Fig. 1.

Hoxa2 expression in the FNC alters gene expression in the developing head. a Experimental design for FNC transfection and implantation into a stage-matched naive embryo at 5ss. a′ Bilateral electroporation of NC at cephalic level with Fluorescein Dextran in a 5ss-embryo. The FNC, which extends from the mide-diencephalon down to r2, is delineated with dotted line; once transfected, the FNC is subjected for transplantation into a naive untransfected embryo. a″ At 7ss, fluorescent FNC cells that have been engrafted have started to migrate (b–e) Head morphology 24 h after eletroporation and surgery. b, c External appearance and d, e internal morphology of cephalic vesicles (dotted lines) in control (b, d) and Hoxa2-transfected (c, e) embryos, showing that Hoxa2 expression in the FNC alters face and brain development. f, g HNK1-Mab labeling of migrating FNC cells; despite morphological changes, the forced expression of Hoxa2 in the FNC does not alter FNC cell migration. h–y Analysis of the expression of Fgf8 (h–j), Bmp4 (k–m), Shh (n–p), Wnt8b (q–s, arrowheads; dotted lines indicate the boundary between the diencephalon posteriorly and the telencephalon anteriorly), Noggin (t–v) and Six2 (w–y) in control (h, k, n, q, t, w), Hoxa2-transfected (i, l, o, r, u, x), and Six2-silenced (j, m, p, s, v, y) embryos. While Six2 expression is inhibited in the FNC-derived mesenchyme, its expression in the pharyngeal endoderm persists after Hoxa2 activation or Six2 silencing in FNC cells. Changes in gene expression are shown with arrowheads (z) RT-PCR analysis of Hoxa2, Noggin, Dan, and Six2 expression in NF-derived cells grown in vitro for 24 h, taken from r4 to r8 NC, FNC, and Hoxa2-transfected and Six2-silenced FNC. Di diencephalon, Mes mesencephalon, Rh rhombencephalon, Tel telencephalon