Regulation of barx1+ condensations by Edn1 and Notch.
(A) At 36 hpf, the intermediate sox9a domain (green) only partially overlaps with zones of barx1 expression (magenta) at the ventral and dorsal poles of each arch. The oral ectoderm (oe) and first pharyngeal pouch (p1) are shown for reference. (B, C) jag1b (green) and barx1 (magenta) are anti-correlated in dorsal NCCs at 36 and 48 hpf. (D-J) barx1 expression at 36 hpf in the first and second arches of wild-type controls, mutants, and overexpression embryos. Open arrowheads show the loss of ventral barx1 in edn1 mutants (E) and its restoration in 5/6 jag1b; edn1 mutants (J). The blue arrow in E indicates weak upregulation of barx1 in the intermediate domain of edn1 mutants. Upregulation of barx1 in the dorsal first arch (white arrowhead) and dorsal second arch (white arrow) is seen in Edn1-overexpressing embryos (F), jag1b mutants (G), notch2; notch3 mutants (H), and jag1b; edn1 mutants (J). Dotted lines in (I) show the arches of NICD-overexpression embryos in which barx1 is nearly absent. (K, L) Ectopic barx1 persists in jag1b mutants at least until 48 hpf, but no ectopic expression of sox9a is observed. (M, N) Representative barx1 expression patterns and skeletal preparations in embryos treated with the Notch inhibitor DBZ (10 μM) starting at the indicated time points. Earlier exposure to the DBZ inhibitor correlated with stronger ectopic barx1 expression (M) and more severe and penetrant Notch-type skeletal phenotypes (N). Fractions indicate the number of embryos in each treatment that exhibited unambiguous ectopic barx1 expression in the dorsal first arch (arrowheads in M) or showed posterior Pq malformations (arrowheads in N. DBZ treatment also caused systemic effects, including spinal curvature and cardiac edema, which reduced bone mineralization and led to a general reduction in the size of the craniofacial skeleton. Scale bars in B, C, J, L, M = 20 μm; scale bar in N = 100 μm.