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Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid

¹ McLaughlin Research Institute for Biomedical Sciences, Great Falls, MT 59405, USA
² INSERM 129, ICGM, 75014 Paris, France
³ Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA

View larger version (72K): [in a new window]   Fig. 1. Eya1–/– embryos lack thymus and parathyroid glands. (A) A frontal section showing Eya1 expression in pharyngeal arch (a1-4) mesenchyme, pouch endoderm (p1-3) and pharyngeal clefts (arrows) at E9.5. Cranial is up. (B) A transverse section showing strong Eya1 expression in the distal part of the pharyngeal arches and the surface ectoderm of 2nd cleft (arrow) at E10.5. (C,D) Transverse sections showing Eya1 expression in the thymic lobes (th) and parathyroid glands (pt) at E14.5. In addition, Eya1 is also expressed in sympathetic ganglia (sg) and trachea (tr). Eya1 is not expressed in the thyroid lobe (ty). (E-H) H and E-stained transverse sections of the neck region at E14.5. (E,F) In wild-type embryos, two thymic lobes are present and the parathyroid glands are associated with the thyroid gland (ty). (G,H) In Eya1–/– embryos, no thymus and parathyroid formation (*) was found at the same level or in other regions of the neck and upper trunk. For B-H, dorsal is up. aa, aortic arches; ca, carotid artery; es, esophagus.

View larger version (134K): [in a new window]   Fig. 2. The 3rd pharyngeal pouches do not separate as buds to form the primordia of thymus/parathyroid. (A,B) Transverse sections showing that in wild-type embryos (A), the 3rd pouches evaginate and then separate as buds to form the primordia of thymus/parathyroid (th/pt) at around E12.5, while the 4th pouches separate to form the rudiments of the ultimobranchial bodies (ub) at the same time; however in Eya1–/– embryos (B), the primordia of thymus/parathyroid failed to form, while the rudiments of ultimobranchial bodies (ub) were formed but with a slight indentation (arrow in B). (C-F) Transverse sections showing Pax1 and Pax9 expression in the rudiments of the thymus/parathyroid and ultimobranchial bodies at E12.0 of wild-type embryos (C,E). In Eya1–/– embryos, the expression of Pax1 and Pax9 was only detected in the rudiments of ultimobranchial bodies (arrows). The absence of Pax1 and Pax9 expression in the prospective region of the thymus/parathyroid rudiments confirms the absence of these structures in Eya1–/– embryos (D,F). Dorsal is up. ph, pharynx; tr, trachea.

View larger version (74K): [in a new window]   Fig. 3. Ultimobranchial body and thyroid lobe defects in Eya1–/– mice. (A,B) The embryos of E10.5, after completion of neural crest migration, were stained with Hoxb1 antibody to label the 4th pharyngeal pouches (p4). The 4th pharyngeal pouches express Hoxb1 (brown stain) in both wild-type (A) and Eya1–/– embryos (B). No significant difference of Hoxb1 expression in the 4th pouch endoderm was observed in Eya1 mutants. (C,D) Transverse sections through newborns, stained with anti-calcitonin antibody (brown staining). (C) A wild-type thyroid with numerous calcitonin-positive cells and follicular cells (f, arrow) throughout the lobe. (D) An Eya1–/– animal with bilateral persistent ultimobranchial bodies and malformed thyroid lobes. Only a few follicles are formed in the main body of the thyroid (ty, arrow). Note that the dorsally placed vesicle which is strongly positive for calcitonin represents a persistent ultimobranchial body (ub) in Eya1-mutants, not fusing with the thyroid lobe. This ultimobranchial body also contains follicle-like structures (arrow). No isthmus was present in this animal. (E-H) Transverse sections at E15.5 showing Pax8 expression in the thyroid lobes in wild-type and Eya1–/– embryos. (E) The ultimobranchial body cell populations showing weak Pax8 expression (arrow) were visible within the thyroid lobes at this stage. Parathyroid (pt) was also visible. (F) No ultimobranchial body cell population was observed within the Eya1–/– thyroid lobes on the same level and parathyroid was also absent. However, the ultimobranchial bodies showing weak Pax8 expression were observed at the anterior end dorsal to the thyroid lobe in Eya1–/– embryos (H). No ultimobranchial bodies were found in wild-type embryos on the same level (G). (I-L) Transverse sections showing Ttf1 expression in the thyroid lobes in wild-type embryos at E15.5 (I,K) and in both the persistent ultimobranchial bodies and the thyroid lobes of Eya1–/– embryos (J,L). Similarly, the persistent ultimobranchial bodies were observed as separate structures located anterodorsally to the thyroid lobes in Eya1–/– animals (L). For C-L, dorsal is up. tr, trachea; la, larynx.

View larger version (105K): [in a new window]   Fig. 4. Normal development of the thyroid primordia in Eya1–/– embryos. (A-D) Transverse sections showing the thyroid primordia (ty) in wild-type (A,C) and Eya1–/– (B,D) embryos at E10.5. The thyroid primordia were evident in Eya1–/– embryos. (E,F) Transverse sections showing Pax8 expression in the thyroid primordia of wild-type and Eya1–/– embryos at E10.5. (G,H) Sagittal sections showing Ttf1 expression in wild-type and Eya1–/– thyroid primordia at E10.5. (I,J) Transverse sections showing that Eya1 expression was not detectable in the thyroid primordia at E10.5-11.5 in wild-type embryos. ph, pharynx; baa, 2nd branchial arch artery. For transverse sections, dorsal is up; for sagittal sections, cranial is up and dorsal is to the left.

View larger version (122K): [in a new window]   Fig. 5. Neural crest defects in the pharyngeal arches of Eya1–/– embryos. (A,B) Transverse sections of wild-type and Eya1–/– embryos at E9.5 showing Hoxa3 expression in the hindbrain neural tube, migrating neural crest cells (arrow) and 3rd pharyngeal arches (a3). No significant change was found in Eya1 mutants. (C,D) Transverse sections showing Msx1 expression in the neural crest cells in the pharyngeal arches of wild-type and Eya1–/– embryos at E9.5. No significant difference of Msx1 expression was observed at E9.5 in Eya1–/– embryos. (E,F) Transverse sections showing Six1 expression in the distal edge of arch mesenchyme in wild-type and Eya1–/– embryos at E10.5. Six1 expression was not detectable in Eya1–/– embryos (asterisks in F). Dorsal is up.

View larger version (69K): [in a new window]   Fig. 6. Expression of Six1 in the pharyngeal pouch endoderm and the ectoderm of pharyngeal clefts was markedly reduced in Eya1–/– embryos. (A,B) Coronal sections showing that Hoxa3 is normally expressed in the 3rd and 4th pharyngeal pouches and arches (A), and its expression was unaffected in Eya1–/– embryos at E9.5 (B). (C,D) Coronal sections showing that Pax1 is normally expressed in the pouch endoderm at E9.5-10.5 (C), and its expression is preserved in Eya1–/– embryos (D). (E,F) Coronal sections showing that Pax9 is also expressed in the pouch endoderm at E9.5-10.5 (E) and its expression is also preserved in Eya1–/– embryos (F). (G,H) Coronal sections showing that Fgf3 is normally expressed in the posterior half of the pouch endoderm at E10.5 (G) and its expression was unaffected in Eya1–/– embryos (H). (I,J) Coronal sections showing that Six1 is normally co-expressed with Eya1 in the 3rd pouch endoderm (arrow in I) and the surface ectoderm of 3rd clefts (arrowhead in I), and its expression was markedly reduced in both structures in Eya1–/– embryos (arrows and arrowheads in J). (K,L) Coronal sections showing strong Six1lacZ expression in wild type (K) in the pouch endoderm and surface ectoderm including 2nd, 3rd and 4th pharyngeal clefts; however, in Eya1–/– embryos, Six1lacZ expression was significantly reduced in the pouch endoderm (arrows) and surface ectoderm (arrowheads, L) in the 2nd, 3rd and 4th pharyngeal regions (a2-a4). In contrast, its expression surrounding the arteries of pharyngeal arches remains unaffected (open arrowheads).

View larger version (141K): [in a new window]   Fig. 7. Gcm2 and Foxn1 expression was not detectable in Eya1–/– embryos. (A,B) Coronal sections through the 3rd pharyngeal pouch of E10.5 wild-type and Eya1–/– embryos showing that Gcm2 is strongly expressed in the 3rd pouch endoderm in wild-type embryos (arrow, A) and its expression was not detectable in Eya1–/– embryos (arrow, B). (C,D) Sagittal sections through the 3rd pharyngeal region showing that Foxn1 is expressed in the ventral region of the thymus/parathyroid primordia at E11.5 of wild-type embryos (arrow, C), but its expression was not detectable in Eya1–/– embryos (arrow, D). Note that the 3rd pouch is attached to the pharyngeal endoderm and does not separate as buds to form the primordia of thymus/parathyroid in Eya1–/– embryos. Cranial is up (A-D) and dorsal is to the left (C,D). a, pharyngeal arches; baa3, 3rd branchial arch arteries; ph, pharynx.

View larger version (115K): [in a new window]   Fig. 8. TUNEL analysis of transverse sections through the pharyngeal region in wild-type and Eya1–/– embryos at E10.5. (A-C) Apoptotic cells were observed in the pharyngeal arches (a) and pouches (p) in wild-type embryos. (D-F) In Eya1–/– embryos, there were more apoptotic cells in the proximal region of the 1st and 2nd arches (arrows in D,E, other data not shown). No significant changes of apoptotic cells were observed either in the neural crest mesenchyme or the pouch endoderm in the 3rd and 4th pharyngeal regions (E,F and other data not shown). Note that numerous apoptotic cells were detected in the surface ectoderm (se) of the 3rd (reflexed arrows in E,F) and 4th pharyngeal regions (data not shown). The panels below C and F are higher magnification of the boxed areas. Dorsal or cranial is up. fg, pharyngeal region of foregut.