(A) Schematic of expression patterns of progenitor and neuronal subtype marker proteins in the ventral neural tube. FP, floor plate. (B) Quantification of cells expressing Evx1, En1, Chx10, and MNR2, markers of V0, V1, V2, and MNs, respectively, in [i] explants cultured for 48 h in the indicated concentrations of Shh (n≥4; cells/unit ± s.d.). (C) Summary of experimental scheme for (D) and (E). Explants were exposed to 0.5 nM Shh for 6–48 h (blue columns), then transferred to fresh media lacking Shh (gray columns) and incubation continued until a total of 48 h had elapsed. (D) Quantification of cells expressing Evx1, En1, Chx10, and MNR2 after 48 h of ex vivo culture of [i] explants exposed to 0.5 nM Shh for the indicated times (n≥4; cells/unit ± s.d.). (E) Quantification of cells expressing the progenitor markers Pax7, Dbx1, Nkx6.1, and Olig2 after 48 h of ex vivo culture of [i] explants exposed to 0.5 nM Shh for the indicated times (n≥4; cells/unit ± s.d.).

(A) Scheme for experiments in (B) and (C). Explants were exposed to 0.5 nM Shh (blue columns) for 36 h, either continuously (a) or after 18 h some were transferred to media containing 500 nM cyclopamine (Cyc.) and 0.5 nM Shh (b; red columns) to block Shh signalling. All explants were assayed 36 h after the start of culture. (B) Representative images of explants assayed for Dbx1, Nkx6.1, and Olig2 expression in each condition. (C) Quantification of cells expressing Dbx1, Nkx6.1, and Olig2 in [i] explants exposed to 0.5 nM Shh for 36 h (a) or 0.5 nM Shh for 18 h followed by 500 nM cyclopamine and 0.5 nM Shh (b) for an additional 18 h (n≥4; cells/unit ± s.d.). (D) Scheme of the experiments in (E) and (F). Explants were exposed to 4 nM Shh (blue columns) for 18 h or 36 h. After 18 h, some explants were transferred to media containing 500 nM cyclopamine and 4 nM Shh (red columns); others were transferred to basal media lacking added factors (gray columns). Explants were assayed for marker gene expression at 18 h (i) or 36 h (ii–iv) and for Gli activity at 24 h after the start of culture. (E) Fold increase in Gli activity, compared to untreated explants (relative Gli activity ± s.e.m.) measured with GBS-Luc, in [i] explants treated as indicated in the schema. At 18 h (i) and 24 h (ii) 4 nM Shh induced ∼20–25-fold increase in Gli activity. Within 6 h of blockade of Shh signalling with cyclopamine, Gli activity levels returned to pre-stimulus levels (iv). Removal of Shh resulted in a reduction of Shh signalling at 6 h (iii), however levels 3–4-fold above basal were still present at this time point. (F) Explants, cultured in the conditions indicated in (D), assayed for Olig2 and Nkx2.2 expression. After 18 h exposure to 4 nM Shh (i) explants expressed a mixture of Olig2 and Nkx2.2 with some cells co-expressing both markers (inset). Treatment with 4 nM Shh for 36 h resulted in the repression of Olig2 and the exclusive expression of Nkx2.2 (ii). In explants assayed at 36 h in which 4 nM Shh had been removed 18 h earlier, Nkx2.2 expression had largely been lost while Olig2 expression was maintained (iii). In contrast, blockade of Shh signalling with cyclopamine at 18 h resulted in the loss of both Olig2 and Nkx2.2 (iv); in this condition progenitors expressed high levels of Pax6.

(A) A Shh induced negative feedback loop involving Gli mediated transcriptional induction of the Shh receptor Ptc1 produces a temporally dynamic output of Gli activity in response to fixed Shh input. (B) A state space model of ventral neural tube patterning. The range of time (x-axis) and levels (y-axis) of Gli activity necessary to induce each positional identity (p0–p3 and pMN) are illustrated by the coloured regions on the graph. This indicates that more ventral progenitor identities require higher levels and longer durations of Gli activity. On to this state space representation, the profile of Gli activity generated by different concentrations of Shh is indicated by the black lines. The incrementally thicker lines represent the profile generated by increasingly higher concentrations of Shh. The diagram shows how the differing profiles of Gli activity generate different positional identities in a progressive manner in the neural tube. (C) Positional information corresponds to the time integral of Gli activity. The time integral of Gli activity, equivalent to the area under the curves in (B), provides the correlate of positional information. Increasingly more ventral progenitors are generated at correspondingly higher time integrals of Gli activity.

(A) Experimental scheme. The reporter plasmid, which carries the coding sequence of the firefly luciferase gene (FF-luciferase) driven by octameric Gli-binding sites (8xGBS), was introduced with a normalization plasmid, containing Renilla luciferase, into HH st.10 chick embryos by in ovo electroporation. [i] Explants were removed and cultured ex vivo in the presence of different concentrations of recombinant Shh. Dual luciferase assays were performed at appropriate time points. (B) Gli activity, measured with GBS-Luc, in neural cells exposed to the indicated concentrations of Shh (nM) for 6 h. Graph shows the fold induction of Gli activity in response to Shh compared to the level of Gli activity in untreated explants (relative Gli activity ± s.e.m.). Between 0.1 nM and 1 nM Shh the level of Gli activity at 6 h is a function of Shh concentration. (C) Temporal dynamics of Gli activity in explants exposed to Shh. The fold increase in Gli activity (relative Gli activity ± s.e.m.) measured with GBS-Luc in [i] explants treated with either 0.25 nM Shh (blue) or 2 nM Shh (green) for the indicated times. Data for 1 nM Shh (red dashed line) taken from Dessaud et al. (2007) [24] is included for comparison. Cells become adapted to these concentrations of Shh; moreover the time taken for Gli activity to return to less than 4-fold above pre-stimulus levels (inset) when exposed to the indicated concentrations of Shh is proportional to Shh concentration.

(A) Experimental scheme. Explants were exposed to 0.5 nM Shh for 6–36 h (blue columns), then fixed directly and assayed for the expression of Dbx1, Nkx6.1, and Olig2. (B) Quantification of cells expressing Dbx1, Nkx6.1, and Olig2 in [i] explants exposed to 0.5 nM Shh for the indicated time (n≥4; cells/unit ± s.d.). Dbx1 expression peaked by 18 h, while the number of cells expressing Nkx6.1 and Olig2 continued to increase over time. (C) Strategy for permanently marking cells that have expressed Dbx1. Mice harbouring a Cre recombinase in the Dbx1 locus were crossed with ROSA26^floxSTOP^floxYFP mice. In cells that express Dbx1, expression of Cre results in a recombination event removing the STOP cassette from ROSA26^floxSTOP^floxYFP, thus allowing LacZ expression. The ubiquitously expressed ROSA26 locus directs continued YFP expression even after Dbx1 is downregulated. (D, E) Two examples of e11.5 embryos containing Dbx1cre and ROSA26^floxSTOP^floxYFP assayed for YFP and either cre (D) or Olig2 (E) expression. The expression of cre from the Dbx1 locus is limited to the p0 progenitor domain in the intermediate region of the neural tube. YFP expression is observed within the cre expressing progenitors but also in more ventrally located progenitors including some that express the pMN marker Olig2. This indicates that Dbx1 is transiently expressed in progenitors ventral to later expression pattern.

(A) Expression of Olig2 (red), Nkx2.2 (green), and Arx (which marks specifically floor plate cells; white) [84] in HH st.18 chick embryos. (B) In ovo blockade of Shh signalling using cyclopamine (Cyc). HH st.18 embryos were treated with vehicle (HBC; i) or cyclopamine (ii, iii) in ovo and incubated for 24 h. Assay for Nkx2.2 (green), Olig2 (red), and Arx (white) expression at anterior thoracic levels revealed a ventral shift and marked decrease in the number of cells expressing Nkx2.2 and Olig2 compared to HBC control embryos and HH st.18 embryos. In addition, some Olig2 and Nkx2.2 double-positive cells can be seen, close to the floor plate, in the Nkx2.2-positive domain (arrowheads) in embryos exposed to cyclopamine. (C) In ovo inhibition of Shh signalling by expression of Ptc1^Δloop2. HH st.18 embryos in ovo electroporated with Ptc1^Δloop2IRES-GFP were assayed 24 h after transfection for the expression of Olig2 and Nkx2.2 and GFP, which marks transfected cells. Cells expressing Ptc1^Δloop2 downregulated Olig2 and Nkx2.2 expression in a cell autonomous manner. (D–I) Expression of Olig2 (red) and Nkx2.2 (green) at the brachial levels in the mouse embryos in which the conditional allele of Shh^flox/flox has been deleted with Brn4cre (conditional knockout of Shh; Shh^CKO). At e9.5, expression of both Olig2 and Nkx2.2 in Shh^CKO embryos (E) is indistinguishable from that in wild type (D). At e10.5, however, there was a marked reduction in the mutants (G), compared to the control littermates (F), and at e12.5, the expression had completely declined (H, I). (J) Lineage tracing of Olig2 progeny. E10.5 embryos containing Olig2-Cre and ROSA26^floxSTOP^floxLacZ assayed for β-Gal and Olig2 expression. β-Gal expression is observed in a small number of progenitors dorsal to the pMN domain that do not express Olig2 (inset j). This suggests that Olig2 is transiently expressed dorsal to its normal domain of expression. (K) Assaying Olig2-Cre; ROSA26^floxSTOP^floxGFP embryos at e11.5 revealed some GFP cells co-expressing the V2 marker Chx10 (arrowheads; inset k). This is consistent with the transient expression of Olig2 in progenitor cells of the p2 domain that then generate V2 neurons.