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pax2.1 is required for the development of thyroid follicles in zebrafish

Thomas Wendl1, Klaus Lun2, Marina Mione3, Jack Favor4, Michael Brand2, Stephen W. Wilson3 and Klaus B. Rohr3,*,{dagger}

1 Institute for Developmental Biology, University of Cologne, Gyrhofstrasse 17, D-50923 Köln, Germany
2 Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany
3 Department of Anatomy and Developmental Biology, UCL, Gower Street, London WC1E 6BT, UK
4 GSF-National Research Center for Environment and Health, Institute for Human Genetics, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany
* Present address: Institute for Developmental Biology, University of Cologne, Gyrhofstrasse 17, D-50923 Köln, Germany



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  		Fig. 1. Reconstruction of the thyroid gland in adult zebrafish. (A) Cross-section of an adult zebrafish head in the ventral gill region at the level of the second branchial arch (Haematoxylin and Eosin staining). The arrows indicate the thyroid follicles. (B-F) High magnification views of thyroid follicles of adult zebrafish. (B) Haematoxylin and Eosin staining. The arrow shows the follicular epithelium. (C,D) Giemsa staining. The arrows show the follicular epithelium in C and the nuclei of the epithelium in D, the arrowhead indicates a vesicle at the apical surface of the follicular cells. (E) PAS staining visualising the follicular epithelium (arrow) in medium pink and the colloid in light pink; the arrowheads indicate vesicles at the apical surface of the follicular cells. Cartilage is stained strongly. (F) Immunostaining with an antibody against T4 reveals strong reactivity of the follicular cells (arrow). (G) Reconstruction of the distribution of thyroid follicles in the adult zebrafish head. Thyroid follicles in blue, heart and ventral aorta in red, gill arches in yellow. ca, cartilage; co, colloid; g, gills; mo, mouth/pharynx; va, ventral aorta.

 


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  		Fig. 2. T4 immunostaining in the early (4.5-5 dpf) zebrafish larva. (A-I) Selected cross-sections at 4.5 dpf, T4 immunostained (H,I, high magnification). (A) Overview. Cross-section through the head at an anteroposterior level between D and E (compare with schematic drawing in J). Cartilage is highlighted by colours as in J (yellow, first branchial arch; magenta, second branchial arch; green, basibranchial cartilage that is composed of derivatives of all branchial arches). The arrow shows immunostaining in the anterior non-follicular domain, the arrowhead a pigment cell. Insert indicates adjacent control section, processed for immunostaining without first antibody. Note absence of immunostaining (arrow). The arrowhead indicates the same pigment cell as in the adjacent section. (B-G) Selected sections from one embryo at different anteroposterior levels as indicated in J. Arrows indicate immunostaining in the thyroid. (H) Close up of the anterior non-follicular T4 domain. (I) Close up showing a follicle further posterior. (J) Schematic drawing of a zebrafish larval head of 4.5 to 6 dpf, ventral view, showing the skeleton, parts of the circulatory system and the thyroid. The approximate positions of the sections shown in B-G are indicated as grey bars. Blue, thyroid/T4 immunostaining; brown, third to sixth branchial arches; green, basibranchial cartilage; magenta, second branchial arch; orange, heart and ventral aorta; yellow, first branchial arch. bb, basibranchial cartilage; ch, ceratohyale; co, colloid; h, heart; me, Meckel’s cartilage; pq, palatoquadrate; va, ventral aorta.

 


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  		Fig. 3. Marker gene expression in the zebrafish thyroid primordium. (A,D,G) hhex (A,G) or hhex plus nk2.1a (D) expression in the thyroid (arrows). (B,E,H) pax8 (B, H) or pax8 plus hhex (E) expression in the thyroid (arrows). (C,F,I) pax2.1 (C,I) or pax2.1 plus pax8 (F) expression in the thyroid (arrows). (J) Lower jaw area of 4 dpf wild-type larva, lateral view. Arrows indicate the nk2.1a expression in patches along the ventral aorta (arrowheads). (K) Cryosection of a 7 dpf wild-type larva, showing pax8 (arrow) expression in the area of a thyroid follicle. (L) Cryosection of a 7 dpf wild-type larva, showing pax2.1 expression (arrow) in the area of a thyroid follicle. The position of the sections in K,L corresponds roughly to the right arrow in J. e, Pax gene expression in the eye; h, heart, H, cartilage of the hyoid arch (second branchial arch); M, cartilage of the mandibular arch (first branchial arch); m, Pax gene expression in the midbrain-hindbrain boundary; mo, mouth cavity/pharynx.

 


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  		Fig. 4. Histological analysis and T4 immunostaining in wild-type and noitu29–/– mutant zebrafish larvae at 7-8 dpf. (A,B) Haematoxylin and Eosin staining: selected section of a wild-type sibling (A) showing a thyroid follicle and noitu29–/– larva (B), in the area where normally follicles would form. (C,D) PAS staining: selected section of a wild-type sibling (C) and noitu29–/– larva (D), in the area where normally follicles would form. (E-L) T4 immunostaining. (E,G,I,K) Selected sections of a wild-type sibling with the anterior domain of T4 localisation (E,G) and three follicles out of a total of seven follicles present in this specimen (I,K). The follicles are distributed along the ventral aorta, ventral to the basibranchial cartilage (compare with Fig. 2J). (F,H,J,L) Four selected sections of a 7 dpf noitu29–/– larva, showing the presence of the anterior domain of T4 localisation (F,H). No follicles were found in any of the noi–/– mutants (J,L). Arrows point to follicles and/or T4 immunostaining, the asterisks mark the heard oedema that is forming in noitu29–/–. bb, basibranchial cartilage; ch, ceratohyale.

 


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  		Fig. 5. Expression of molecular markers in noi–/– mutant and sibling embryos. (A,B,E,F) hhex expression, (C,D,G,H) nk2.1a expression, (I,J) pax8 expression, (K-N) pax2.1 expression. Embryo age is indicated in the bottom left-hand corner. The arrows indicate the thyroid primordium.

 


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  		Fig. 6. Expression of Pax2 in wild-type mouse embryos. (A-C) E9 embryos, whole-mount in situ hybridisation. (A,B) Pax8 expression. Arrows show thyroid primordium, arrowheads indicate Pax8 expression in the visceral arches. (A) Lateral view, (B) Cross-section at the level of the thyroid primordium. (C) Pax2 expression, lateral view as in A. (D-F) Neighbouring sections of E15.5 wild-type embryo. (D) Nkx2.1 expression, arrow shows thyroid gland, arrowhead Nkx2.1 expression in the tracheal epithelium. (E) Pax8 expression (arrow shows thyroid gland), (F) Pax2 expression (insert shows positive staining in the CNS on the same section as a positive control). e, Pax2 expression in the eye; fb, Pax2 expression in the forebrain; m, Pax2 expression in the midbrain; n, neural tube; ot, otic vesicle; p, pharynx.

 

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