Gene expression profiles within midline cells of mouse neural tube. (A) Schematic depicting the dynamics of gene expression and Shh gradient within the ventral neural tube. (nc) Notochord. (B–K) Expression of FoxA2, Arx, Shh, Nkx2.2, and Olig2 (B–J), and Shh (K) mRNA showing the progressive appearance of FP markers at the midline of the neural tube from 7ss embryos. (B–K) Transverse sections at brachial levels of the neural tube. (D–D″) Lateral view of a whole embryo. (C–D″) FoxA2 protein is first observed from 7ss in ventral midline cells that express Nkx2.2. (D–D″) FoxA2 expression appears more robust than the scattered Nkx2.2 expression at posterior levels of the neural tube. (B) At these stages, FoxA2 expression domain abuts Olig2-expressing cells. (E,E″) From E9.5 onward, the dorsal limit of FoxA2 is no longer adjacent to Olig2-expressing cells. Its expression is stronger at the midline, and weaker in cells that express Nkx2.2. (E–F′) This pattern persists until E10.5. From this stage, additional FP markers, Arx (G–I) and Shh (J,K), appear in cells expressing high levels of FoxA2. (G–H′) Concomitant to the induction of these markers, Nkx2.2 is no longer seen in the midline; hence, Arx and Shh are barely coexpressed with Nkx2.2-expressing cells.

Induction of FP markers by transient high levels of Shh signaling. (A) Schematic of HH10 chick embryos indicating the position of [i] neural plate explants. (B–E) Expression of Arx and Nkx2.2 in [i] neural plate explants after 48 h of culture. Cells in explants treated with 1 nM Shh exclusively express Nkx2.2 (B), while 4 nM Shh promotes FP fate, identified by Arx expression (C). In explants first exposed for 24 h to 4 nM Shh and then placed in a media either devoid of Shh (D) or containing 500 nM cyclopamine (cyclo) (E), FP induction is retained. (F) Quantification of cells expressing Arx and Nkx2.2 in [i] neural plate explants after 24 or 48 h of culture (n ≥ 4 explants, number of cells per unit ± SD). (G) Temporal dynamics of Nkx2.2, Arx, FoxA2, and Ptch1 expression relative to Actin transcripts and normalized between sets of experiments assayed by quantitative PCR in [i] neural plate explants treated with 1 nM (pale green) or 4 nM (pink) Shh (n ≥ 2 experiments, mean ± SD). As definitive FP markers are induced, p3 markers and Ptch1 are down-regulated. (H–P) Expression of Ptch1 (H–J,N–O) and Gli1 (K–M,P) in mouse embryos (H–M) and chick embryos (N–P). Insets in H and I indicate Arx, Nkx2.2, and Olig2 expression on sections adjacent to the main panel. Midline cells transiently express high levels of Ptch1 and Gli1. The asterisks indicate the notochord.

Requirement for Shh signaling prior to somitogenesis for FP specification. (A–L) Arx, FoxA2, Nkx2.2, Olig2, and Pax6 expression at forelimb levels of the neural tube in embryos cultured with vehicle, EtOH (A,C,E,I,G,K), 5 μM cyclopamine (B,D,F,H), or 10 μM cyclopamine (J,L) from the headfold stage (A–D), 3–5ss (E–H), and E9.5 (I–L) for 48, 36, and 24 h, respectively. Cyclopamine treatment affected the expression of p3 and pMN markers in all conditions, but only inhibited the emergence of FP markers if added prior to 3ss. (M–N) Transfection of Ptc1^Δloop2 in the ventral neural tube of HH12 chick embryos assayed for Arx, FoxA2, and Nkx2.2. Ptc1^Δloop2 does not affect the expression of FP markers (M,M″), but down-regulates Nkx2.2 expression (N). (O–V) Zebrafish embryos assayed for foxa2 and shh expression 48 hpf treated with vehicle, EtOH + DMSO (O,S), 800 μM SB431542 alone from 30% epiboly (P,T), or a combination of 5 μM cyclopamine and 800 μM SB431542 added at either 30% epiboly (Q,U) or 16ss (R,V). (O,S) In control embryos, shh and foxa2 were expressed within the medial FP (mFP) and the lateral and medial FP (l+mFP), respectively. While in embryos treated with SB431542 alone, both genes are expressed in the spinal cord (P,T; foxa2: n = 24 of 26; shh: n = 27 of 29), embryos treated with SB431542 and cyclopamine from 30% epiboly lack cells expressing these genes (Q,U; foxa2: 25 of 33; in eight of 33 a few cells were observed; shh: 23 of 31). The addition of cyclopmaine at 16ss in SB431542-treated embryos does not impair shh or foxa2 expression in the medial FP, but down-regulates foxa2 expression in the lateral FP (foxa2: 34 of 37; in three embryos the expression was weak; shh: 21 of 28; see also Supplemental Fig. S3). (W–Y) Zebrafish cyclops mutant embryos assayed for foxa2 expression at 48 hpf treated with vehicle, EtOH (W), or 5 μM cyclopamine added at either 30% epiboly (X) or 16ss (T). Embryos treated with cyclopamine from 30% epiboly lack cells expressing foxa2 (n = 9 of 9), while those treated from 16ss or with vehicle do express foxa2 (n = 8 of 9 and 7 of 7, respectively). The asterisks indicate absence of expression.

Temporal restriction to FP induction in vivo. (A,E) Schematic of electroporation performed in chick embryos at HH12 and HH8, respectively. (B–D,E–F″) Expression of Arx, FoxA2, Nkx2.2, and Gfp in chick embryos electroporated with ShhN. ShhN induces a p3 identity at brachial levels of HH12 embryos (B–D), but only induces FP markers more caudally (inset in D). (E–G″) In contrast, in HH8 embryos, it activates FP markers along the rostral–caudal axis. (H) Schematic of HH10 chick embryos indicating the position of [i-nt] and neural plate [i] explants. (I) Quantification of cells expressing Arx and Nkx2.2 in [i] or [i-nt] explants after 36 or 48 h of culture (n ≥ 4, number of cells per unit ± SD). [i-nt] explants do not express FP markers when exposed to high concentrations of Shh. Likewise, [i] explants do not express Arx when cultured in media devoid of Shh for 12 h before being transferred to high concentrations of Shh. (J) Temporal dynamics of Ptch1 expression relative to Actin levels assayed by RT–PCR in [i] explants exposed to 4 nM Shh from the start of the culture (pink) or after 12 h (pale green) (n = 2, mean ± SD). [i] neural plate explants do not produce the high peak of Ptch1 expression when initially exposed to a media devoid of Shh.

Down-regulation of Shh signaling is required for the expression of definitive FP markers. (A–F) Transfection of Gli3^HIGH and SmoM2 in the ventral midline of HH11–12 chick embryos assayed for Arx, Nkx2.2, FoxA2, GFP, and Ptch1 expression 48 hpe. Gli3^HIGH switches FP cells into a p3 identity (A–C), while SmoM2 does not affect the fate of these cells (D–F). (G–J) Expression of Gli2 in E9.5 and E10.5 mouse (G–I) and HH18 chick (J) embryos. Gli2 expression is progressively down-regulated from ventral midline cells in amniotes. (K–L″) Gli2 electroporated into ventral midline cells of HH12 chick embryos, assayed for Arx, Nkx2.2, FoxA2, and GFP expression 48 hpe. Similar to Gli3^HIGH, Gli2 down-regulates the expression of FP markers and induces Nkx2.2 expression.

Shh signaling relayed by FoxA2 during FP specification. (A–F). Transfection of FoxA2 and FoxA2^EnR in HH11–12 chick embryos assayed for Arx, Nkx2.2, Shh, Ptch1, and GFP expression 24 and 48 hpe. FoxA2^EnR blocks FP specification and induces Ptch1 expression. Conversely, FoxA2 is sufficient to induce Arx and Shh expression and to down-regulate Ptch1 expression. (G) Coelectroporation of FoxA2 and Ptc1^Δloop2 in HH11–12 chick embryos assayed for Arx after 48 hpe indicates that FoxA2 is able to induce FP identity independently of Shh signaling. (H,I) Electroporation of SmoM2 alone or together with FoxA2 assayed for Arx and Nkx2.2 48 hpe. FoxA2 inhibits the ability of SmoM2 to induce ectopic p3 cells. (J) Gli activity (relative activity ± SEM) measured with GBS-Luc reporter in HH11–12 chick neural tube electroporated with GFP or SmoM2, either alone or together with FoxA2. FoxA2 inhibits the ability of SmoM2 to induce Gli activity.

Dynamics of FP specification. (A) A schematic of the dynamics of signaling and gene expression profiles within the ventral neural tube. At early times (t0), the Shh target gene Ptch1 is restricted to the most ventral cells, and the pMN marker Olig2 is initiated in cells at a distance from the ventral midline. At later times (t1), Ptch1 and Olig2 expression expand dorsally; the p3 progenitor markers Nkx2.2 and low levels (L) of FoxA2 are induced in the ventral midline. Subsequently (t2), p3 and pMN domains shift dorsally, and Ptch1 is down-regulated in midline cells; at the same time, midline cells adopt a definitive FP identity expressing high (H) levels of FoxA2, Arx, and Shh. (B) State space map of neural progenitors' response to Shh signaling. FP (red) requires high but transient Shh signaling early in development, while p3 identity (green) can be induced by Shh signaling at later times. The dashed line represents the path of a p3 precursor cell, and the solid line represents the path of a FP precursor cell. Cells exposed to high levels of Shh signaling early in development initially express Nkx2.2 before inducing definitive FP markers such as Arx. Importantly, the acquisition of a FP state results in cells becoming refractory to Shh signaling (represented by the solid circle); however, if signaling is maintained artificially, these cells acquire a p3 identity. In contrast, a neural progenitor initially exposed to a lower level of signaling first adopts a pMN identity (blue). If the level of signaling is appropriate, the cell then transitions into a p3 identity, and this identity is kept if sufficient signaling levels are maintained.