RESUMO
Pituitary cells that express the transcription factor SOX2 are stem cells because they can self-renew and differentiate into multiple pituitary hormone-producing cell types as organoids. Wounding and physiological challenges can activate pituitary stem cells, but cell numbers are not fully restored, and the ability to mobilize stem cells decreases with increasing age. The basis of these limitations is still unknown. The regulation of stem cell quiescence and activation involves many different signalling pathways, including those mediated by WNT, Hippo and several cytokines; more research is needed to understand the interactions between these pathways. Pituitary organoids can be formed from human or mouse embryonic stem cells, or from human induced pluripotent stem cells. Human pituitary organoid transplantation is sufficient to induce corticosterone release in hypophysectomized mice, raising the possibility of therapeutic applications. Today, pituitary organoids have the potential to assess the role of individual genes and genetic variants on hormone production ex vivo, providing an important tool for the advancement of exciting frontiers in pituitary stem cell biology and pituitary organogenesis. In this article, we provide an overview of notable discoveries in pituitary stem cell function and highlight important areas for future research.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Hipófise/metabolismo , Fatores de Transcrição/metabolismo , Transdução de Sinais , Diferenciação CelularRESUMO
BACKGROUND: Congenital hypopituitarism (CH) and its associated syndromes, septo-optic dysplasia (SOD) and holoprosencephaly (HPE), are midline defects that cause significant morbidity for affected people. Variants in 67 genes are associated with CH, but a vast majority of CH cases lack a genetic diagnosis. Whole exome and whole genome sequencing of CH patients identifies sequence variants in genes known to cause CH, and in new candidate genes, but many of these are variants of uncertain significance (VUS). METHODS: The International Mouse Phenotyping Consortium (IMPC) is an effort to establish gene function by knocking-out all genes in the mouse genome and generating corresponding phenotype data. We used mouse embryonic imaging data generated by the Deciphering Mechanisms of Developmental Disorders (DMDD) project to screen 209 embryonic lethal and sub-viable knockout mouse lines for pituitary malformations. RESULTS: Of the 209 knockout mouse lines, we identified 51 that have embryonic pituitary malformations. These genes not only represent new candidates for CH, but also reveal new molecular pathways not previously associated with pituitary organogenesis. We used this list of candidate genes to mine whole exome sequencing data of a cohort of patients with CH, and we identified variants in two unrelated cases for two genes, MORC2 and SETD5, with CH and other syndromic features. CONCLUSIONS: The screening and analysis of IMPC phenotyping data provide proof-of-principle that recessive lethal mouse mutants generated by the knockout mouse project are an excellent source of candidate genes for congenital hypopituitarism in children.
Assuntos
Hipopituitarismo , Camundongos Knockout , Hipófise , Hipopituitarismo/genética , Animais , Humanos , Hipófise/metabolismo , Hipófise/anormalidades , Hipófise/patologia , Camundongos , Fenótipo , Feminino , Masculino , Modelos Animais de Doenças , Sequenciamento do Exoma , Displasia Septo-Óptica/genéticaRESUMO
The pituitary gland regulates growth, metabolism, reproduction, the stress response, uterine contractions, lactation, and water retention. It secretes hormones in response to hypothalamic input, end organ feedback, and diurnal cues. The mechanisms by which pituitary stem cells are recruited to proliferate, maintain quiescence, or differentiate into specific cell types, especially thyrotropes, are not well understood. We used single-cell RNA sequencing in juvenile P7 mouse pituitary cells to identify novel factors in pituitary cell populations, with a focus on thyrotropes and rare subtypes. We first observed cells coexpressing markers of both thyrotropes and gonadotropes, such as Pou1f1 and Nr5a1. This was validated in vivo by both immunohistochemistry and lineage tracing of thyrotropes derived from Nr5a1-Cre; mTmG mice and demonstrates that Nr5a1-progenitors give rise to a proportion of thyrotropes during development. Our data set also identifies novel factors expressed in pars distalis and pars tuberalis thyrotropes, including the Shox2b isoform in all thyrotropes and Sox14 specifically in Pou1f1-negative pars tuberalis thyrotropes. We have therefore used single-cell transcriptomics to determine a novel developmental trajectory for thyrotropes and potential novel regulators of thyrotrope populations.
Assuntos
Doenças da Hipófise , Adeno-Hipófise , Gravidez , Feminino , Camundongos , Animais , Tireotropina/metabolismo , Hipófise/metabolismo , Fatores de Transcrição/metabolismo , Doenças da Hipófise/metabolismo , Imuno-Histoquímica , Adeno-Hipófise/metabolismo , Fatores de Transcrição SOXB2/metabolismoRESUMO
Dietary vitamin A is metabolized into bioactive retinoic acid (RA) in vivo and regulates the development of many embryonic tissues. RA signaling is active in the oral ectoderm-derived tissues of the neuroendocrine system, but its role there has not yet been fully explored. We show here that RA signaling is active during pituitary organogenesis and dependent on the pituitary transcription factor Prop1. Prop1-mutant mice show reduced expression of the aldehyde dehydrogenase gene Aldh1a2, which metabolizes the vitamin A-intermediate retinaldehyde into RA. To elucidate the specific function of RA signaling during neuroendocrine development, we studied a conditional deletion of Aldh1a2 and a dominant-negative mouse model of inhibited RA signaling during pituitary organogenesis. These models partially phenocopy Prop1-mutant mice by exhibiting embryonic pituitary dysmorphology and reduced hormone expression, especially thyrotropin. These findings establish the role of RA in embryonic pituitary stem cell progression to differentiated hormone cells and raise the question of gene-by-environment interactions as contributors to pituitary development and disease.
Assuntos
Família Aldeído Desidrogenase 1/metabolismo , Proteínas de Homeodomínio/metabolismo , Hipófise/embriologia , Retinal Desidrogenase/metabolismo , Tretinoína/metabolismo , Família Aldeído Desidrogenase 1/genética , Animais , Camundongos Endogâmicos C57BL , Organogênese , Hipófise/metabolismo , Retinal Desidrogenase/genética , Transdução de Sinais , Células-Tronco/metabolismo , Tireotropina/metabolismoRESUMO
In the last 15 years, single-cell technologies have become robust and indispensable tools to investigate cell heterogeneity. Beyond transcriptomic, genomic and epigenome analyses, technologies are constantly evolving, in particular toward multi-omics, where analyses of different source materials from a single cell are combined, and spatial transcriptomics, where resolution of cellular heterogeneity can be detected in situ. While some of these techniques are still being optimized, single-cell RNAseq has commonly been used because the examination of transcriptomes allows characterization of cell identity and, therefore, unravel previously uncharacterized diversity within cell populations. Most endocrine organs have now been investigated using this technique, and this has given new insights into organ embryonic development, characterization of rare cell types, and disease mechanisms. Here, we highlight recent studies, particularly on the hypothalamus and pituitary, and examine recent findings on the pancreas and reproductive organs where many single-cell experiments have been performed.
Assuntos
Sistema Endócrino/citologia , Análise de Célula Única , Animais , Humanos , Morfogênese , Reprodução , Processos de Determinação Sexual , Transcriptoma/genéticaRESUMO
Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on â¼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3' transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.
Assuntos
Biomarcadores/análise , Hipófise/citologia , Hipófise/metabolismo , Hormônios Hipofisários/metabolismo , Análise de Célula Única/métodos , Células-Tronco/classificação , Células-Tronco/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular/genética , Separação Celular/métodos , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/genética , Hipófise/embriologia , Hormônios Hipofisários/genética , Análise de Sequência de RNA/métodos , Células-Tronco/citologia , TranscriptomaRESUMO
The hypothalamic-pituitary axes are the coordinating centers for multiple endocrine gland functions and physiological processes. Defects in the hypothalamus or pituitary gland can cause reduced growth and severe short stature, affecting approximately 1 in 4000 children, and a large percentage of cases of pituitary hormone deficiencies do not have an identified genetic cause. SOX21 is a protein that regulates hair, neural, and trophoblast stem cell differentiation. Mice lacking Sox21 have reduced growth, but the etiology of this growth defect has not been described. We studied the expression of Sox21 in hypothalamic-pituitary development and examined multiple endocrine axes in these mice. We find no evidence of reduced intrauterine growth, food intake, or physical activity, but there is evidence for increased energy expenditure in mutants. In addition, despite changes in pituitary hormone expression, hypothalamic-pituitary axes appear to be functional. Therefore, SOX21 variants may be a cause of non-endocrine short stature in humans.
Assuntos
Deleção de Genes , Hipotálamo/metabolismo , Hipófise/metabolismo , Fatores de Transcrição SOXB2/genética , Animais , Animais Recém-Nascidos , Metabolismo Basal , Peso Corporal , Diencéfalo/metabolismo , Feminino , Hormônio do Crescimento/metabolismo , Crescimento e Desenvolvimento , Masculino , Camundongos Knockout , Especificidade de Órgãos , Tiroxina/sangueRESUMO
The anterior pituitary gland is comprised of specialized cell-types that produce and secrete polypeptide hormones in response to hypothalamic input and feedback from target organs. These specialized cells arise from stem cells that express SOX2 and the pituitary transcription factor PROP1, which is necessary to establish the stem cell pool and promote an epithelial to mesenchymal-like transition, releasing progenitors from the niche. The adult anterior pituitary responds to physiological challenge by mobilizing the SOX2-expressing progenitor pool and producing additional hormone-producing cells. Knowledge of the role of signaling pathways and extracellular matrix components in these processes may lead to improvements in the efficiency of differentiation of embryonic stem cells or induced pluripotent stem cells into hormone producing cells in vitro. Advances in our basic understanding of pituitary stem cell regulation and differentiation may lead to improved diagnosis and treatment for patients with hypopituitarism.
Assuntos
Proteínas de Homeodomínio/metabolismo , Adeno-Hipófise/citologia , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Animais , Diferenciação Celular , Transição Epitelial-Mesenquimal , Matriz Extracelular/metabolismo , Humanos , Adeno-Hipófise/metabolismo , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
The genetic basis for combined pituitary hormone deficiency (CPHD) is complex, involving 30 genes in a variety of syndromic and nonsyndromic presentations. Molecular diagnosis of this disorder is valuable for predicting disease progression, avoiding unnecessary surgery, and family planning. We expect that the application of high throughput sequencing will uncover additional contributing genes and eventually become a valuable tool for molecular diagnosis. For example, in the last 3 years, six new genes have been implicated in CPHD using whole-exome sequencing. In this review, we present a historical perspective on gene discovery for CPHD and predict approaches that may facilitate future gene identification projects conducted by clinicians and basic scientists. Guidelines for systematic reporting of genetic variants and assigning causality are emerging. We apply these guidelines retrospectively to reports of the genetic basis of CPHD and summarize modes of inheritance and penetrance for each of the known genes. In recent years, there have been great improvements in databases of genetic information for diverse populations. Some issues remain that make molecular diagnosis challenging in some cases. These include the inherent genetic complexity of this disorder, technical challenges like uneven coverage, differing results from variant calling and interpretation pipelines, the number of tolerated genetic alterations, and imperfect methods for predicting pathogenicity. We discuss approaches for future research in the genetics of CPHD.