RESUMO
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
Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.
Assuntos
Mutação com Ganho de Função , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófise/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Corticotrofos/citologia , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Fácies , Insuficiência de Crescimento/genética , Células HEK293 , Cardiopatias Congênitas/genética , Humanos , Lactente , Sistema de Sinalização das MAP Quinases/genética , Melanotrofos/citologia , Melanotrofos/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sequenciamento do Exoma/métodosRESUMO
Smoking is the largest preventable cause of death and disease in the United States. However, <5% of quit attempts are successful, underscoring the urgent need for novel therapeutics. Microglia are one untapped therapeutic target. While previous studies have shown that microglia mediate both inflammatory responses in the brain and brain plasticity, little is known regarding their role in nicotine dependence and withdrawal phenotypes. Here, we examined microglial changes in the striatum-a mesolimbic region implicated in the rewarding effects of drugs and the affective disruptions occurring during withdrawal. We show that both nicotine and withdrawal induce microglial morphological changes; however, proinflammatory effects and anxiogenic behaviors were observed only during nicotine withdrawal. Pharmacological microglial depletion during withdrawal prevented these effects. These results define differential effects of nicotine and withdrawal on inflammatory signaling in the brain, laying the groundwork for development of future smoking cessation therapeutics.
Assuntos
Microglia/patologia , Núcleo Accumbens/metabolismo , Síndrome de Abstinência a Substâncias/patologia , Animais , Ansiedade/etiologia , Modelos Animais de Doenças , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , NADPH Oxidase 2/metabolismo , Nicotina/administração & dosagem , Compostos Orgânicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/complicações , Síndrome de Abstinência a Substâncias/metabolismoRESUMO
The pituitary gland is a critical organ that is necessary for many physiological processes, including growth, reproduction, and stress response. The secretion of pituitary hormones from specific cell types regulates these essential processes. Pituitary hormone cell types arise from a common pool of pituitary progenitors, and mutations that disrupt the formation and differentiation of pituitary progenitors result in hypopituitarism. Canonical WNT signaling through CTNNB1 (ß-catenin) is known to regulate the formation of the POU1F1 lineage of pituitary cell types. When ß-catenin is deleted during the initial formation of the pituitary progenitors, Pou1f1 is not transcribed, which leads to the loss of the POU1F1 lineage. However, when ß-catenin is deleted after lineage specification, there is no observable effect. Similarly, the generation of a ß-catenin gain-of-function allele in early pituitary progenitors or stem cells results in the formation of craniopharyngiomas, whereas stimulating ß-catenin in differentiated cell types has no effect. PROP1 is a pituitary-specific transcription factor, and the peak of PROP1 expression coincides with a critical time point in pituitary organogenesis-that is, after pituitary progenitor formation but before lineage specification. We used a Prop1-cre to conduct both loss- and gain-of-function studies on ß-catenin during this critical time point. Our results demonstrate that pituitary progenitors remain sensitive to both loss and gain of ß-catenin at this time point, and that either manipulation results in hypopituitarism.
Assuntos
Craniofaringioma/genética , Hipopituitarismo/genética , Hipófise/embriologia , Células-Tronco/metabolismo , Fator de Transcrição Pit-1/genética , beta Catenina/genética , Animais , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Lactotrofos/citologia , Camundongos , Organogênese , Hipófise/metabolismo , Somatotrofos/citologia , Células-Tronco/citologia , Tireotrofos/citologia , Fator de Transcrição Pit-1/metabolismo , Via de Sinalização WntRESUMO
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
BACKGROUND: The pituitary gland is a highly vascularized tissue that requires coordinated interactions between the neural ectoderm, oral ectoderm, and head mesenchyme during development for proper physiological function. The interactions between the neural ectoderm and oral ectoderm, especially the role of the pituitary organizer in shaping the pituitary precursor, Rathke's pouch, are well described. However, less is known about the role of head mesenchyme in pituitary organogenesis. The head mesenchyme is derived from definitive mesoderm and neural crest, but the relative contributions of these tissues to the mesenchyme adjacent to the pituitary are not known. RESULTS: We carried out lineage tracing experiments using two neural crest-specific mouse cre lines, Wnt1-cre and P0-cre, and determined that the head mesenchyme rostral to the pituitary gland is neural crest derived. To assess the role of the neural crest in pituitary development we ablated it, using Wnt1-cre to delete Ctnnb1 (ß-catenin), which is required for neural crest development. The Wnt1-cre is active in the neural ectoderm, principally in the mesencephalon, but also in the posterior diencephalon. Loss of ß-catenin in this domain causes a rostral shift in the ventral diencephalon, including the pituitary organizer, resulting in pituitary dysmorphology. The neural crest deficient embryos have abnormally dilated pituitary vasculature due to a loss of neural crest derived pericytes. CONCLUSIONS: ß-catenin in the Wnt1 expression domain, including the neural crest, plays a critical role in regulation of pituitary gland growth, development, and vascularization.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mesencéfalo/metabolismo , Crista Neural/metabolismo , Organogênese/genética , Hipófise/metabolismo , beta Catenina/genética , Animais , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Imuno-Histoquímica , Hibridização In Situ , Masculino , Mesencéfalo/embriologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Crista Neural/embriologia , Hipófise/embriologia , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMO
Mutations in PROP1, the most common known cause of combined pituitary hormone deficiency in humans, can result in the progressive loss of all hormones of the pituitary anterior lobe. In mice, Prop1 mutations result in the failure to initiate transcription of Pou1f1 (also known as Pit1) and lack somatotropins, lactotropins, and thyrotropins. The basis for this species difference is unknown. We hypothesized that Prop1 is expressed in a progenitor cell that can develop into all anterior lobe cell types, and not just the somatotropes, thyrotropes, and lactotropes, which are collectively known as the PIT1 lineage. To test this idea, we produced a transgenic Prop1-cre mouse line and conducted lineage-tracing experiments of Prop1-expressing cells. The results reveal that all hormone-secreting cell types of both the anterior and intermediate lobes are descended from Prop1-expressing progenitors. The Prop1-cre mice also provide a valuable genetic reagent with a unique spatial and temporal expression for generating tissue-specific gene rearrangements early in pituitary gland development. We also determined that the minimal essential sequences for reliable Prop1 expression lie within 10 kilobases of the mouse gene and demonstrated that human PROP1 can substitute functionally for mouse Prop1. These studies enhance our understanding of the pathophysiology of disease in patients with PROP1 mutations.
Assuntos
Proteínas de Homeodomínio/metabolismo , Adeno-Hipófise/metabolismo , Hipófise/metabolismo , Células-Tronco/metabolismo , Animais , Linhagem da Célula/genética , Teste de Complementação Genética , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Mutação , Hipófise/citologia , Hipófise/embriologia , Adeno-Hipófise/citologia , Adeno-Hipófise/embriologiaRESUMO
Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific ß-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.
Assuntos
Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Gonadotrofos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transativadores/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Gonadotrofos/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Masculino , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Fatores de Transcrição Box Pareados/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroides/farmacologia , Transativadores/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Proteína Homeobox SIX3RESUMO
Many aspects of pituitary development have become better understood in the past two decades. The signaling pathways regulating pituitary growth and shape have emerged, and the balancing interactions between the pathways are now appreciated. Markers for multipotent progenitor cells are being identified, and signature transcription factors have been discovered for most hormone-producing cell types. We now realize that pulsatile hormone secretion involves a 3D integration of cellular networks. About a dozen genes are known to cause pituitary hypoplasia when mutated due to their essential roles in pituitary development. Similarly, a few genes are known that predispose to familial endocrine neoplasia, and several genes mutated in sporadic pituitary adenomas are documented. In the next decade, we anticipate gleaning a deeper appreciation of these processes at the molecular level, insight into the development of the hypophyseal portal blood system, and evolution of better therapeutics for congenital and acquired hormone deficiencies and for common craniopharyngiomas and pituitary adenomas.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Mutação , Doenças da Hipófise/genética , Hipófise/metabolismo , Animais , Humanos , Modelos Genéticos , Doenças da Hipófise/metabolismo , Doenças da Hipófise/fisiopatologia , Hipófise/crescimento & desenvolvimento , Hormônios Hipofisários/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Tissue-specific expression of cre recombinase is a well-established genetic tool to analyze gene function, and it is limited only by the efficiency and specificity of available cre mouse strains. Here, we report the generation of a transgenic line containing a cre cassette with codon usage optimized for mammalian cells (iCre) under the control of a mouse glycoprotein hormone α-subunit (αGSU) regulatory sequences in a bacterial artificial chromosome genomic clone. Initial analysis of this transgenic line, Tg(αGSU-iCre), with cre reporter strains reveals onset of cre activity in the differentiating cells of the developing anterior pituitary gland at embryonic day 12.5, with a pattern characteristic of endogenous αGSU. In adult mice, αGSU-iCre was active in the anterior lobe of the pituitary gland and in the cells that produce αGSU (gonadotropes and thyrotropes) with high penetrance. Little or no activity was observed in other tissues, including skeletal and cardiac muscle, brain, kidney, lungs, testis, ovary, and liver. This αGSU-iCre line is suitable for efficient, specific, and developmentally regulated deletion of floxed alleles in anterior pituitary gonadotropes and thyrotropes.
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/genética , Gonadotrofos/metabolismo , Integrases/metabolismo , Recombinação Genética , Tireotrofos/metabolismo , Alelos , Animais , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Embrião de Mamíferos , Feminino , Genótipo , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Integrases/genética , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos , Sequências Reguladoras de Ácido NucleicoRESUMO
Stem cells have been identified in organs with both low and high cell turnover rates. They are characterized by the expression of key marker genes for undifferentiated cells, the ability to self-renew, and the ability to regenerate tissue after cell loss. Several recent reports present evidence for the presence of pituitary stem cells. Here we offer a critical review of the field and suggest additional studies that could resolve points of debate. Recent reports have relied on different markers, including SOX2, nestin, GFRa2, and SCA1, to identify pituitary stem cells and progenitors. Future studies will be needed to resolve the relationships between cells expressing these markers. Members of the Sox family of transcription factors are likely involved in the earliest steps of pituitary stem cell proliferation and the earliest transitions to differentiation. The transcription factor PROP1 and the NOTCH signaling pathway may regulate the transition to differentiation. Identification of the stem cell niche is an important step in understanding organ development. The niche may be the marginal zone around the lumen of Rathke's pouch, between the anterior and intermediate lobes of mouse pituitary, because cells in this region apparently give birth to all six pituitary hormone cell lineages. Stem cells have been shown to play a role in recurrent malignancies in some tissues, and their role in pituitary hyperplasia, pituitary adenomas, and tumors is an important area for future investigation. From a therapeutic viewpoint, the ability to cultivate and grow stem cells in a pituitary predifferentiation state might also be helpful for the long-term treatment of pituitary deficiencies.
Assuntos
Células-Tronco Adultas , Hipopituitarismo/terapia , Hipófise/citologia , Animais , Diferenciação Celular , Humanos , Hipopituitarismo/etiologia , Camundongos , Hipófise/crescimento & desenvolvimento , RegeneraçãoRESUMO
The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke's pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the postnatal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke's pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke's pouch are necessary for cell specification between e11.5 and e13.5 and that cell-cell communication likely plays an important role in regulating this process.
Assuntos
Modelos Neurológicos , Hipófise/embriologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Idade Gestacional , Camundongos , Hipófise/citologia , Hipófise/fisiologia , Adeno-Hipófise/citologia , Adeno-Hipófise/embriologia , Adeno-Hipófise/fisiologia , Hormônios Hipofisários/biossíntese , Gravidez , Transdução de SinaisRESUMO
Genetic cases of congenital pituitary hormone deficiency are common and many are caused by transcription factor defects. Mouse models with orthologous mutations are invaluable for uncovering the molecular mechanisms that lead to problems in organ development and typical patient characteristics. We are using mutant mice defective in the transcription factors PROP1 and POU1F1 for gene expression profiling to identify target genes for these critical transcription factors and candidates for cases of pituitary hormone deficiency of unknown aetiology. These studies reveal critical roles for Wnt signalling pathways, including the TCF/LEF transcription factors and interacting proteins of the groucho family, bone morphogenetic protein antagonists and targets of notch signalling. Current studies are investigating the roles of novel homeobox genes and pathways that regulate the transition from proliferation to differentiation, cell adhesion and cell migration. Pituitary adenomas are a common human health problem, yet most cases are sporadic, necessitating alternative approaches to traditional Mendelian genetic studies. Mouse models of adenoma formation offer the opportunity for gene expression profiling during progressive stages of hyperplasia, adenoma and tumorigenesis. This approach holds promise for the identification of relevant pathways and candidate genes as risk factors for adenoma formation, understanding mechanisms of progression, and identifying drug targets and clinically relevant biomarkers.