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doi: 10.1242/10.1242/dev.00427

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Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b

¹ Institute of Life Science, National Defense Medical Center, Taipei, Taiwan
² Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan

View larger version (126K): [in a new window]   Fig. 1. The structure and ultrastructure of adult zebrafish head kidney. (A) The zebrafish head kidney is located in the anterior part of kidney. (B) In situ hybridization of scc, which identifies the interrenal gland of the head kidney (arrows, ventral view). Asterisks indicate pigment. (C) Higher magnification of the interrenal gland of the right lobe. (D) A section of the gland. (E,F) Higher magnification of the boxed regions in G and D, respectively. (G) Interrenal epithelial cells (arrows) lie adjacent to a blood vessel (asterisks). rt, renal tube. (H) Transmission electron micrographs showing interrenal epithelial cells (i) associated with adrenaline (a) and noradrenaline (n) chromaffin cells. (I) The interrenal epithelial cell has many mitochondria (m). Nu, nucleus. (J) The adrenaline cell (a) contains electron-lucent vesicles and the noradrenaline cell (n) contains vesicles with strong electron-dense granules. Scale bars; 10 µm (C,D,G), 5 µm (E,F), 1.1 µm (H), 0.6 µm (I), and 0.4 µm (J).

View larger version (73K): [in a new window]   Fig. 2. Marker gene expression in the interrenal primordium. In situ hyridization showing expression of scc (A), 3ß-HSD (B) and ff1b (C) in the region ventral to the third somite and wt1 (D) expression in a wider region ventral to the second and third somites at 36 hpf. (E-J) Co-expression of ff1b and scc at 30 (E-G) and 33 hpf (H-J). Green indicates scc expression, red indicates ff1b expression and yellow indicates the merged signal of scc and ff1b.

View larger version (145K): [in a new window]   Fig. 3. Histological analysis of the interrenal primordium at 3 dpf. (A-C) Interrenal primordium (irp) is located ventral to the notochord (n) and dorsal to the gut (g). (A,B) Hematoxylin and Eosin staining and (C) hybridization of the cross sections of interrenal primordium with ff1b. (B,C) Higher magnification of A. The blue arrowhead indicates the capsule like structure. (D) The interrenal primordial cells have many mitochondria in their cytoplasm. (E) Higher magnification of the dashed square in D. g, gut; m, mitochondria; n, notochord; Nu, nucleus; p, pigment; pt, pronephric duct. Scale bar: 1.1 µm (D), 0.25 µm (E).

View larger version (100K): [in a new window]   Fig. 4. Morphogenetic movement of the interrenal gland from 20 hpf to 3 dpf. (A-E) Cartoons showing development of pronephric (black) and interrenal (red) primordia; ventral view. The notochord is represented in light blue; the second and third somites are shown as dark blue rectangles. (F-O) wt1 (black) and ff1b (red) double in situ hybridization; (F-J) lateral view, (K-O) ventral view. (A,F,K) At 20-22 hpf, the wt-expressing cells lie on both sides of the notochord and some regions are also labeled with ff1b. (B,G,L) At 24 hpf, the ff1b-expressing cells are separated from the wt1-expressing cells on both sides of the notochord. (C,H,M) At 30 hpf, the wt1-expressing cells are still separate, whereas the ff1b-expressing cells have assembled towards the right of the notochord (left when viewed ventrally). (D,I,N) At 33 hpf, the wt1-expressing cells start to fuse. (E,J,O) At 3 dpf, the wt1 cells occupy the midline, while the ff1b-expressing cells continue to proliferate and redistribute on both sides of the notochord.

View larger version (104K): [in a new window]   Fig. 5. Migration of the interrenal primordium in mutants defective in midline signaling. The internal primordia are labeled by scc. A group of fused interrenal primordial cells are located slightly to the right of the notochord (left in this ventral view) in wild-type (wt; A), syu (B), smu (C), and cyc (F) embryos. In the flh (D) and oep (E) embryos, the two groups of interrenal primordial cells do not fuse.

View larger version (106K): [in a new window]   Fig. 6. Knockdown of wt1 by morpholino affects early development of both the pronephros and interrenal glands. The pronephric and interrenal primordia of wild-type (A,D,G) and wt1 morphants (mo; C,F,I) are labeled with wt1 (black) and ff1b (red) (ventral view). (B,E,H) The pronephric primordia of ff1a morphants are labeled with wt1 (brown). (A-C) 24 hpf, (D-F) 36 hpf, (G-I) 2 dpf. The pronephric primordia in C are more spread out than in A and B. The pronephric primordia in F,I are smaller and unfused. The sizes of interrenal primordia and ff1b expression in wt1 morphants (C,F,I) are also reduced.

View larger version (112K): [in a new window]   Fig. 7. Disruption of ff1b expression by antisense morpholinos reduces interrenal primordia and decreases ff1b/scc expression. The interrenal primordia of the wild-type embryos (A,D,G), ff1a morphants (B,E,H) and ff1b morphants (C,F,I) are labeled with ff1b (brown color) and also (in A,C,D,F) scc (red color). (A-F) Ventral view, (G-I) side view. The sizes of the interrenal primordia (arrows) and the expression of ff1b and scc are reduced in ff1b morphants at 24 hpf (C), 33 hpf (F) and 3 dpf (I), while the hypothalamic region, marked by ff1b (arrowheads) becomes larger (I).

View larger version (14K): [in a new window]   Fig. 8. ff1b can activate human SCC transcription directly. Zebrafish ff1b was transiently co-transfected into H1299 cells with either wild-type or mutated SCC/lacZ reporter. The activity of the mutated SCC/lacZ reporter alone was set as 100.