Figure 7. Loss of Rax results in abnormal VMH nuclei formation. A–D, In situ hybridization analyses of adjacent coronally sectioned E10.5 brains with Rax and Shh. A, Whole-brain image denotes the area of interest. D, Digitally merged image shows the overlap of Rax and Shh expression domains at E10.5. E, F, In situ hybridization using Sf1 riboprobe and immunofluorescence for GFP in adjacent coronal sections of Shh::CreGFP at E12.5. G, H, Immunofluorescence for Nkx2.1 and GFP in adjacent coronal sections of Shh::CreGFP at E17.5. Cre expression in the ARC (red arrow) is highlighted. Dotted oval denotes VMH. I–P, In situ hybridization using Sf1 riboprobe across neurogenesis, E12.5 (I, M), E13.5 (J, N), and E14.5 (K, O), in Rax CKO mutants and WT controls generated by crossing Shh::CreGFP; Rax+/null × Raxfloxed/floxed. The void of Sf1+ cells in the central region of the VMH is noted (L, P, black brackets and diagram). Q–X, In situ hybridization using the broad ventral hypothalamic marker Nkx2.1 is shown for both WT and Rax CKO generated as per above across the three hypothalamic regions in adjacent coronal sections at E14.5. The Nkx2.1-free region with the tuberal hypothalamus is noted (V, W, black brackets). Y–H', In situ hybridization analyses of WT (Y–C') and Rax CKO (D'–H') neonatal brains generated as per above. A region void of VMH markers in Rax CKO (D'–F', black brackets) and a region with misexpression of non-VMH markers (Gad67; G', black brackets) are noted. ARC-specific riboprobe is also shown (Pomc, C', H').