A mouse model for hereditary thyroid dysgenesis and cleft palate.

1Stazione Zoologica Anton Dohrn, Napoli, Italy. 2European Molecular Biology Laboratory, Heidelberg, Germany. 3Istituto Nazionale dei Tumori Fondazione Senatore Pascale, Naples, Italy. 4Faculte de Medicine de la Timone, Marseille, France. 5Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita Federico II, Napoli, Italy. M.D. & C.O contributed equally to this work. Correspondence should be addressed to R.D.L. (e-mail: rdilauro@unina.it). Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue1. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described2. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathke’s pouch3. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2–/– results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans. Thyroid gland organogenesis involves the dorso-caudal migration of a median endodermal bud that originates from the posterior region of the pharyngeal floor. The thyroid primordium migrates to the area located between the fourth pharyngeal pouches and eventually fuses with them. The adult thyroid gland is comprised of cells derived from all three germ layers, but thyroid follicular cells (TFCs), which are responsible for thyroid hormone biosynthesis, are thought to derive primarily from the median primordium, though a contribution from the endoderm of the pharyngeal pouches has been proposed4. Functional differentiation, as shown by the expression of thyroglobulin, occurs in TFCs following migration5, suggesting that migration and functional differentiation may be mutually exclusive. Three transcription factors, TTF-1, TTF-2 and Pax8, are present from the start of thyroid morphogenesis6. TTF-2, which is also expressed in most of the foregut endoderm, in the craniopharyngeal ectoderm involved in palate formation and in Rathke’s pouch, is transiently expressed at these sites from embryonic day (E) 8−8.5 to E13.5 (ref. 3). The mRNA encoding TTF-2 is down-regulated in TFC precursors following their migration and just before their differentiation. We suggested that TTF-2 is involved either in promoting the migration process or in repressing differentiation of the TFCs until migration has occurred3. Thus, we predicted that the absence of TTF-2 would result in alteration of thyroid primordium migration and/or precocious functional differentiation. The TTF-2 genetic locus, designated Titf2, is located on mouse chromosome 4C2 (ref. 3) and on band 9q22 of the human genome (data not shown). A genomic fragment encoding TTF-2 was isolated and used to construct a targeting vector for Titf2 (Fig. 1a). Targeted ES cell clones were injected into blastocysts to introduce the mutation into the germline. Heterozygous Titf2+/– mice showed no overt phenotype. Homozygous Titf2–/–offspring were detected at the expected mendelian ratios (Fig. 1b), however, they died within 48 hours of birth and examination of these mice revealed severe cleft palate (Fig. 1d,e), which was probably responsible for their perinatal death. In addition, Titf2–/– mice had no thyroid gland in its normal location (Fig. 1f,g) and showed a corresponding absence of thyroid hormones, measured as free thyroxine, in the bloodstream (FT4, Fig. 1c). A compensatory elevation of circulating thyroid stimulating hormone (TSH, Fig. 1c) demonstrates a normal pituitary response. In fact, measurements of several additional pituitary hormones revealed no significant differences between wild-type and Titf2–/– mice (data not shown), indicating that the absence of TTF-2 during development of the anterior pituitary does not affect its function. Similarly, thymus, ultimobranchial body and C cells, structures derived from the pharyngeal pouches where mRNA encoding TTF-2 is present, appear normal (data not shown). To determine when the absence of TTF-2 blocks thyroid morphogenesis, we compared the early steps of this process in Titf2+/– and Titf2–/– embryos. TFC precursors were traced by staining with an antibody to TTF-1 (ref. 5). In mice, budding of thyroid primordium begins at E8−8.5, and does not require TTF-2 as it occurred similarly in Titf2+/– and Titf2–/– embryos (data not shown). In E9.5 Titf2+/− embryos, the thyroid primordium begins to descend and TFC precursors staining for TTF-1 detach from the pharyngeal cavity (Fig. 2a,c), whereas in Titf2–/– embryos, the TTF-1 expressing cells remain contiguous with the pharyngeal endoderm (Fig. 2b,d) suggesting an inability of TFC precursors to migrate. At E11.5, continued migration is evident in Titf2+/– embryos (Fig. 3a), and two different phenotypes are observed in Titf2–/– embryos. In half of the Titf2–/– embryos, TFC precursors remain in their original position (Fig. 3b), whereas in the remaining half, TTF-1-positive TFC precursors are undetectable (Fig. 3c). The detectable non-migratory precursors represent ectopic thyroid, which we investigated for their ability to differentiate. In Titf2+/− embryos at E15.5 (identical results obtained with Titf2+/+ mice, data not shown), migration is complete, with the thyroid positioned dorsal to the cricoid cartilage, ventral to the trachea and cranial to the thymus (Fig. 4a). © 1998 Nature America Inc. • http://genetics.nature.com