During vertebrate development, the thyroid gland undergoes a unique relocalisation from its site of induction to a distant speciesspecific position in the cervical mesenchyme. We have analysed thyroid morphogenesis in wild-type and mutant zebrafish and mice, and find that localisation of growing thyroid tissue along the anteroposterior axis in zebrafish is linked to the development of the ventral aorta. In grafting experiments, ectopic vascular cells influence the localisation of thyroid tissue cell non-autonomously, showing that vessels provide guidance cues in zebrafish thyroid morphogenesis. In mouse thyroid development, the midline primordium bifurcates and two lobes relocalise cranially along the bilateral pair of carotid arteries. In hedgehog-deficient mice, thyroid tissue always develops along the ectopically and asymmetrically positioned carotid arteries, suggesting that, in mice (as in zebrafish), co-developing major arteries define the position of the thyroid. The similarity between zebrafish and mouse mutant phenotypes further indicates that thyroid relocalisation involves two morphogenetic phases, and that variation in the second phase accounts for species-specific differences in thyroid morphology. Moreover, the involvement of vessels in thyroid relocalisation sheds new light on the interpretation of congenital thyroid defects in humans.KEY WORDS: Thyroid, Zebrafish, Mouse, Arteries, Vegf, Scl, Hedgehog Development 133, 3797-3804 (2006) DEVELOPMENT MATERIALS AND METHODS AnimalsZebrafish work was carried out according to standard procedures and staging in hours post fertilisation (hpf) refers to development at 28.5 to 29°C. The term 'larva' is used in the text for fry that have hatched from the chorion and generally refers to an age older than 72 hpf. The mutant zebrafish line kdr y17 (Covassin et al., 2006) is allelic to the kdr (flk1) lines described previously (Habeck et al., 2002) and develops heart oedema owing to compromised circulation or other specific defects at stages older than 40 hours, so that determination of mutant phenotypes was possible based on morphology. Homozygous cloche mutant embryos (clo s5 ) (Stainier et al., 1995) were also identified before fixation according to their phenotype at 24 hpf. Phenotypic details described in this study were always evident in all homozygous specimens (more than 10 in each experiment).For analysis of short digits (Dsh/Dsh) (Niedermaier et al., 2005) and Xt J /Xt J mutant mouse embryos (Persson et al., 2002) timed matings of mutants were generated. The homozygous phenotypes are distinguishable from wild type owing to severe morphological defects, and in the case of Dsh-Xt J matings we determined the genotype by PCR from extra-embryonic membranes (primer information available upon request). Preparation of specimensIn situ hybridisation on zebrafish was carried out according to standard procedures, using nk2.1a (Rohr and Concha, 2000) as molecular marker for the thyroid primordium. Whole-mount immunohistochemistry with antibodies against thyroid h...

Background NKX2-1 encodes a transcription factor with large impact on the development of brain, lung and thyroid. Germline mutations of NKX2-1 can lead to dysfunction and malformations of these organs. Starting from the largest coherent collection of patients with a suspected phenotype to date, we systematically evaluated frequency, quality and spectrum of phenotypic consequences of NKX2-1 mutations. Methods After identifying mutations by Sanger sequencing and array CGH, we comprehensively reanalysed the phenotype of affected patients and their relatives. We employed electrophoretic mobility shift assay (EMSA) to detect alterations of NKX2-1 DNA binding. Gene expression was monitored by means of in situ hybridisation and compared with the expression level of MBIP, a candidate gene presumably involved in the disorders and closely located in close genomic proximity to NKX2-1. Results Within 101 index patients, we detected 17 point mutations and 10 deletions. Neurological symptoms were the most consistent finding (100%), followed by lung affection (78%) and thyroidal dysfunction (75%). Novel symptoms associated with NKX2-1 mutations comprise abnormal height, bouts of fever and cardiac septum defects. In contrast to previous reports, our data suggest that missense mutations in the homeodomain of NKX2-1 not necessarily modify its DNA binding capacity and that this specific type of mutations may be associated with mild pulmonary phenotypes such as asthma. Two deletions did not include NKX2-1, but MBIP, whose expression spatially and temporarily coincides with NKX2-1 in early murine development. Conclusions The high incidence of NKX2-1 mutations strongly recommends the routine screen for mutations in patients with corresponding symptoms. However, this analysis should not be confined to the exonic sequence alone, but should take advantage of affordable NGS technology to expand the target to adjacent regulatory sequences and the NKX2-1 interactome in order to maximise the yield of this diagnostic effort.

Objective: Steroidogenic factor 1 (SF1, NR5A1) is a key transcriptional regulator of genes involved in the hypothalamicpituitary-gonadal axis. Recently, SF1 mutations were found to be a frequent cause of 46,XY disorders of sex development (DSD) in humans. We investigate the frequency of NR5A1 mutations in an Egyptian cohort of XY DSD. Design: Clinical assessment, endocrine evaluation and genetic analysis of 50 Egyptian XY DSD patients (without adrenal insufficiency) with a wide phenotypic spectrum. Methods: Molecular analysis of NR5A1 gene by direct sequencing followed by in vitro functional analysis of the two novel missense mutations detected. Results: Three novel heterozygous mutations of the coding region in patients with hypospadias were detected. p.Glu121AlafsX25 results in severely truncated protein, p.Arg62Cys lies in DNA-binding zinc finger, whereas p.Ala154Thr lies in the hinge region of SF1 protein. Transactivation assays using reporter constructs carrying promoters of anti-Mü llerian hormone (AMH), CYP11A1 and TESCO core enhancer of Sox9 showed that p.Ala154Thr and p.Arg62Cys mutations result in aberrant biological activity of NR5A1. A total of 17 patients (34%) harboured the p.Gly146Ala polymorphism. Conclusion: We identified two novel NR5A1 mutations showing impaired function in 23 Egyptian XY DSD patients with hypospadias (8.5%). This is the first study searching for NR5A1 mutations in oriental patients from the Middle East and Arab region with XY DSD and no adrenal insufficiency, revealing a frequency similar to that in European patients (6.5-15%). We recommend screening of NR5A1 in patients with hypospadias and gonadal dysgenesis. Yearly follow-ups of gonadal function and early cryoconservation of sperms should be performed in XY DSD patients with NR5A1 mutations given the risk of future fertility problems due to early gonadal failure.

Der Fremdenverkehr ist an der ganzen Riviera in den letzten 10 Jahren merklich zuriickgegangen. Da dieser Riickgang zum Tell auf s prinzipiell sehr wichtige Ursachen zur[ickzufiihren ist, so halte ich es fiir angebracht, dieselben zu besprechen.Bis zum Schluss des vorigen Jahrhunderts rief das Wort Riviera sowohl in _~rzte-wie in Laienkreisen das Assoziationswort ,,Lungenkrank" hervor. Denn die Riviera war seit Jahrzehnten fiir die, die es sich leisten konnten, die auser]esenste Gegend fiir die Behandlung der Tuberkulose. Auf italienischer Seite erfreuten sich San Remo, Ospedaletti, Nervi, auf franzSsischer Seite Mentone, Cannes einer besonderen Beliebtheit, da diese Oi:te vor Nordwind geschiitzt waren. Die grosse Mehrzahl der Besucher dieser Stationen, vonPassanten abgesehen, waren Tuberkul5se~ und dank ihren arbeiteten dieselben ausgezeichnet. Es war dies aber zu einer Zeit, wo man das Wort ,,Tuberkulose" nicht so oft gebrauchte wie heute; Friihdiagnosen wurden welt seltener gestellt, und so kamen viel TuberkulSse unter der Diagnose ,,Katarrhe", ,,Wachstumsan~mie" usw. an die Riviera. Endlich war man sowohl in J~rzte-wie in Laienkreisen noeh nicht yon der Infektiosit~t der Tuberkulose so fiberzeugt wie heute. TuberkulSse waren bedauernswerte Kranke, die oft reizende Gesellschafter waren und mit denen man gern verkehrte. Das Publikum glaubte an die Heredit~it, dagegen nicht, oder kaum an die Kontagiosit~t des Leidens. Die ernster Kranken besuchten mit Vorliebe die oben genannten Stationen; leichter Kranke gingen auch gern 1) Nach einem auf dem intern. Kongress filr Physiotherapie (Berlin, M~rz 1913) gehaltenen Vortrage.