Fig. 2

Failure of serotonergic differentiation in VPA-treated embryos is mimicked by blockade of Hdac1 activity and is associated with downregulation of ascl1b. (A) hdac1 mutation or pharmacological blockade mimics the effect of VPA on the differentiation of brainstem neuronal subtypes. At 48 hpf, hdac1^s436 mutants lack 5HT neurons (n=15/15, P<0.0001) and show a severe depletion of Isl1⁺ motor neurons. Treatment with TSA from 24 to 48 hpf [condition (vii)] selectively affects 5HT expression (n=30/30, P<0.0001), but other neuronal subtypes are retained. Scale bar: 50 µm. (B) Partial recovery of 5HT neuronal differentiation in hdac1^s436 mutants at 72 hpf (n=14/14). Scale bar: 50 µm. (C) Short duration (8 hour) exposure to VPA from 24 hpf [condition (vi)] leads to the downregulation of ascl1b expression (n=15/15), and similar loss of ascl1b expression is observed in hdac1^s436 mutants (n=10/10). Rhombomeres (r) marked by arrowheads. Scale bar: 50 µm. (D) Double in situ hybridisation showing ascl1b expression in serotonergic progenitors, marked by expression of nkx2.2. Co-labelled cells are indicated by white arrowheads. Scale bar: 20 µm. (E) Short-duration VPA treatment [condition (viii)] does not affect the expression of the paralogous gene ascl1a, or other progenitor-expressed determinants of serotonergic fate, nkx2.2, foxa2 or shh (arrowheads).