Neurog2 Acts as a Classical Proneural Gene in the Ventromedial Hypothalamus and Is Required for the Early Phase of Neurogenesis

Abstract

The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU⁺ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.

Keywords: Neurog2; VMH; neurogenesis; proneural genes; specification.

Figure 1.

Neurogenesis within the tuberal hypothalamus has an outside-in trend. Immunostaining showing BrdU expression in the sectioned E19.5 mouse brain injected with BrdU at the following: E9.5 (A-A′′), E10.5 (B-B′′), E11.5 (C-C′′), E12.5 (D-D′′), E13.5 (E-E′′), and E14.5 (F-F′′). A′′′–F′′′, Schematic figure of tuberal hypothalamic neurogenesis at E9.5 (A′′′), E10.5 (B′′′), E11.5 (C′′′), E12.5 (D′′′), E13.5 (E′′′), and E14.5 (F′′′). A′′′′–F′′′′, E14.5 immunostaining showing Fezf1 expression in the sectioned E19.5 mouse brain injected with BrdU at the following: E9.5 (A′′′′), E10.5 (B′′′′), E11.5 (C′′′′), E12.5 (D′′′′), E13.5 (E′′′′), and E14.5 (F′′′′). Scale bars, 100 μm.

Figure 2.

Proneural genes are expressed within VMH progenitors across embryonic development. ISH results demonstrating Neurog1 (A-A′′′′), Neurog2 (B-B′′′′), Neurog3 (C-C′′′′), and Ascl1 (D-D′′′′) expression at E10.5, E12.5, E14.5, E16.5, and P0. E, E′, RNAScope results for Ascl1 (red) and Neurog2 (green) on a rostral (E) and caudal (E′) section of E12.5 hypothalamus. Scale bars, 100 μm.

Figure 3.

Neurog2 is required for proper neurogenesis within the embryonic tuberal hypothalamus. Immunostaining results for anti-BrdU on E19.5.5 coronal sections of control and Neurog2^−/− brains injected with BrdU at E9.5 (A,B), E10.5 (C,D), or E11.5 (E,F). G–L, Binary images of the data presented in A–F. Histogram plots of these binary images demonstrate the location of BrdU⁺ cells following injection at E9.5 (M,N), E10.5 (O,P), or E11.5 (Q,R). Cell counts of BrdU⁺ cells within control and Neurog2^−/− tuberal hypothalamus injected with BrdU at E19.5 (S), E10.5 (T), or E11.5 (U). Bar graphs represent mean ± SEM (n = 3 embryos per group; 3 brain sections per embryo). **p < 0.007; ***p < 0.0004; ****p < 0.0001; unpaired t test. Scale bars, 100 μm.

Figure 4.

Neurog2 is required for proper neurogenesis within the embryonic tuberal hypothalamus. Immunostaining results for anti-BrdU on E19.5.5 coronal sections of control and Neurog2^−/− brains injected with BrdU at E12.5 (A,B), EC13.5 (C,D), or E14.5 (E,F). G–L, Binary images of the data presented in A–F. Histogram plots of these binary images demonstrate the location of BrdU⁺ cells following injection at E12.5 (M,N), E13.5 (O,P), or E14.5 (Q,R). Cell counts of BrdU⁺ cells within control and Neurog2^−/− tuberal hypothalamus injected with BrdU at E12.5 (S), E13.5 (T), or E14.5 (U). Bar graphs represent mean ± SEM (n = 3 embryos per group; 3 brain sections per embryo). **p < 0.009 (unpaired t test), ns - not significant. Scale bars, 100 μm.

Figure 5.

Neurog2 is required for specification of VMH neurons. A, B, Immunostaining results for anti-Fezf1 on E12.5 mouse coronal sections on control and Neurog2^−/−. C, Fezf1⁺ cell counts for whole VMH at E12.5 on both control and Neurog2^−/− brains. D, E, Immunostaining results for anti-Fezf1 on E15.5 mouse coronal sections on control and Neurog2^−/−. F, Fezf1⁺ cell counts for whole VMH at E15.5 on both control and Neurog2^−/− brains. G, H, Immunostaining results for anti-Nkx2.1 on E12.5 mouse coronal sections on control and Neurog2^−/−. I, Nkx2.1⁺ cell counts for whole VMH at E12.5 on both control and Neurog2^−/− brains. J, K, Immunostaining results for anti-Nkx2.1 on E15.5 mouse coronal sections on control and Neurog2^−/−. L, Nkx2.1⁺ cell counts for whole VMH at E15.5 on both control and Neurog2^−/− brains. M, N, Immunostaining results for anti-Stab2 on E15.5 mouse coronal sections on control and Neurog2^−/−. O, Satb2⁺ cell counts for whole VMH at E15.5 on both control and Neurog2^−/− brains. P, Q, ISH results for Vgll2 ribo-probe on E15.5 mouse coronal sections on control and Neurog2^−/−. Bar graphs represent mean ± SEM (n = 3 embryos per group; 3 brain sections per embryo). *p < 0. 01; **p < 0.005; ***p < 0.0001; unpaired t test. Dashed oval represents VMH nucleus and its three subdomains. Scale bars, 50 μm.

Figure 6.

Neurog2 is required for proper neurogenesis of VMH-specific neurons. Double-immunostaining results for anti-BrdU and anti-Fezf1 on E19.5.5 mouse coronal sections injected with BrdU on control and Neurog2^−/− at E9.5 (A,B), E10.5 (C,D), E11.5 (E,F), 12.5 (G,H), E13.5 (I,J), and E14.5 (K,L). Double-immunostaining results for anti-BrdU and anti-Nkx2.1 on E19.5.5 mouse coronal sections injected with BrdU on control and Neurog2^−/− at E9.5 (M,N), E10.5 (O,P), E11.5 (Q,R), E12.5 on control (S,T), E13.5 (U,V), and E14.5 (W,X). Y, Number of Fezf1⁺ cells colabeled with BrdU in control compared with Neurog2^−/− backgrounds. Z, Number of Nkx2.1⁺ cells colabeled with BrdU in control compared with Neurog2^−/− backgrounds. Bar graphs represent mean ± SEM (n = 3 embryos per group; 3 brain sections per embryo). **p < 0.001; ***p < 0.0002; ****p < 0.0001, ns - not significant; unpaired t test. Dashed oval represents VMH nucleus and its three subdomains. Scale bars, 50 μm.

Figure 7.

Ascl1 is required for proper neurogenesis within embryonic tuberal hypothalamus. Immunostaining results for anti-BrdU on E19.5.5 mouse coronal sections injected with BrdU on control and Ascl1^−/− at E9.5 (A,B), E10.5 (C,D), E11.5 (E,F), E12.5 (G,H), and E13.5 (I,J). A′–J′, Binary images of the data presented in A–J. Histogram plots of these binary images demonstrate the location of BrdU⁺ cells following injection at E9.5 (K,L), E10.5 (M,N), E11.5 (O,P), E12.5 (Q,R), and E13.5 (S,T). BrdU⁺ cell counts for tuberal hypothalamus on E19.5 mouse brains injected with BrdU at E9.5 (U), E10.5 (V), E11.5 (W), E12.5 (X), and E13.5 (Y) for both control and Ascl1^−/−. Bar graphs represent mean ± SEM (n = 3 embryos per group; 3 brain sections per embryo). **p < 0.005; ****p < 0.0001, ns - not significant; unpaired t test. Scale bars, 100 μm.

Figure 8.

Neurog2 does not regulate expression of Neurog1, Neurog3, or Ascl1 within the tuberal hypothalamus. Neurog1(A,B), Neurog3 (C,D), and Ascl1 (E,F) expression levels in E12.5 mouse brain sections in control and Neurog2^−/− backgrounds. Scale bar, 100 μm.

Figure 9.

Schematic figure depicting neurogenesis within the tuberal hypothalamus and VMH. A, Schematic curve for the timing of neurogenesis within the tuberal hypothalamus, in control compared with Neurog2^−/− and Ascl1^−/− brains. B, Diagram summarizing the pattern of neurogenesis within the VMH nucleus.