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The role of Phox2b in synchronizing pan-neuronal and type-specific aspects of neurogenesis

¹ CNRS UMR 8542, Département de Biologie, Ecole Normale Supérieure, 46, rue d'Ulm, 75230 Paris Cedex 05, France
² Laboratoire de Génétique et Physiologie du Développement, IBDM, CNRS-INSERM-Université de la Méditerranée, Campus de Luminy case 907, 13288 Marseille Cedex 9, France

View larger version (76K): [in a new window]   Fig. 1. Phox2b misexpression promotes Ngn2 but not Cash1 expression. In this and the following figures, light stippled lines demarcate the transfected area. A,A',A'' means that these are either consecutive sections from the same embryo (for in situ hybridization) or the same sections photographed through different filters (for GFP fluorescence and fluorescent antibody labeling). A broken line indicates the lumen of the neural tube. The time of incubation after electroporation (h.a.e.) is indicated above each panel. (A-D) Ngn2 and Mash1 have proneural activity in the chick neural tube. Overexpression of mouse Ngn2 or Mash1 in spinal regions of the chick neural tube upregulates Delta1 (A,A',C,C'). At 24 h.a.e. of mouse Ngn2, the transfected cells start to relocate to the mantle layer (ML) (A), and migration is complete at 48 h.a.e. (B). At 18 h.a.e. of Mash1, the transfected cells are still in the neuroepithelium (C), but they have relocated to the ML at 48 h.a.e. (D). In control transfections, most cells were still in the neuroepithelium at 48 h.a.e. (see Dubreuil et al., 2000). (E-H') In situ hybridization with Ngn2 and Cash1 probes on spinal cord sections at different times after electroporation of mPhox2b. GFP fluorescence shows the extent of electroporation. (F',I) Forced expression of mPhox2b expands the Ngn2 expression domain at 6 h.a.e.; the effect becomes less pronounced at 18 h.a.e. (H'). Double-labeling with a Ngn2 probe and anti-Phox2b antibodies (I) shows that most cells expressing Ngn2 ectopically also express mPhox2b (arrowheads in right panel). The dorsalmost region of the neural tube appears refractory to Ngn2 induction. Cash1 is not induced at 6 h.a.e. (E') or at 24 h.a.e. (G'). Note that at the earlier time point (HH 15/16), Cash1 is not yet expressed at spinal levels of the neural tube (E'). (J) Quantitative analysis of Ngn2 induction by mPhox2b at different times after electroporation. Results are expressed as the difference in Ngn2 signal intensity between the transfected and the non-transfected sides (arbitrary units). Data points represent mean values±s.e.m. from more than 35 sections from at least five transfected embryos.

View larger version (115K): [in a new window]   Fig. 2. Phox2b induces Cash1 in combination with Nkx2.2. In situ hybridization on transverse sections of the spinal cord with Cash1 (A',B',C',D') and Ngn2 (A'',B'') probes at 6 (A-A'') and 20 (B-B'') h.a.e. of mPhox2b plus mouse Nkx2.2 (0.5 mg/ml) or at 20 h.a.e. of 0.5 mg/ml (C,C') or 1 mg/ml (D,D') mouse Nkx2.2 alone. The extent of transfection is shown by in situ hybridization with a GFP probe (A-C) or by GFP fluorescence (D). Mouse Nkx2.2 (0.5 mg/ml) does not induce Cash1 (C'), but does so in combination with mPhox2b (B'). Ngn2 is repressed in the area where Cash1 has been induced (B''). At 1 mg/ml, mNkx2.2 transfection alone elicits a modest increase in Cash1 expression (D'). No effects on Cash1 or Ngn2 expression were observed at 6 h.a.e. (A' and A'').

View larger version (94K): [in a new window]   Fig. 3. Effects of Phox2b overexpression in the hindbrain. (A-C'') Phox2b promotes migration to the ML and induces endogenous Islet1/2 and Phox2b expression. In situ hybridization on transverse sections of the caudal hindbrain with GFP (A), mPhox2b (B) and chicken Phox2b (B') probes and double-labeling with anti-Islet1/2 (C) and anti-mPhox2b (C') antibodies. At 48 h.a.e. of mPhox2b into the hindbrain, the transfected cells have relocated to the ML (B), an effect not seen in control transfections with GFP alone (A). The mPhox2b-expressing cells also express the endogenous Phox2b gene (B'). The ventral expression domain of endogenous Phox2b corresponds to the vm precursors. Islet1/2 is expressed in ventral motoneurons and by the cells that ectopically express mPhox2b (C,C'). (D-G') Phox2b upregulates Ngn2 and Cash1 in combination with Nkx2.2. In situ hybridization on transverse sections of the caudal hindbrain using mPhox2b (D-F), mouse Nkx2.2 (G), Ngn2 (D') and Cash1 (E',F',G') probes. mPhox2b alone upregulates Ngn2 (D'), but not Cash1 (E'). Nkx2.2 (0.5 mg/ml) alone does not induce Cash1 (G'), but does so when co-electroporated with Phox2b (F').

View larger version (80K): [in a new window]   Fig. 5. Regulatory interactions between Phox2b and transcription factors expressed in the progenitor domain. (A-A''') Mash1 induces Islet1, but not other motoneuronal markers. Anti-Islet1/2 immunostaining (A') and in situ hybridization with Islet2 (A'') and ChAT (A''') probes at 48 h.a.e. of Mash1 on spinal cord sections. At this time point, the transfected cells have relocated to the mantle layer, as shown by in situ hybridization with a GFP probe. (A) Forced expression of Mash1 in the spinal cord induces ectopic Islet 1/2+ (A') cells, but neither Islet2 (A'') nor ChAT (A''). The effect of Mash1 overexpression on Islet1/2 expression has been quantitated by counting the Islet1/2+ cells in the transfected area and in an equivalent area of the non-transfected side, excluding the region of the Islet1/2+ spinal motoneurons. A total of 692 Islet1/2+ cells were counted on the transfected versus 296 cells on the control side, which amounts to an average difference of 9.7±0.27 cells/section (mean±s.e.m., P<0.0001, 40 sections from six embryos). (B-D'') Phox2b upregulates Nkx6.1 and Nkx6.2 and represses Pax6 and Olig2. In situ hybridization on spinal cord sections with Nkx2.2 (B'), chick Phox2b (B''), Nkx6.2 (C'), Olig2 (C''), Nkx6.1 (D') and Pax6 (D'') probes at 24 h.a.e. of mPhox2b. The extent of electroporation is shown on adjacent sections by anti-mPhox2b immunohistochemistry (B) or in situ hybridization with a GFP probe as indicated. mPhox2b misexpression induces the endogenous Phox2b gene (B'') and expands the expression domains of Nkx6.1 (D') and Nkx6.2 (C'). By contrast, Olig2 (C'') and Pax6 (D'') expression is repressed. Nkx2.2 expression is not induced by mPhox2b (B').

View larger version (125K): [in a new window]   Fig. 4. Phox2b represses, while Ngn2 upregulates negative regulators of neurogenesis. (A-F) Hes5 overexpression inhibits neuronal differentiation and counteracts the increase in Delta1 expression in response to Phox2b. In situ hybridization with Delta1 (A',C',E') and NeuroM (B',D') probes on transverse spinal cord sections. The extent of transfection is shown by GFP in situ hybridization as indicated. Hes5 overexpression downregulates Delta1 (C') and NeuroM (D') expression. The promotion of Delta1 expression by mPhox2b (A') is reduced by co-transfecting Hes5 (E'). (F) Quantitative analysis of Delta1 expression in response to mPhox2b, Hes5 or mPhox2b plus Hes5. Results are expressed as the difference in Delta1 signal intensity between the transfected and the non-transfected sides. Data points represent mean values±s.e.m. (arbitrary units) from more than 35 sections from at least four transfected embryos. *P<0.05, **P<0.001 with respect to transfection of mPhox2b alone. (G-J') Id2 overexpression inhibits neuronal differentiation and counteracts the increase in Delta1 and NeuroM expression in response to Phox2b. In situ hybridization with Delta1 (G',I') and NeuroM (H',J') probes on transverse spinal cord sections. The extent of transfection is shown by GFP in situ hybridization as indicated. Id2 overexpression downregulates NeuroM expression (H'); expression of Delta1 is only marginally affected (G'). The promotion of Delta1 and NeuroM expression by mPhox2b (A',B') is reduced by co-transfecting Id2 (compare A' and B' with I' and J'). (K-N'') Phox2b represses chick Hes5b and Id2, while Ngn2 upregulates their expression. In situ hybridization with chick Hes5b (K',L',N') and Id2 (K'',L'',N'') probes alone or combined with anti-BrdU immunohistochemistry (M) (brown nuclei) on spinal cord sections of embryos electroporated with either mPhox2b (K-M) or mouse Ngn2 (N-N''). In situ hybridization with a GFP probe (L,N) or GFP fluorescence (K) show the transfected area. Repression of chick Hes5b and Id2 is already observed at 6 h.a.e. of mPhox2b (K',K'') and has become pronounced at 24 h.a.e (L',L''). (M) After an 1 hour BrdU pulse, virtually all BrdU-positive cells on the untransfected (left) side are chick Hes5b- or Id2-positive, while in the transfected area (right) chick Hes5b or Id2 expression is absent in both BrdU-positive and -negative cells. The broken lines in M outline the lumen of the neural tube. In contrast to mPhox2b, mouse Ngn2 overexpression promotes chick Hes5b (N') and Id2 (N'') expression.

View larger version (65K): [in a new window]   Fig. 6. Phox2b acts as an activator in promoting neurogenesis and inducing Islet1, Nkx6.1 and endogenous Phox2b. (A-B') at 48 h.a.e. of PHDVP16 (A) but not of PHDEnR (B), the transfected cells have emigrated to the mantle layer as shown by GFP fluorescence. Anti-Islet1/2+ cells are found in ectopic location after expressing the PHDVP16 (arrowheads, A') but not the PHDEnR construct (B'). (C-J') in situ hybridization with Ngn2 (C' ,E' ,F'), Ngn1 (D'), Phox2b (G' ,J') probes at 6 h.a.e. of PHDVP16 (C'), PHDEnR (E') and SHDVP16 (F'), at 20 h.a.e. of PHDVP16 (D'), or at 24 h.a.e. of PHDVP16 (G' ,H') or PHDEnR (I' ,J'). GFP fluorescence (A-C) or in situ hybridization with a GFP probe (D-J) shows the extent of electroporation. At 6 h.a.e. of PHDVP16, Ngn2 is upregulated in the transfected area (C') while PHDEnR or SHDVP16 constructs have no effect (E' ,F'). Forced expression of PHDVP16 also promotes expression of Ngn1 (D'), induces the endogenous Phox2b gene (G') and expands Nkx6.1 expression (H'), while PHDEnR does not induce Phox2b (I') and represses Nkx6.1 (J').

View larger version (82K): [in a new window]   Fig. 7. Chick Hes5b and Id2 are repressed by activator and repressor forms of Phox2b and by expressing the native homeodomain. (A-D'') In situ hybridization with chick Hes5b (A'-D') and Id2 (A''-D'') probes at 24 h.a.e. of PHDVP16 (A-A''), PHDEnR (B-B''), PHD (C-C'') and SHDVP16 (D-D'') constructs. The extent of electroporation is shown by GFP fluorescence (C) or in situ hybridization with a GFP probe (all others). Both, Phox2b transcriptional activator (PHDVP16) or repressor (PHDEnR) forms repress chick Hes5b (A',B') and Id2 (A'',B''). The repressive activity is shared by the Phox2 HD (C',C'), but not by control constructs with the Siamois HD (SHDVP16) (D',D'').

View larger version (29K): [in a new window]   Fig. 8. Postulated genetic interactions during cranial motoneuron differentiation in the ventral hindbrain and upon misexpression of Phox2b in the dorsolateral neural tube. (A) Within the bm/vm progenitor domain, Phox2b in combination with Nkx2.2 drives Mash1 expression, which in turn promotes generic neuronal differentiation. Repression of Hes and Id gene expression by Phox2b may be necessary to overcome a negative feedback on neuronal differentiation triggered by Mash1. In the progenitor domain, Nkx2.2 and Phox2b repress Pax6 and Olig2, which can be supposed to inhibit bm/vm specification. Phox2b is postulated to boost expression of the Nkx6 genes that are required for proper development of bm/vm neurons. The inhibitory interaction between Pax6 and Phox2b is reciprocal. (B) Upon misexpression in the dorsolateral spinal cord and caudal hindbrain, Phox2b is postulated to cooperate with an unknown HD factor (X) to upregulate Ngn2, which in turn drives pan-neuronal differentiation. By boosting expression of Hes5 and Id2, Ngn2 triggers a negative feedback loop that is relieved by Phox2b. In addition, Phox2b directs a bm/vm fate, in part by repressing Pax6 and Olig2, which are not permissive for this fate, and by upregulating expression of the Nkx6 genes, which are compatible with it. In A,B, the positive autoregulation of Phox2b has been omitted for clarity.