. (J,K) Chordin blocks prechordal mesoderm-mediated specification of Nkx2.1+/Shh+ progenitors. (J) A stage 5 pHyp explant, co-cultured with prechordal mesoderm in the presence of chordin. Very few Nkx2.1+/Shh+ cells are detected. (K) Significantly more Nkx2.1+/Shh+ cells form in control cultures compared with cultures treated with chordin (control, 100%±23.2%; +Chd, 15.9%±4.3%, P<0.0001; unpaired t-test). (L) A stage 5 pHyp explant co-cultured with prechordal mesoderm and chordin for 6 days. Th+ cells differentiate (blue arrowheads), but these do not co-express Nkx2.1. (M) Stage 5 aLNP explants, incubated with Shh. After 22 hours, Shh but not Nkx2.1 (M, inset) was induced. (N) Stage 5 aLNP explants, incubated with Bmp7. After 22 hours, Msx/2 was detected but no Nkx2.1 was observed. (O) Stage 5 aLNP explants, transiently incubated with Bmp7 (day 0-1), then Shh (day1-7). On day 7, Th+ cells differentiate but they do not co-express Nkx2.1 (blue arrowheads). (P) Stage 5 aLNP explant, transiently incubated with Shh (day 0-1), then Bmp7 (days 1-7). On day 7, Nkx2.1+/Th+ cells differentiate (arrows). (Q) Stage 5 aLNP explant, transiently incubated with Shh (day 0-1), then transiently incubated with Bmp7 (day1-2). After 7 days, Nkx2.1+/Th+ cells differentiate (arrows). (R) Stage 5 aLNP explants, transiently incubated with Shh (day 0-1), then transiently exposed to Bmp7 (day 5-6). On day 7, Nkx2.1+/Th+ cells differentiate (arrows). (S) Stage 5 aLNP explants, transiently incubated with Shh (day 0-1), then transiently exposed to Bmp7 (day 6-7). On day 7, Th+ cells differentiate but they do not co-express Nkx2.1 (blue arrowheads). (T) Stage 5 pHyp explants, incubated with BrdU on day 5. On day 7, no BrdU+/Th+ cells are observed (blue arrowheads). Inset: stage 5 pHyp explants, incubated with BrdU on day 3. On day 7, BrdU+/Th+ cells are observed. (U) Stage 5 pHyp explants, transiently incubated with Bmp7 on day 5. On day 7, Nkx2.1+/Th+ cells differentiate (arrows). Scale bars: 40 µm in A,B,E-G,J,L,M-U,W; 30 µm in C,I.