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Fig. 6. Shh and Bmp7 are necessary and sufficient to induce Nkx2.1+/Shh+ cells and
Nkx2.1+/Th+ neurons in a Six3-dependent manner in vivo. (A-D) Transverse
section through tuberal hypothalamus (A-C) or anterior hypothalamus/dorsal
telencephalon (D) of HH stage 15 embryos, immunolabelled to detect Nkx2.1 and
Shh. (A) Control embryo showing Nkx2.1+/Shh+ cells in the lateral tuberal
hypothalamus (arrow) and Nkx2.1+ ventral tuberal cells (arrowhead) after
implantation of a control bead (bead has been displaced). (B) A chordin
(Chd)-soaked bead implanted at stage 5, adjacent to the prechordal
mesoderm/ventral tuberal hypothalamus. Nkx2.1 expression is abolished from
lateral tuberal hypothalamic cells (arrow). Concomitantly, Shh expression is
expanded (arrowhead in D). (C,D,D') Implanting a Shh/Bmp7-soaked bead at
stage 5 (schematic above: bead displaced laterally) results in ectopic
Nkx2.1+/Shh+ cells in the dorsal telencephalon (arrow in D,D'). Ventral
cells in the anterior hypothalamus also co-express Shh and Nkx2.1. (C) Normal
expression of Nkx2.1+/Shh+ lateral hypothalamic progenitors (arrow). (E,F)
Electroporation of the RD Six3 construct results in the ectopic expression of
Six3 in mesencephalic lateral neural tube explants (F, arrow). No Six3
expression is detected in control (RFP) electroporated explants (E). (G,H)
Nkx2.1+/Th+ cells are induced in RD Six3-electroporated lateral mesencephalic
explants, exposed to Shh and Bmp7 (G, arrows), whereas mock-electroporated
explants give rise only to Nkx2.1–/Th+ cells after exposure to Shh and
Bmp7 (H, blue arrowheads). (I-K) In AD Six3-electroporated mesencephalic
tissue, exposed to Shh and Bmp7, Th+ cells do not co-express Nkx2.1 (I, blue
arrowheads) but co-express En1 and Lmx (J,K, light blue arrows). Scale bars:
75 µm in A,B; 150 µm in C,D; 40 µm in G-K.
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