Rax is a selector gene for mediobasal hypothalamic cell types

Abstract

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

Figure 1.

Rax expression in the developing optic stalk and hypothalamus during preneurogenesis. A–E, In situ hybridization analysis of coronally sectioned E10.5 mouse brains with Pax6 (A), Rax (B, D), and Nkx2.1 (C, E) riboprobes. A, Pax6 expression in the optic stalk. B, Rax expression in the optic stalk and in the developing hypothalamus. A Rax-free region of progenitors between the optic stalk–hypothalamus boundary is noted (star). C, Nkx2.1 expression in the developing hypothalamus. D, E, Coronal rostral-caudal adjacent sections of the developing forebrain show the onset of Rax expression (black arrow) compared with Nkx2.1 expression throughout the ventral prosencephalon. Diagram illustrates the plane of sectioning. OS, optic stalk; Hypo, hypothalamus.

Figure 2.

Rax is expressed in ARC–VMH progenitors at mid-neurogenesis. In situ hybridization analyses of coronally sectioned E12.5 mouse brains with hypothalamic markers. Diagram, Top, Left, Section plane. Whole-brain image, Top, Right, Hypothalamic region. A–C, Adjacent sections of Rax (A), Sf-1 (B), and Rax/Sf-1 dual-label (C) riboprobes. The dashed black line denotes the developing tuberal hypothalamic nuclear structures in the mantle zone. Star denotes Rax-free zone. The hypothalamic sulcus is highlighted (black arrow; A, L). Dual-label in situ hybridization for Rax (purple) and Sf-1 (brown) demonstrates their adjacent but restricted expression profiles to VZ or MZ, respectively. D–F, In situ hybridization for ARC (D) and DMH (E, F) markers. G, Diagram representation of images shown in A, B, D–F. H–M, In situ hybridization for other transcription factors (H, I, K–M) and secreted morphogen (J) that contribute to ARC–VMH development. N, Diagram representation of the three domains defined by Rax expression in the developing tuberal hypothalamic neuroepithelium.

Figure 3.

Rax expression boundaries relative to other hypothalamic markers in serial sections from the anterior through the mammillary hypothalamic regions. In situ hybridization analyses of coronally sectioned E12.5 mouse brains probed with several hypothalamic markers. The dashed black line denotes the developing nuclear structures in the mantle zone. Black box outlines the onset of the tuberal hypothalamic region (rostral tuberal hypothalamus). Numbers on the top row refer to the column.

Figure 4.

Rax-expressing progenitors give rise to VMH neurons. Immunofluorescence analyses of sectioned P0 brains with α-βgal, α-SF1, and α-Nkx2.1 is shown. The VMH (dotted oval) and ARC (dotted triangle) are highlighted. D–F, Double immunofluorescence of β-gal (D), SF1 (E), and merged (F) on P0 Rax::Cre x Rosa26R brains. Dual-labeled Rax+ lineages and SF1+ neurons are noted (yellow cells, white arrowheads).

Figure 5.

Molecular mapping of Rax-expressing lineages. Schema illustrates the genetic cross and the compartmentalization of β-gal staining in the soma and AP staining in the projections. Here for illustrative purposes, we show β-gal staining and AP staining within the cell, but this double staining would not occur in vivo. A–D, β-Gal and AP staining on neonatal brains for Rax::Cre- and Rax::Cre+ animals crossed with Z/AP. Dashed oval marks the VMH. E, AP staining on E12.5 brains generated crossing Rax::Cre × Z/AP. The developing VMH nucleus consisting of the cell bodies (black dashed oval) and the axonal projections (red arrows) are noted. Clonal populations of Rax+ cells are highlighted (brown arrowhead). F–H, Double-label immunofluorescence of Tuj1 and Nkx2.1 of E12.5 Rax::Cre × Z/AP brains. Merged image illustrates the proximity of Nkx2.1+ cell bodies and Tuj1+ projections. I–K, AP-stained projections generated using Rax::Cre × Z/AP are shown for the anterior, tuberal, and mammillary hypothalamic regions. L, Diagram showing locations of planes shown in I–K. White boxes denote regions highlighted in M–Q. M–Q, Immunofluorescence of GFP-labeled VMH projections in neonatal brains from Sf1^eGFPKI animals. A, amygdala; OT, optic tract; micro, microcircuits; VTA, ventral tegmental area; SN, substantia nigra; MN, mammillary nucleus.

Figure 6.

Rax is required for ventral hypothalamic lineages. A, B, In situ hybridization analysis of adjacent coronally sectioned E10.5 brains with Rax and Six3 riboprobes. C, Anti-GFP labeling of Six3::Cre lineages and neuronal cell bodies (DAPI, blue) in the neonatal brain. The VMH (dotted oval) and ARC (dotted triangle) are highlighted. D–K, In situ hybridization using Fezf1 (D, E), Nkx2.1 (F, G), Pomc (H, I), and Gad67 (J, K) riboprobes on Rax neonatal (P0) controls (D, F, H, J) and conditional mutants (E, G, I, K) generated using Six3::Cre. The expansion of Gad67 into VMH territory is noted (J, K, black brackets).

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::Cre^GFP at E12.5. G, H, Immunofluorescence for Nkx2.1 and GFP in adjacent coronal sections of Shh::Cre^GFP 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::Cre^GFP; Rax^+/null × Rax^{floxed/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').

Figure 8.

Elimination of Rax in a subset of VMH progenitors results in a cell fate change. A–L, In situ hybridization analyses using VMH markers (Sf1, VGlut2, Nkx2.1) and DMH markers (Gad67, Lhx1, Dlx2) in both WT (Rax^flox/+) and Rax CKO (Rax^flox/null; K, L) E12.5 brains are shown. Black brackets mark regions void of VMH+ cellular identities and dotted black oval highlights regions where non-VMH markers become misexpressed. M–U, The localization of Cre+ cells in relation to VMH markers in Rax CKO generated by crossing Shh::Cre^GFP; Rax^+/null × Rax^{floxed/floxed}. In situ hybridization using Sf1 riboprobe (M) and immunofluorescence of Nkx2.1 (P, S) and GFP (N, Q, T) on Rax CKO brains (E12.5, E13.5, and P0) is demonstrated. Photoshop was used to digitally merge M and N, P and Q, and S and T to aid in visualization of the proximity of GFP+ cells and VMH neurons. White oval denotes VMH doublet (P, Q) and white bracket illustrates the location of Rax mutant cells (N, O, T, U).