Enhanced Canonical Wnt Signaling During Early Zebrafish Development Perturbs the Interaction of Cardiac Mesoderm and Pharyngeal Endoderm and Causes Thyroid Specification Defects.

Background: Congenital hypothyroidism due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the majority of cases. This situation reflects, in part, our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. Methods: We treated zebrafish embryos during different developmental periods with small-molecule compounds known to manipulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. We used the antisense morpholino (MO) technique to create a zebrafish acardiac model. For thyroid rescue experiments, bone morphogenetic protein (BMP) pathway induction in zebrafish embryos was obtained by manipulation of heat-shock inducible transgenic lines. Results: Combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of MO-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. Conclusion: Collectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

Novel inactivating follicle-stimulating hormone receptor mutations in a patient with premature ovarian insufficiency identified by next-generation sequencing gene panel analysis.

OBJECTIVE: To find the genetic etiology of premature ovarian insufficiency (POI) in a patient with primary amenorrhea and hypergonadotropic hypogonadism. DESIGN: Case report. SETTING: University hospital. PATIENTS: A Belgian woman aged 32 years with POI at the age of 17, her parents, and her sister whose POI was diagnosed at age 29. INTERVENTIONS: Analysis of a panel of 31 genes implicated in POI (POIGP) using next-generation sequencing (NGS), Sanger sequencing, and in vitro functional study. MAIN OUTCOME MEASURES: Gene variants, family mutational segregation, and in vitro functional impact of the mutant proteins. RESULTS: The analysis of the gene panel using NGS identified the presence of two novel follicle-stimulating hormone receptor (FSHR) missense mutations at a compound heterozygous state in the affected patient: c.646 G>A, p.Gly216Arg, and c.1313C>T, p.Thr438Ile. Sanger sequencing showed the presence of each mutation at heterozygous state in the patient's parents and at heterozygous compound state in the affected sister. Both substituted amino acids (Gly216 and Thr438) were conserved in FSHR of several vertebrate species as well as in other glycoproteins receptors (TSHR and LHCGHR), suggesting a potentially important role in glycoprotein receptor function. An in vitro functional study showed similar results for both variants with more than 90% reduction of their cell surface expression and a 55% reduction of their FSH-induced cyclic adenosine 3':5' monophosphate (cAMP) production compared with the wild-type FSHR. CONCLUSIONS: The analysis of a gene panel of 31 genes implicated in POI allowed us to identify two novel partially inactivating mutations of FSHR that are likely responsible for the POI phenotype of the proband and of her affected sister.

Transgenic zebrafish illuminate the dynamics of thyroid morphogenesis and its relationship to cardiovascular development.

Among the various organs derived from foregut endoderm, the thyroid gland is unique in that major morphogenic events such as budding from foregut endoderm, descent into subpharyngeal mesenchyme and growth expansion occur in close proximity to cardiovascular tissues. To date, research on thyroid organogenesis was missing one vital tool-a transgenic model that allows to track the dynamic changes in thyroid size, shape and location relative to adjacent cardiovascular tissues in live embryos. In this study, we generated a novel transgenic zebrafish line, tg(tg:mCherry), in which robust and thyroid-specific expression of a membrane version of mCherry enables live imaging of thyroid development in embryos from budding stage throughout formation of functional thyroid follicles. By using various double transgenic models in which EGFP expression additionally labels cardiovascular structures, a high coordination was revealed between thyroid organogenesis and cardiovascular development. Early thyroid development was found to proceed in intimate contact with the distal ventricular myocardium and live imaging confirmed that thyroid budding from the pharyngeal floor is tightly coordinated with the descent of the heart. Four-dimensional imaging of live embryos by selective plane illumination microscopy and 3D-reconstruction of confocal images of stained embryos yielded novel insights into the role of specific pharyngeal vessels, such as the hypobranchial artery (HA), in guiding late thyroid expansion along the pharyngeal midline. An important role of the HA was corroborated by the detailed examination of thyroid development in various zebrafish models showing defective cardiovascular development. In combination, our results from live imaging as well es from 3D-reconstruction of thyroid development in tg(tg:mCherry) embryos provided a first dynamic view of late thyroid organogenesis in zebrafish-a critical resource for the design of future studies addressing the molecular mechanisms of these thyroid-vasculature interactions.

Deletion of the RNaseIII enzyme dicer in thyroid follicular cells causes hypothyroidism with signs of neoplastic alterations.

Micro-RNAs (miRNAs) are small non-coding RNAs that regulate gene expression, mainly at mRNA post-transcriptional level. Functional maturation of most miRNAs requires processing of the primary transcript by Dicer, an RNaseIII-type enzyme. To date, the importance of miRNA function for normal organogenesis has been demonstrated in several mouse models of tissue-specific Dicer inactivation. However, the role of miRNAs in thyroid development has not yet been addressed. For the present study, we generated mouse models in which Dicer expression has been inactivated at two different stages of thyroid development in thyroid follicular cells. Regardless of the time of Dicer invalidation, the early stages of thyroid organogenesis, preceding folliculogenesis, were unaffected by the loss of small RNAs, with a bilobate gland in place. Nevertheless, Dicer mutant mice were severely hypothyroid and died soon after weaning unless they were substituted with T4. A conspicuous follicular disorganization was observed in Dicer mutant thyroids together with a strong down regulation of Nis expression. With increasing age, the thyroid tissue showed characteristics of neoplastic alterations as suggested by a marked proliferation of follicular cells and an ongoing de-differentiation in the center of the thyroid gland, with a loss of Pax8, FoxE1, Nis and Tpo expression. Together, our data show that loss of miRNA maturation due to Dicer inactivation severely disturbs functional thyroid differentiation. This suggests that miRNAs are mandatory to fine-tune the expression of thyroid specific genes and to maintain thyroid tissue homeostasis.

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity.

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of beta-strands and alpha-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the beta-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the beta-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.

Human body composition: A review of adult dissection data.

Although body composition analysis is popular, dissection data are sparse and sometimes difficult to access. Published data that include the weights of skin, adipose tissue, muscle, and bone, along with body weight, are reviewed. The 31 men and 20 women include 34 cadavers from three separate dissection studies in Brussels, 12 from 19th century reports, and 5 from the United States. The age range was 16-94y. Men differed from women in that they had less adipose tissue and more muscle in both absolute and relative terms. The body mass index (BMI) did not differ between the sexes, because lower weights of muscle and bone compensated for the greater adiposity in women. The relationship between the BMI and relative adiposity was significant, but the BMI explained only about one-third of the variance in adiposity, indicating that in this sample it is a poor predictor of fatness. The composition of the fat-free weight (FFW) and adipose tissue free weight (ATFW), though less variable than body weight, showed enough variability that the assumption of constancy of the fat-free body required for densitometry and other indirect methods of fat estimation, could not be supported. In the few dissections that did fat extraction, essential on non-adipose fat, varied from 4-14% of the FFW, thus undermining the concept of lean body weight. More dissection data are needed, especially in children and adolescents, and especially in conjunction with in vivo body composition methods to help in their validation. Am. J. Hum. Biol. 11:167-174, 1999. Copyright 1999 Wiley-Liss, Inc.