Neurogenin3 restricts serotonergic neuron differentiation to the hindbrain

Abstract

The development of the nervous system is critically dependent on the production of functionally diverse neuronal cell types at their correct locations. In the embryonic neural tube, dorsoventral signaling has emerged as a fundamental mechanism for generating neuronal diversity. In contrast, far less is known about how different neuronal cell types are organized along the rostrocaudal axis. In the developing mouse and chick neural tube, hindbrain serotonergic neurons and spinal glutamatergic V3 interneurons are produced from ventral p3 progenitors, which possess a common transcriptional identity but are confined to distinct anterior-posterior territories. In this study, we show that the expression of the transcription factor Neurogenin3 (Neurog3) in the spinal cord controls the correct specification of p3-derived neurons. Gain- and loss-of-function manipulations in the chick and mouse embryo show that Neurog3 switches ventral progenitors from a serotonergic to V3 differentiation program by repressing Ascl1 in spinal p3 progenitors through a mechanism dependent on Hes proteins. In this way, Neurog3 establishes the posterior boundary of the serotonergic system by actively suppressing serotonergic specification in the spinal cord. These results explain how equivalent p3 progenitors within the hindbrain and the spinal cord produce functionally distinct neuron cell types.

Keywords: hindbrain; neural tube; neuronal specification; serotonergic system; spinal cord; transcription factor.

Figure 1.

Neurog3 is expressed in the ventral spinal cord p3 domain. A–I, Hindbrain serotonergic and spinal V3 interneurons arise from ventral p3 progenitors. Hindbrain and spinal cord E11.5 cross sections were hybridized with probes against the p3 marker Nkx2.2 (A); serotonergic-specific transcription factors Gata2 (B), Lmx1b (C), and Pet1 (D); and V3 transcription factors Sim1 (E) and Uncx (F). Serotonergic hindbrain neurons were identified by immunostaining against 5-HT (G), and a probe against vGluT2 (H) was used to identify glutamatergic neurons in the spinal cord. Arrowheads point to p3 cells or p3-derived postmitotic neurons. I, Schematic representation of p3-derived cells in distinct regions of the neural tube: serotonergic neurons in the hindbrain (5HT-N) and glutamatergic V3 interneurons in the spinal cord (top). The transcription factors expressed in serotonergic and V3 neuron differentiation are shown (bottom). J–N, Neurog3 is expressed in the spinal p3 ventricular zone but is absent in the ventral hindbrain. Cross sections of E10.5 hindbrain and spinal cord were hybridized with a Neurog3 probe (J, K) and immunolabeled with Neurog3 and Nkx2.2 antibodies (L, M). Note that while Neurog3 is robustly expressed in Nkx2.2⁺ spinal cord ventral cells (arrowhead in K, M), it is missing in the hindbrain (arrowhead in J, L). Low levels of Neurog3 expression were found dorsally to the p3 domain (arrow in K). The number of Neurog3⁺ cells per section that coexpress Nkx2.2 is shown (N). Bars are mean ± SD. Scale bars: 50 μm.

Figure 2.

Neurog3 delineates spinal cord p3 precursors. A–D, Neurog3 is expressed in the spinal p3 domain. Immunostainings on E10.5 spinal cord sections revealed that Neurog3⁺ cells are restricted to Nkx6.1⁺ territories (A), ventral to the domain that gives rise to motoneurons, marked by Olig2 (B) and Lhx3 (C), and excluded from the Foxa2^HIGH floor plate (D). Dotted lines represent dorsoventral boundaries. E, F, Neurog3 is expressed before neuronal differentiation. Immunohistochemistry against Neurog3 on E10.5 spinal cord sections show coexpression with the progenitor marker Sox2 (E), but absence in β-III-tubulin newborn neurons (F). Ventricle to the left. G–J, Neurog3 precedes V3 differentiation. Sim1 (anti-Cre antibody, G, H) and Uncx (anti-β-galactosidase antibody, I, J) were analyzed in Sim1^Cre/+ and Uncx^lacZ/+ E11.5 spinal cords, respectively. Ventricle to the left in H and J. K, Neurog3 expression depends on retinoid activity. In situ hybridization with a cNeurog3 probe on E4 chick spinal cord sections that were electroporated with a dominant-negative RAR403-IRES-GFP expression construct at E3. GFP labeling shows the targeted region. Arrowhead points to Neurog3 reduced expression. L, M, Neurog3 expression is regulated by Nkx2.2. Immunostaining against Neurog3 and Nkx2.2 on E10.5 spinal cord sections from wild-type, Nkx2.2, or Pax6 mutant embryos. Nkx2.2 mutants lack Neurog3 in the ventral spinal cord. Arrow points to one Neurog3⁺ cell found (L, middle). The expanded Nkx2.2⁺ territory in Pax6 mutants induces a dorsal extension of Neurog3⁺ cells (L, right). M, Number of Neurog3⁺ cells per section. Bars are mean ± SD. Scale bars: A–D, G, I, K, L, 50 μm; E, F, H, J, 20 μm.

Figure 3.

Neurog3 and Ascl1 inversely correlate in the ventral neural tube. A–E, Neurog3 and Ascl1 have a dynamic expression pattern in the ventral spinal cord. Immunohistochemistry against Neurog3 and Ascl1 shows that Neurog3⁺ cells increase in the p3 domain from E9.5 to E11.5 (A), while Ascl1 expression decreases (B). Ascl1 in the ventral hindbrain remained constant throughout development (C). D–E, Number of Neurog3⁺ or Ascl1^HIGH cells in the spinal cord at the indicated stages. F, Measurement of Ascl1 intensity in p3 progenitors of the hindbrain or spinal cord at E11.5. The dashed line is the threshold for considering cells Ascl1^HIGH in E (see Materials and Methods); ***p < 0.001, Mann-Whitney test. G, Spinal p3 cells express Ascl1 at E9.5 but not at E10.5. In Ascl1^CreER;CAG:floxstop-tdTomato embryos; Cre was induced at E9.5 or at E10.5 with TAM. E11.5 spinal cord sections were stained against tdTomato. At E11.5, spinal p3-derived tdTomato⁺ cells were found only when Cre was activated at E9.5 (arrowhead). H–K, Serotonergic and V3 differentiation strictly correlates with Neurog3 and Ascl1-expressing territories. H, I, Relative numbers of p3-derived neuron types: serotonergic (5HT-N) or V3 interneurons (H) and p3 cells expressing Neurog3 or Ascl1^HIGH (I) along the rostrocaudal axis at E11.5. Serotonergic differentiation was determined by GFP-expressing cells in the Nkx2.2⁺ domain of Gata2^GFP embryos (immunostaining), while V3 interneurons were identified by Sim1 expression (in situ hybridization). The pink area denotes the transition between the hindbrain and the spinal cord shown in the scheme. Cell numbers or signal intensity along the anterior–posterior axis were made relative to the sections with maximum number or maximum intensity. Points are mean ± SD. J, K, Representative images of Ascl1 (J) and Neurog3 (K) immunostainings at indicated rostrocaudal coordinates. L–O, Serotonergic specification is impaired in Ascl1 mutants, while spinal V3 neurons are not affected. In situ hybridizations on E11.5 cross sections. The serotonergic-related transcription factors Gata2 (L) and Pet1 (M) were not detected in the hindbrain of Ascl1^−/−, whereas the V3 identity markers Sim1 (N) and Uncx (O) remained unmodified in the spinal cord of Ascl1 mutants. Arrowheads point to p3 cells o3 p3-derived postmitotic neurons. Bars are mean ± SD. Dotted lines represent the dorsal boundary of the Nkx2.2⁺ domain in the same section. Scale bars: 50 μm.

Figure 4.

Neurog3 regulates Ascl1 expression by a mechanism involving Hes5. A–C, The spatial and temporal Neurog3 expression pattern is similar in chick and mouse embryos. Sections of chick E3–E5 ventral hindbrain (A) and spinal cord (B) were hybridized with a probe against cNeurog3. Neurog3 expression was found in the spinal cord beginning at E4, but absent in the ventral hindbrain at all stages. Neurog3 expression pattern in the mouse spinal cord (E9.5–E11.5; C) is included for comparison. D, E, Neurog3 represses Ascl1 expression. E2 chick embryos electroporated with Neurog3-IRES-GFP into the ventral spinal cord (D) or hindbrain (E) showed reduced Ascl1 mRNA levels analyzed at E3 (arrowheads). No changes were found in Nkx2.2 expression assessed by immunohistochemistry (inset in E). GFP labeling shows targeted region. F, Neurog3 misexpression changes neuronal identity in the hindbrain. Cross sections of E5 ventral hindbrain electroporated with Neurog3 were stained with antibodies against GFP and 5-HT, and hybridized with probes against Ascl1 and Sim1. The electroporated side showed reduced Ascl1 levels, suppressed serotonergic specification, and a moderate induction of the V3 neuronal marker Sim1 (arrowheads). G, Coelectroporation of Neurog3 and Ascl1 expression vectors restores 5-HT neuron differentiation in the ventral hindbrain (arrowhead). Cotransfected cross sections of E5 hindbrain were stained with antibodies against GFP and 5-HT. H, Altered neuronal differentiation in the hindbrain after Hes5 misexpression. Cross sections of E5 chick hindbrain electroporated with Hes5-IRES-GFP were hybridized with an Ascl1 probe and labeled with an antibody against 5-HT. The electroporated side showed reduced Ascl1 expression and a suppression of serotonergic differentiation. I, Increased Hes5 levels in the ventral hindbrain after Neurog3 misexpression. E2 chick embryos were electroporated with Neurog3-IRES-GFP and hybridized with cHes5-1 and cHes5-2 probes at E3. In the control side, Hes5 is mildly expressed in the p3 domain (arrow) while induced in the electroporated side (arrowhead). GFP staining reveals the targeted region. J, K, Hes5 expression in the ventral spinal cord depends on Neurog3. E11.5 spinal cord cross sections from wild-type and Neurog3 mutant mice showed reduced Hes5 mRNA levels in the Neurog3^−/− spinal p3 domain (J). Arrowheads point to p3 domain, arrows point to intermediate dorsal progenitors. Quantitation of Hes5 levels in the p3 domain (K). Bars are mean ± SD; **p < 0.01, Mann–Whitney test. Schemes of relations between Neurog3, Hes5, Ascl1, and differentiation of 5-HT neurons are shown. Scale bars: 50 μm.

Figure 5.

Sustained Ascl1 expression in the Neurog3 mutant spinal cord without changes in dorsoventral patterning. A–E, Neurog3 is necessary to downregulate Ascl1 in the spinal cord. Wild-type and Neurog3^−/− E11.5 sections were stained with antibodies against Ascl1 (A) and Nkx2.2 (D). The number of Ascl1⁺ cells was increased in Neurog3^−/− p3 spinal cord (arrowhead, A), without changes in the number of Nkx2.2⁺ cells (D). Quantitation of the number of Ascl1^HIGH (B) and Nkx2.2⁺ (E) cells in the p3 spinal cord. Bars are mean ± SD. C, Quantitative differences in Ascl1 intensity in individual p3 progenitor cells of wild-type and Neurog3-null spinal cords. Dashed line indicates the threshold for considering cells as Ascl1^HIGH in B; ***p < 0.001, Mann–Whitney test. F, G, Dorsoventral patterning is not affected in Neurog3 mutants. The expression of Olig2 (pMN domain) and Nkx2.2 (p3 domain) were assessed by immunohistochemistry in Neurog3 mutant E10.5 spinal cords. Dorsoventral positioning of the boundaries was made relative to the length of the ventricular aperture (100%) in the same sections (G). Boxes are mean limits ± SD. H–J, Motoneuron development is not modified in Neurog3^−/−. Immunohistochemistry against Isl1/2 (H), in situ hybridization with a probe against the vesicular acetylcholine transporter (vAChT; I), and quantitation of the number of Isl1/2⁺ cells per hemisection (J) of wild-type and Neurog3^−/− E10.5 spinal cord. K, L, Foxa2 expression was unaffected in Neurog3^−/− spinal cord. Foxa2 was detected by immunohistochemistry on E11.5 hindbrain and spinal cord sections. In the wild-type hindbrain high levels of Foxa2 are found in the p3 domain (K, left), while it is expressed at low levels in the spinal p3 domain of wild-type and Neurog3 mutants (K, center and right). Quantitation of Foxa2 levels relative to the expression in the floor plate of the same sections (L). Boxes are mean ± SD. Dotted lines indicate the boundaries of the Nkx2.2⁺ territory in the same section. Scale bars: 50 μm.

Figure 6.

Neurog3-Ascl1 repression is unidirectional. A–D, Neurog3 is not expressed in the hindbrain of Ascl1 mutants. E11.5 (A) or E10.5 (B) cross sections of wild-type and Ascl1^−/− hindbrain were stained with an antibody against Neurog3. C, Acute deletion of Ascl1 by TAM at E10 in CAG:CreER;Ascl1^flox/flox embryos did not result in Neurog3 expression in the E11.5 hindbrain (C, left). Immunostaining of Ascl1 after TAM induction shows a reduction in Ascl1-expressing cells (arrows, C, right; see Fig. 3C for comparison). D, The analysis of Neurog3 expression along the anterior–posterior axis of E11.5 wild-type and Ascl1 mutant mice do not show signs of rostral shift in Neurog3 expression. Points are mean ± SD. Nonsignificant differences were found between groups. E–H, Ascl1 does not control the onset of Neurog3 in the p3 spinal cord. Cross sections of E9.5, E10.5, and E11.5 spinal cords of wild-type (E) and Ascl1^−/− (G) mice were stained with an anti-Neurog3 antibody. The appearance of Neurog3 (approximately E10) was not significantly affected in Ascl1 mutants. F, H, Quantitation of the number of Neurog3⁺ cells at indicated stages in wild-type (F) and Ascl1^−/− (H) spinal cords. Bars are mean ± SD. Dotted lines represent the dorsal boundary of the Nkx2.2⁺ domain in the same section. Scale bars: 50 μm.

Figure 7.

Ectopic serotonergic specification in the spinal cord of Neurog3 mutants. A–D, Induction of 5-HT differentiation program in the spinal cord of Neurog3 mutants. E11.5 or E12.5 spinal cord sections were hybridized with probes that recognize the serotonergic-related transcription factors Ascl1 (A), Gata2 (B), Lmx1b (C), and Pet1 (D). In all cases a significantly increased expression was found in the mutant spinal p3 domain. E–H, The heterotopic serotonergic specification in the spinal cord of Neurog3 mutants requires Nkx2.2. Cross sections of E11.5 spinal cord from wild-type, Neurog3^−/−, Nkx2.2/Neurog3, and Pax6/Neurog3 double mutants were analyzed for Ascl1 (E), Gata2 (F), Lmx1b (G), and Pet1 (H) expression by in situ hybridization. Quantitations of the signals are shown on the right. Pax6^−/−;Neurog3^−/− embryos show a ∼2-fold increase in ectopic differentiation of 5-HT neurons in the spinal cord respect to Neurog3 single mutants. Bars are mean ± SD. Arrowheads point to p3 progenitors or postmitotic p3-derived neurons. I–M, Heterotopic 5-HT⁺ neurons in the Neurog3^−/− spinal cord. Cross sections of E12.5 (I, arrowhead) and P0 (J) Neurog3^−/− and wild-type spinal cords were stained with an antibody against 5-HT. Arrows in I point to cellular processes in the ventrolateral funiculus. K, Topological map of serotonergic neurons (n = 98 cells) found in P0 Neurog3^−/− ventral spinal cord. L, M, Cross sections of P0 spinal cords showing an increased density of 5-HT⁺ fibers in ventral funiculus (M) but not in lateral funiculus (L; see boxes in K). N, O, Cross sections of wild-type and Neurog3-null E12.5 spinal cords were hybridized with probes against the serotonin transporter SerT (Slc6a4, N) and the vesicular glutamate transporter vGluT3 (Slc17a8, O), which is also expressed in serotonergic neurons. Arrowheads point to p3-derived neurons. Scale bars: 50 μm.

Figure 8.

Respecification of ventral neurons in Neurog3 mutant spinal cord. A–F, Serotonergic neurons in the Neurog3-null spinal cord are produced at the expense of V3 interneurons. In situ hybridization of Sim1 (A), Uncx (B), and vGluT2 (Slc17a6, C) on E11.5 spinal cord cross sections. Reduced expression was detected in Neurog3^−/− spinal cords compared with wild-type littermates (D–F). Bars are mean ± SD; ***p < 0.001, Mann–Whitney test. Arrowheads point to p3 progenitor cells or postmitotic neurons generated from this domain. G, H, Ectopic 5-HT neurons as well as a reduced V3 population arise from Neurog3^−/− p3 domain, and segregate afterward. Wild-type and Neurog3^−/− spinal cord cross sections were hybridized with Gata2, Sim1, and Pet1 probes at E11.5 (G) and E12.5 (H). Signals from adjacent sections were overlaid and pseudocolored. At E11.5 newborn ectopic Gata2/Pet1-expressing cells and Sim1⁺ neurons emanate both from p3 progenitors (G). By E12.5 each population occupies different regions: Pet1⁺ neurons are found adjacent to the ventrolateral funiculus while Sim1⁺ neurons distribute throughout the ventromedial spinal cord (H). Scale bars: 50 μm. I, Scheme of neuronal specification in the p3 domain of the hindbrain and spinal cord, and the role of the transcription factor Neurog3 in restricting serotonergic neuron differentiation to the hindbrain. Ventral signals (Shh) induce the expression of common transcriptional regulators in both the hindbrain and the spinal cord p3 early progenitors. Caudalizing signals, such as RA, induce the onset of Neurog3 in p3 precursors of the spinal cord. Neurog3 represses Ascl1 through a process mediated by Hes genes and thus prevents serotonergic specification in the spinal cord. Neurog3-dependent downregulation of Ascl1 is likely to involve cell–cell interactions within the spinal p3 domain.