Fig. 3. Forced expression of Hoxa2 in the entire Hox-negative neural fold and contribution of Hoxa2 neural fold-derived cells to facial development. (A) The experimental procedure consists in the unilateral co-electroporation of Hoxa2 RCAS (BP)B (red) and GFP (green) constructs in the Hox-negative neural fold, performed alternatively on each side of two different 5 ss chick embryos. The transfected neural folds are excised and bilaterally transposed to homotopic level into a stage-matched recipient chick embryo. Concurrently, the untransfected contralateral neural folds are bilaterally engrafted in a chick embryo that serves as a control. (B) Dorsal view of E3 embryo (HH11) showing the migration of GFP-labeled cells. (C) E3 embryo (HH14) after hybridization for Hoxa2, showing (1) endogenous expression in the second branchial arch (open arrow); and (2) ectopic expression in mesectodermal cells that both fill the first arch and nasofrontal bud and overlay the prosencephalic, mesencephalic and metencephalic vesicles (arrows). At E7, control embryos, in which the contralateral non transfected neural folds have been grafted, (D,D') show a normal development of both upper and lower beaks. By contrast, embryos engrafted with Hoxa2 transfected neural crest (E,E') do not develop facial structures. At E4, embryos in which the diencephalic neural fold has been transfected with the Hoxa2 construct (F), show a severe misdevelopment of the forebrain vesicles compared with control embryos (G). (H) Hoxa2 neural fold-derived cells form glial cells in peripheral nerves, as observed after HNK1 immunolabeling. At diencephalic level (I), neural crest cells give rise to pericytes (that accumulate smooth -actin, detected by 1A4 Ab) to the forebrain vasculature (J), and differentiate into cilliary muscles and corneal endothelium (arrows, K). Di, diencephalon; Mes, mesencephalon; Met, metencephalon. Scale bars: 350 µm in F; 400 µm in G; 50 µm in H; 600 µm in I; 60 µm in J; 75 µm in K.

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