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An expanded domain of fgf3 expression in the hindbrain of zebrafish valentino mutants results in mis-patterning of the otic vesicle

Biology Department, Texas A&M University, College Station, TX 77843-3258, USA

View larger version (98K): [in a new window]   Fig. 1. Patterns of hair cells in the otic vesicle. Lateral view of otic vesicles of live val/val (A,B) and wild-type (C) embryos viewed under DIC optics at 21 h. val/val mutants have small, round otic vesicles, and otoliths vary in number and position. (D,E) Dorsolateral view of deltaA expression in the otic vesicle at 19 h in val/val (D) and wild-type (E) embryos. Arrowheads indicate nascent tether cells. (F-H) Dorsolateral view of otic vesicles showing hair cells stained with anti-Pax2 (red) and anti-acetylated tubulin (green) antibodies. (F) val/val mutant at 24 h. Seven hair cells are distributed along the length of the anteroposterior axis of the otic vesicle. (G) val/val mutant at 30 h. An ectopic patch of hair cells (arrowhead) is evident between the anterior and posterior maculae. (H) Wild-type embryo at 30 h. (I-K) Dorsolateral view of val/val mutants at 27 h stained with anti-Pax2 to visualize hair cell nuclei. The number and distribution of hair cells are variable. Anterior is towards the left in all specimens. Scale bar: 20 µm in A-C; 15 µm in D,E; 30 µm in F-H; 40 µm in I-K.

View larger version (11K): [in a new window]   Fig. 2. Time course of hair cell formation in the otic vesicle. Embryos were fixed at the indicated times and hair cells were visualized by Pax2 staining. Each datum is the mean number of hair cells per ear (±s.d.) of 10 or more specimens. val/val mutants produce excess hair cells throughout the time course. •, wild type; val/val embryos.

View larger version (96K): [in a new window]   Fig. 3. Expression of AP markers in the inner ear. Lateral or dorsolateral views of the otic vesicle (anterior towards the left). (A-D) pax5 expression at 24 h (A,B) and 30 h (C,D). Staining is limited to the anterior end of the otic vesicle in wild-type embryos (A,C) but is greatly expanded in val/val mutants (B,D). The midbrain-hindbrain border (mhb) is indicated. (E,F) Expression of nkx5.1 at 24 h in wild-type (E) and val/val (F) embryos. Expression is expanded posteriorly in val/val mutants. (G,H) Expression of zp23 at 24 h in wild-type (G) and val/val (H) embryos. No expression is detectable in the ear in val/val mutants. Relative positions of rhombomeres are indicated. Scale bar: 25 µm in A,B,G,H; 75 µm in C,D; 50 µm in E,F.

View larger version (118K): [in a new window]   Fig. 4. DV and ML patterning in the inner ear. (A-D) Expression of otx1 at 24 h in wild-type (A,C) and val/val (B,D) embryos. Dorsal views (A,B) show expression in the lateral epithelium of the otic vesicle (arrowheads) and lateral views (C,D) show expression in the ventral epithelium. (E,F) Dorsolateral views showing expression of dlx3 at 24 h in wild-type (E) and val/val (F) embryos. Gene expression patterns are normal. (G-I) Lateral views of the inner ear at 72 h in wild-type (G) and val/val (H,I) embryos. Morphology ranges from nearly normal to highly aberrant. Anterior is towards the left in all specimens. Abbreviations: a, anterior semicircular canal; 1, lateral semicircular canal; p, posterior semicircular canal; u, utricle. Scale bar: 100 µm in A,B,G-I; 50 µm in C-F.

View larger version (73K): [in a new window]   Fig. 5. Expression of fgf8 and fgf3 in the hindbrain. Dorsal view (anterior towards the left) of specimens double stained for Fgf gene expression (blue) and krox20 (red). Loss of krox20 staining in r5 identifies val/val mutants. (A,B) fgf8 expression at 12 h in wild-type (A) and val/val (B) embryos. Brackets indicate the r4 domain of fgf8. No change is detected in the mutant. (C,D) fgf3 expression at 12 h in wild-type (C) and val/val (D) embryos. (E,F) fgf3 expression at 14 h in wild-type (E) and val/val (F) embryo. Brackets indicate the domain of fgf3 corresponding to either r4 (C,E) or r4 to rX (D,F). fgf3 is ectopically expressed in the rX region in val/val embryos. Scale bar: 80 µm.

View larger version (52K): [in a new window]   Fig. 6. Effects of mis-expressing val. (A,B) Dorsal views showing expression of fgf3 (blue) and krox20 (red) at 14 h in a normal embryo (A) and an embryo injected with val RNA (B). The val-injected embryo shows little or no fgf3 expression in the r4 domain (arrowheads) and has undergone less convergence than normal. (C,D) Lateral view of a val-injected embryo at 24 h. Trunk and tail tissues are ablated (C) and no otic vesicle is visible (D). (E,F) Dorsal views of val-injected embryos with relatively normal axial development. (E) Expression of fgf3 (blue) and krox20 (red) at 14 h. The left side of r4 shows little fgf3 expression (arrowhead) whereas the right side is nearly normal (bracket). (F) Expression of pax2.1 at 24 h in the midbrain-hindbrain border (mhb) and otic vesicles (ov). The left otic vesicle (broken circle) is severely disrupted. (G,H) Expression of fgf8 at 12 h in a normal wild-type embryo (G) and a val-injected embryo (H). The val-injected embryo has a truncated axis (not shown) and has undergone less convergence than normal. Nevertheless, fgf8 is expressed relatively normally in the prechordal plate (p), midbrain-hindbrain border (mhb) and rhombomere 4 (r4). Anterior is towards the left in all panels. Scale bar: 100 µm in A,B,D-H; 250 µm in C.

View larger version (98K): [in a new window]   Fig. 7. Effects of fgf3 knockdown on inner ear development. Dorsolateral view (anterior towards the left) of otic vesicles in embryos injected with fgf3-MO. (A-C) In situ hybridization of pax5 at 24 h in injected wild-type (A) and injected val/val (B,C) embryos. Expression levels are greatly reduced in half to two-thirds of embryos (see text for details). (D,E) Expression of zp23 at 24 h in injected wild-type (D) and injected val/val (E) embryos. Expression is detected throughout the medial wall of the otic vesicle, including cells adjacent to r4. (F) In situ hybridization of nkx5.1 at 24 h in an injected val/val embryo. No expression is detected in the otic vesicle. (G-I) Anti-Pax2 staining at 30 h in injected wild-type (G) and injected val/val (H,I) embryos. The number of hair cells is reduced relative to uninjected controls, and the majority (19/25) of val/val embryos do not produce ectopic hair cells. fgf3-depleted val/val embryos with extremely small otic vesicles (I) produced anterior hair cells only. Relative positions of rhombomeres are indicated. Scale bar: 70 µm in A-C,F; 50 µm in D,E; 30 µm in G-I.

View larger version (70K): [in a new window]   Fig. 8. Effects of fgf8 dysfunction on inner ear development. (A,B) Dorsal view of the hindbrain at 14 h showing expression of fgf3 (blue, with brackets) and krox20 (red) in ace/ace (A) and ace/ace; val/val (B) embryos. (C,D) Dorsolateral view showing pax5 expression in the otic vesicle at 24 h in ace/ace (C) and ace/ace; val/val (D) embryos. (E,F) Dorsolateral view showing anti-Pax2 staining in the otic vesicle at 30 h in ace/ace (E) and ace/ace; val/val (F) embryos. Relative positions of rhombomeres are indicated. Double mutants show ectopic expression of fgf3 in rX (B), ectopic expression of pax5 (D) and ectopic hair cells in the otic vesicle (F). Anterior is towards the left in all specimens. Scale bar: 80 µm in A,B; 30 µm in C-F.