NH innervation and vascularisation are compromised in zebrafish fgf3 mutants. Whole-mount in situ hybridisation (A-H), or immunohistochemical analyses (whole-mounts: I,J,M,N; sections: K,L,O,P), and confocal images (Q-T) of wild-type (wt) and fgf3–/– zebrafish; lateral (A-P) or ventral (Q-T) views; anterior to the left. (A-H) fgf3–/– display robust crabp1a expression in the NH anlage at 33 hpf (B) and 48 hpf (D); prl+ AH cells are largely (B) or completely (D) lost. At 72 hpf, crapb1a expression is moderately reduced (E,F); only a remnant of diffuse expression detected at 120 hpf (G,H). (I-L) Axons enter the NH and then extend throughout its antero-posterior length in wt larvae, but remain outside the fgf3 mutant NH. AH and NH outlined by yellow dots. (M-P) At 120 hpf, AcTub+ axons in the wt NH include both TH+ (M) and Avpl+ (O) axons. Neither is detected in fgf3 mutant NH (N,P). (Q-T) fgf3 mutants display normal numbers of Avpl+ neurons in the hypothalamus (Q,R), but lose Avpl+ axonal NH innervation (T), the hypophyseal artery and the two bilateral hypophyseal capillaries (T). The hypophyseal vein remains present (T). Q and R display maximal confocal projections through midbrain area. Arrow (Q) points to pituitary region, magnified and intensified in S. Panels S and T show single confocal sections at higher magnification in same specimens as in Q and R. Red arrows (S) point to hypophyseal capillaries; green arrowheads to GFP+ axons of avpl:egfp+ neurons projecting to NH; green arrow to GFP+ axonal termini within NH. ah, adenohypophysis; cadi, caudal division of internal carotid artery; hyv, hypophyseal vein; nihl, nucleus of the inferior hypothalamic lobe; nh, neurohypophysis; oc, oral cavity; po, preoptic area. Scale bars: 50 μm.
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