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First published online 30 June 2004
doi: 10.1242/dev.01235

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Fgf3 signaling from the ventral diencephalon is required for early specification and subsequent survival of the zebrafish adenohypophysis

Wiebke Herzog^1,*, Carmen Sonntag¹, Sophia von der Hardt¹, Henry H. Roehl^2,, Zoltan M. Varga³ and Matthias Hammerschmidt^1,

¹ Max-Planck Institute for Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany
² Max-Planck Institute for Developmental Biology, Spemannstraße 35, 72076 Tübingen, Germany
³ University of Freiburg, Department of Biology I, Hauptstraße 1, 79104 Freiburg, Germany

View larger version (58K): [in a new window]   Fig. 1. The four lia mutations cause exchanges or deletions of conserved amino acid residues of Fgf3 protein, leading to a complete loss of Fgf3 activity. (A) Amino acid alignment of Fgf3 proteins from zebrafish, rat, mouse and human. Conserved amino acids are boxed, amino acid residues mutated in the different lia alleles are highlighted in black, with the new amino acid residues indicated in red. The premature protein termination caused by the t24152 mutation is indicated with a star (*). (B) Phenotypic classes (F1-F4) and frequencies (%) obtained upon injection of wild-type or mutant fgf3 mRNAs at concentrations of 0.1 or 10 ng/µl. F1 embryos are characterized by a loss of the eyes and a normal tail; F2 by a loss of eyes, fore- and midbrain, and a curled up, truncated tail; F3 embryos by a complete loss of head and tail; and F4 embryos by lysis around the 10-15 somite stage. Data were obtained in two independent experiments. n, numbers of scored embryos.

View larger version (88K): [in a new window]   Fig. 2. fgf3 mutant embryos display defects in otic vesicles, ventral head skeleton and pharyngeal endoderm. Probes used in whole-mount in-situ hybridizations are indicated in the lower right corners, ages of embryos in the upper right corners. Left panels (A,C,E,G) show wild-type siblings (wt), right panels (B,D,F,H) fgf3 mutants (lia). Embryos were genotyped after photography. (A-F) Lateral views. (G,H) Dorsal views on heads. `k' in C marks krox20 in rhombomeres 3 and 5. Arrows and numbers in C,D mark neural crest streams to the corresponding pharyngeal arches (I, mandibular; II, hyoid; III, IV, V, gill arches). Arrows in E mark pharyngeal pouches of gill arches. (G,H) Alcian Blue staining of craniofacial cartilage; arrows in G indicate ceratobranchials of the gill arches, arrow in H remaining part of first ceratobranchial of fgf3 mutant. Arrowheads mark otoliths, which are fused in the mutant. bb, basibranchial; cb, ceratobranchials; ch, ceratohyal; i, isthmus; mb, mandibulare; nc, neural crest; ppe, pharyngeal pouch endoderm; pt, pallial (dorsal) telencephalon; os, optic stalk; spt, subpallial telencephalon; vt, ventral thalamus; pvh, posterior-ventral hypothalamus (infundibulum; presumptive neurohypophysis).

View larger version (132K): [in a new window]   Fig. 3. The adenohypophyseal anlage is faced by fgf3-expressing telencephalic cells early, and ventral diencephalic cells later. Probes used for in-situ hybridizations are indicated in the corresponding colors in the lower right corners, ages of embryos in the upper right corners. (A-C,F-K,N) Lateral views on head region. (D,E,L,O) Transverse optical sections. (N) Sagittal section. White arrows in B indicate levels of optical cross sections shown in (D,E). Numbers in G mark pharyngeal arches (see Fig. 2). Arrow in H indicates fgf8-positive cells in posteriormost regions of the posterior-ventral hypothalamus. Arrow in I indicates level of optical cross section shown in L, arrow in J indicates sagittal section shown in M, arrow in N cross section shown in O. ad, adenohypophysis; ep, epiphysis; fe, facial ectoderm; h, hypothalamus; i, isthmus; os, optic stalk; pe, placodal ectoderm; po, polster; pvh, posterior-ventral hypothalamus; ppe, pharyngeal pouch endoderm.

View larger version (98K): [in a new window]   Fig. 4. fgf3 mutants fail to initiate the expression of early pituitary-specifying genes, whereas the hypothalamus develops normally. Probes used for in situ-hybridizations are indicated in the lower right corners. (A,C,E,G,I,K) wild-type siblings (wt). (B,D,F,H,J,L) fgf3 mutants (lia), with age of embryos indicated in the upper right corners. (A,B) Tailbud stage, animal views, dorsal to the right; embryos were genotyped after photography. (C-L) Lateral views on heads. Arrows in (C,E) indicate expression in anterior domain, arrowheads expression in lateral posterior domains of adenohypophyseal anlage [compare with Fig. 3M and Nica et al. (Nica et al., 2004)]. Arrowheads in (K) indicate the two adenohypophyseal pomc expression domains. anc, endorphin-synthesizing arcuate nuclei cells of hypothalamus; fe, facial ectoderm; adh, anterior-dorsal hypothalamus; i, isthmus; os, optic stalk; pvh, posterior-ventral hypothalamus.

View larger version (73K): [in a new window]   Fig. 5. Fgf3 signaling from the diencephalon, but not the telencephalon, is sufficient for adenohypophyseal development. (A,C-H) Embryos at 32 hpf, lateral views (A,C,F,G), or frontal views (D,F,H), on head regions. (A) Embryo stained for fgf3 transcripts. Note the fissure of the eye vesicle as a reference point for the anterior border of the infundibular fgf3 expression domain. (B) Cartoon showing the transplantation sites at shield stages. Consistent with results obtained in fate-mapping experiments (Woo and Fraser, 1995; Varga et al., 1999; Mathieu et al., 2002), telencephalic chimeras as shown in G,H were obtained by transplanting cells from/to the animal pole, diencephalic chimeras as shown in C-F by transplanting cells from/to dorsal regions anterior/animal of the shield. Cells of the adenohypophysis (ad) are supposed to derive from region indicated by arrowhead. (C-H) Chimeras, in-situ hybridized for lim3 transcripts in blue, and with transplanted wild-type cells in brown. Out-of-focus bilateral lim3 domains in D,F,H represent hindbrain motoneurons (Glasgow et al., 1997). (C,D) Wild-type recipients with wild-type donor cells in the diencephalon, displaying normal adenohypophyseal lim3 expression (indicated by arrows). (E,F) fgf3 mutant recipients with infundibular wild-type cells (indicated by arrowheads) and adjacent rescued adenohypophyseal lim3 expression (indicated by arrows). Inset in E shows second rescued embryo. (G,H) fgf3 mutant recipient with many wild-type cells in the telencephalon, still lacking adenohypophyseal lim3 expression.

View larger version (84K): [in a new window]   Fig. 6. Fgf signaling governing adenohypophysis development is required during midsegmentation stages. All panels show embryos after lim3 in-situ hybridization. Adenohypophyseal lim3 staining in (A-C,E,G) is indicated by arrows. (A-D) Bead implantations, performed at 18 hpf. Panels show lateral views on heads of embryos at 26 hpf. Embryos were genotyped after photography. (A) Implant of Fgf3 bead into wild-type sibling embryo. (B,C) Implant of Fgf3 beads into mutant embryos, leading to partial rescue of adenohypophyseal lim3 expression; note bead position far from the telencephalon in (B). (D) Implant of BSA bead into mutant embryo. (E-H) SU5402 treatments. (E,F) Lateral views, (G,H) frontal views on heads of embryos at 34 hpf. Incubation of embryo in 20 µM SU5402 from 18-22 hpf leads to complete loss of adenohypophyseal lim3 expression, while control embryo treated with DMSO is unaffected.

View larger version (133K): [in a new window]   Fig. 7. Shh cannot rescue the normal adenohypophysis of fgf3 mutants, but can induce ectopic adenohypophyseal cell specification in an Fgf-dependent manner. All panels show embryos injected with shh mRNA at the 1-cell stage, frontal views on heads. Probes used for in-situ hybridizations are indicated in the lower right corners, ages of embryos, and genotype or further treatment in the upper right corners. (A,C) Wild-type controls; the regular pomc or prl expression domains are indicated by arrows. (B) fgf3 mutant, displaying pomc-positive cells in ectopic positions, whereas the regular region of the adenohypophyseal anlage remains pomc-negative embryo. (D) Moderately affected embryo treated with 12 µM SU54302 from 15-32 hpf, displaying a partial loss of both endogenous and ectopic prl-positive cells. The effect on ectopic prl expression was weaker after SU5402 treatment from 18-22 hpf (data not shown), suggesting that Fgf signaling promoting adenohypophyseal specification in ectopic positions occurs over a longer time period than that by Fgf3 from the infundibular region required for the regular adenohypophysis (compare with Fig. 6E-H).

View larger version (77K): [in a new window]   Fig. 8. Non-specifying adenohypophyseal cells of fgf3 mutants undergo apoptosis. (A-D,I,J) Nomarski images, (G,H) confocal images; frontal views onto the anterior border of the head, superimposed with fluorescent images of Acridine Orange stainings in green (C,D,J), or rhodamine cell labelings in red (G-J). All images are at same magnification; scale bar shown in (I) = 25 µm. In A-D,I, borders of the adenohypophysis are indicated with white arrowheads. All embryos were genotyped after evaluation and photography. (A) Nomarski images at 25 hpf; wild-type sibling; (B) fgf3 mutant, displaying a normal-sized adenohypophysis. For better contrast, images are not superimposed with Acridine Orange stainings, which showed no positive cells for wild-type (A), but few positive cells for mutant (B). (C,D) Acridine Orange stainings at 28 hpf; (C) wild-type sibling; (D) fgf3 mutant. (E,F) Summary of single cell-tracing experiments: cartoons showing the position of single cells from the anterior neural ridge region labeled at the tailbud stage, relative to mesodermal polster outlined in gray, which served as a landmark for the injections. A square corresponds to 50 µm x 50 µm. The fate of each cell is indicated by shape and color. Circles mark cells whose daughter cells were alive 28-30 hpf, crosses indicate cells that died during the course of the analysis, resulting in labeled cell debris only. Red circles mark adenohypophyseal clones, blue circles olfactory epithelium clones, yellow circles facial ectoderm clones, and salmon circles clones ending up in the head mesenchyme. Mixed clones with descendants in two different tissues are indicated with two-colored circles. Numbers and arrows mark cells with descendants shown in G-J. (E) Cells from wild-type siblings; (F) cells from fgf3 mutants. In F, red crosses represent clones with debris within and outside the shrunken adenohypophysis. Gray crosses are clones with debris outside the adenohypophysis only, which had not been investigated at 24 hpf. Therefore, their initial tissue belongings cannot be stated; however, they most likely derive from adenohypophyseal cells that had died early. (G) Descendants of wild-type cell 54 at 29 hpf. (H) Cell debris deriving from mutant cell 49. (I,J) Time course analyses of mutant cell 84. (I) 25 hpf; (H) same embryo at 30 hpf, counterstained with Acridine Orange.

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