Figure 3.

Axial Identity Is Established in Cells prior to Neural Identity

(A–C) The average accessibility (Z score) of region-specific sites over time in anterior (labeled “A”), hindbrain (labeled “H”), or spinal cord (labeled “SC”) conditions. AP-specific sites become accessible between D3 and D4. Spinal cord progenitors do not transiently open sites corresponding to anterior (A) or hindbrain (B) identity before opening spinal cord-specific sites (C).

(D) Neural sites become accessible in all regions at the same time. Error bars = SD.

(E) Schematic of the differentiation (H+ condition).

(F–N) qRT-PCR of genes at D3 and D5 following the differentiation of cells to hindbrain (D5H), spinal cord (D5SC), or “hindbrain+” (D5H+) identity. The WNT target Notum (F) is observed following WNT signaling treatment at D3 (D3NMP) and D5 (D5H+). Induction of posterior spinal cord Hox genes Hoxb9 and Hoxc8 is dependent on timing: induction in D3NMP follows D2 to D3 treatment with WNT signals, but not at D5 in D5H+ cells following D4 to D5 treatment with the same signals (G and H). Induction of T/Bra and Cdx2 is dependent on timing, responding to early (D2 to D3), but not late (D4 to D5), treatment with WNT signals (I and J). Late treatment of WNT in the D5H+ condition prevents expression of ventral neural genes Phox2b and Olig2 (K and L, compare D5H to D5H+) while dorsal Pax7 (M) and intermediate Dbx1 (N) neural tube genes are induced. Error bars represent the standard deviation.

(O) SOX1 immunofluorescence on D3 versus D4 cells cultured in hindbrain (D3A and D4H) or spinal cord (D3NMP and D4SC) conditions. Scale bars represent 20 μm.

(P) Sox1 expression, detected by mRNA-seq (Gouti et al., 2014), at the indicated times and conditions. Error bars = SEM.