Valeria Scagliotti

Transcription factor 7-like 1 is involved in hypothalamo–pituitary axis development in mice and humans

Significance The relevance of transcription factor 7-like 1 (TCF7L1) during hypothalamo–pituitary (HP) axis development remains unknown. Using mouse genetics, we show that TCF7L1 acts as a transcriptional repressor to regulate the expression of the hypothalamic signals involved in pituitary formation. In addition, we screened a cohort of human patients with forebrain and/or pituitary defects and report two independent missense variants, p.R92P and p.R400Q, in human TCF7L1. Functional studies in vitro and rescue experiments in zebrafish mutants deficient for tcf7l1a and tcf7l1b show that the p.R92P and p.R400Q variants exhibit reduced repressing activity compared with wild-type TCF7L1. In summary, we identify TCF7L1 as a determinant for the establishment of HP axis development and as a potential candidate gene to be mutated in congenital hypopituitarism. Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/β-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo–pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke’s pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with β-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Novel FOXA2 mutation causes Hyperinsulinism, Hypopituitarism with Craniofacial and Endoderm-derived organ abnormalities

Congenital hypopituitarism (CH) is characterized by the deficiency of one or more pituitary hormones and can present alone or in association with complex disorders. Congenital hyperinsulinism (CHI) is a disorder of unregulated insulin secretion despite hypoglycaemia that can occur in isolation or as part of a wider syndrome. Molecular diagnosis is unknown in many cases of CH and CHI. The underlying genetic etiology causing the complex phenotype of CH and CHI is unknown. In this study, we identified a de novo heterozygous mutation in the developmental transcription factor, forkhead box A2, FOXA2 (c.505T>C, p.S169P) in a child with CHI and CH with craniofacial dysmorphic features, choroidal coloboma and endoderm-derived organ malformations in liver, lung and gastrointestinal tract by whole exome sequencing. The mutation is at a highly conserved residue within the DNA binding domain. We demonstrated strong expression of Foxa2 mRNA in the developing hypothalamus, pituitary, pancreas, lungs and oesophagus of mouse embryos using in situ hybridization. Expression profiling on human embryos by immunohistochemistry showed strong expression of hFOXA2 in the neural tube, third ventricle, diencephalon and pancreas. Transient transfection of HEK293T cells with Wt (Wild type) hFOXA2 or mutant hFOXA2 showed an impairment in transcriptional reporter activity by the mutant hFOXA2. Further analyses using western blot assays showed that the FOXA2 p.(S169P) variant is pathogenic resulting in lower expression levels when compared with Wt hFOXA2. Our results show, for the first time, the causative role of FOXA2 in a complex congenital syndrome with hypopituitarism, hyperinsulinism and endoderm-derived organ abnormalities.

Visceral Adipose Tissue Immune Homeostasis Is Regulated by the Crosstalk between Adipocytes and Dendritic Cell Subsets

Summary Visceral adipose tissue (VAT) has multiple roles in orchestrating whole-body energy homeostasis. In addition, VAT is now considered an immune site harboring an array of innate and adaptive immune cells with a direct role in immune surveillance and host defense. We report that conventional dendritic cells (cDCs) in VAT acquire a tolerogenic phenotype through upregulation of pathways involved in adipocyte differentiation. While activation of the Wnt/β-catenin pathway in cDC1 DCs induces IL-10 production, upregulation of the PPARγ pathway in cDC2 DCs directly suppresses their activation. Combined, they promote an anti-inflammatory milieu in vivo delaying the onset of obesity-induced chronic inflammation and insulin resistance. Under long-term over-nutrition, changes in adipocyte biology curtail β-catenin and PPARγ activation, contributing to VAT inflammation.

Cell death and cell proliferation in human spina bifida

BACKGROUND Spina bifida is a multifactorial congenital malformation of the central nervous system. The aim of this study was to ascertain the relevance of cell death/proliferation balance in human spina bifida and to assess autophagy distribution and levels during embryo-fetal development in neural tissue. METHODS Five human cases with myelomeningocoele were compared with 10 healthy human controls and LC3 protein expression was also analyzed in mouse embryos. Cell death was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) assay; cell proliferation was studied by counting Ki67-positive cells, and autophagy was assessed by observing the presence of LC3 punctuate dots. RESULTS Comparing human cases and controls (13 to 21 weeks of gestation), we observed a significant increase in TUNEL-positive cells in human spina bifida associated with a significantly decreased proliferation rate, indicating an alteration of the physiological cell rate homeostasis. LC3 distribution was found to be spatiotemporally regulated in both human and murine embryo-fetuses: in early pregnancy a diffuse and ubiquitous LC3 signal was detected. After neural tube closure, an intense LC3-positive signal, normally associated to extra energy requirement, was confined to the Lissauers tract, the dorsolateral spinal zone containing centrally projecting axons from dorsal root ganglia, at any medullar levels. LC3 signal disappeared from 12 weeks of gestation. CONCLUSION In conclusion, this study confirms the fundamental role of cell death/proliferation balance during central nervous system development and reports the changing expression of LC3 protein in mouse and human neural tube. Birth Defects Research (Part A) 106:104-113, 2016.

Rings and Bricks: Expression of Cohesin Components is Dynamic during Development and Adult Life

Cohesin complex components exert fundamental roles in animal cells, both canonical in cell cycle and non-canonical in gene expression regulation. Germline mutations in genes coding for cohesins result in developmental disorders named cohesinopaties, of which Cornelia de Lange syndrome (CdLS) is the best-known entity. However, a basic description of mammalian expression pattern of cohesins in a physiologic condition is still needed. Hence, we report a detailed analysis of expression in murine and human tissues of cohesin genes defective in CdLS. Using both quantitative and qualitative methods in fetal and adult tissues, cohesin genes were found to be ubiquitously and differentially expressed in human tissues. In particular, abundant expression was observed in hematopoietic and central nervous system organs. Findings of the present study indicate tissues which should be particularly sensitive to mutations, germline and/or somatic, in cohesin genes. Hence, this expression analysis in physiological conditions may represent a first core reference for cohesinopathies.

Ephrin-B2 is critical regulator of endocrine organ development

This switch between stem cell quiescence and proliferation is a central but poorly understood issue in stem cell biology. Drosophila larval neural stem cells, neuroblasts, enter into quiescence at the end of embryogenesis and exit quiescence to be reactivated at early larval stages. Upon food stimulation, glial cells secret insulin to activate the insulin/IGF pathway in underlying neuroblasts and promote their proliferation growth and proliferation from quiescence. However, the intrinsic regulators that control neuroblast reactivation are not well-known. During an RNA interference (RNAi) screen, we uncover Chromator (Chro) as a novel regulator of neuroblast reactivation. In chro RNAi knock-down or chro mutant, neuroblasts displayed a cellular extension and most neuroblasts failed to incorporate with EdU (5-ethynyl-2’-deoxyuridine), suggesting that loss of Chro function induces neuroblast quiescence. We find that Chro is not only required for neuroblast reactivation, but important for maintaining neuroblast in a proliferative state. We further show that Chro functions downstream of insulin/IGF pathway during neuroblast reactivation. From a genome-wide in vivo profiling, we identified candidate genes that function downstream of Chro during neuroblast reactivation. Our data suggest that Chro is a critical intrinsic regulator of neuroblast reactivation.