Buffy S. Ellsworth, Ph.D. - US grants

DESCRIPTION (provided by applicant): Foxl-2 is expressed in the developing eye and in the adult ovary and is required for normal development and function of these organs in humans. Recently, expression of FoxL2 has been detected in the developing mouse pituitary from e10.5 to el 5. To examine the role of FOXL2 in pituitary development, the following studies will be performed: characterize the developmental expression of Foxl2 and place it in the genetic hierarchy of pituitary developmental control, define important regulatory sequences for Foxl2 expression in cell culture and transgenic mice, and examine the role of FOXL2 in the developing anterior pituitary by generation of gain and loss of function alleles. In situ hybridization of pituitaries from normal mice will be used to characterize Foxl2 expression. Normal expression patterns for Foxl2 will be compared to those in mice with mutations in critical pituitary transcription factors. This information will place FOXL2 in the genetic hierarchy. Transient transfections of mammalian cells and electrophoretic mobility shift assays will be used to define the minimal essential sequence for Foxl2 expression and to test for direct regulation by factors that are upstream of Foxl2 in the genetic hierarchy. These findings will be confirmed in transgenic animals. To delineate the role of FOXL2 in pituitary development, FoxL2 will be over-expressed and will be disrupted specifically in the pituitaries of mice, allowing us to separate the role of FOXI.2 in the pituitary from its role in other organs.

DESCRIPTION (provided by applicant): Congenital hormone deficiencies are common, occurring in approximately one in 4,000 live births. Pituitary hormone deficiency can consist of loss of a single hormone (isolated hormone deficiency) or several hormones (combined pituitary hormone deficiency). Absence of anterior pituitary hormones does not interfere with fetal viability, but are required for survival after birth, gonadal differentiation, and maturation of the fetal thyroid. Lesions in the transcription factors PITX1, PITX2, HESX1, LHX3, LHX4, TPIT, PROP1 and PIT1 lead to combined pituitary hormone deficiency in mice and humans. However mutations in these transcription factors account for only a fraction of congenital hormone deficiencies in humans. To identify additional factors that contribute to human congenital hormone deficiencies, we are investigating the forkhead transcription factor, FOXO1, which is important for the normal development of several organs. Mouse knockout models for Foxo1 result in embryonic lethality at embryonic day (e)10.5 due to vascular defects. Our objective is to investigate the contributions of FOXO1 to congenital pituitary hormone deficiency. We hypothesize that eliminating FOXO1 in the pituitary will cause hyperplasia and misorganization of the pituitary gland resulting in loss of hormone production and/or mis-specification of hormone-producing cell types. This hypothesis is based on the following observations. First, our preliminary results show that FOXO1 is present in nuclei of non-dividing pituitary cells starting at e14.5 when these cells are beginning to differentiate. Secondly, FOXO1 inhibits proliferation and regulates differentiation and migration of pancreatic 2-cells, myoblasts, adipocytes, and endothelial cells. To test this hypothesis we propose three specific aims: Specific Aim 1. Determine the pituitary cell specificity for FOXO1 during development. We will perform immunohistochemistry for the hormones that mark the five pituitary cell types in combination with immunohistochemistry for FOXO1 on mouse embryonic tissue at e18.5. Specific Aim 2. Investigate the requirement for Foxo1 in pituitary gland development and function. We will analyze pituitary glands from mouse embryos that have had Foxo1 expression eliminated in the pituitary gland (Foxo1 pit/-). We will assess pituitary morphology, analyze anterior pituitary hormone production, and measure cell proliferation and apoptosis. Specific Aim 3. Place Foxo1 in the genetic hierarchy of pituitary gland developmental control. We will perform immunohistochemistry on Foxo1 pit/- mouse embryos for genes that are required during normal pituitary development. The knowledge generated by these studies will further this field by identifying a candidate gene for congenital pituitary hormone deficiency and by adding to our knowledge of how forkhead factor mutations cause birth defects. Our expertise in both forkhead transcription factor function and pituitary development places us in a unique situation to address this problem. PUBLIC HEALTH RELEVANCE: Pituitary hormones are essential for survival after birth and for the normal development of gonads and thyroid gland. The forkhead transcription factor, FOXO1, is essential for the normal development and function of muscle, blood vessels, and pancreas. The pattern of FOXO1 localization in the pituitary gland during development suggests a role for inhibiting cell division and regulating cell differentiation. Our objective is to determine the requirement of FOXO1 for normal pituitary development by analyzing the pituitary phenotype in mice that lack FOXO1 in the pituitary gland.

DESCRIPTION (provided by applicant): The pituitary gland regulates development and function of many organs to control growth, response to stress, fertility and homeostasis. Congenital pituitary hormone deficiencies are common, occurring in approximately one out of every 4000 live births. Hormone deficiency can exist as loss of a single hormone or combined pituitary hormone deficiency (CPHD). Mutations in a dozen genes have been identified as causes of CPHD, but the etiology of half of cases remains unknown. The long-term goal of the proposed studies is to identify the genes that, when mutated, cause pituitary hormone insufficiency and determine the mechanism of action of those genes. To expand the molecular diagnoses for pituitary hormone insufficiency, we have focused on a family of transcription factors referred to as forkhead factors. Forkhead factors are essential for diverse developmental processes and mutations in the genes encoding these factors are responsible for a number of human developmental disorders. Our previous studies have identified the forkhead transcription factor, Foxo1, as a candidate gene for pituitary hormone insufficiency. FOXO1 is expressed in somatotrope cells at e18.5 and in adult males and females. Mice lacking Foxo1 in the pituitary have a drastically reduced number of growth hormone-containing cells, suggesting that Foxo1 is required for normal growth hormone (GH) production. It is important to investigate the mechanisms of FOXO1 regulation of GH production because of the critical role of GH in growth, healthy metabolism, and heart function. We hypothesize that FOXO1 regulates GH production beginning early in development by regulating expression of the growth hormone gene or upstream regulators that control GH production. Our hypothesis will be tested by the following aims: 1) Determine the mechanism by which FOXO1 regulates GH production. This aim will employ luciferase reporter assays and chromatin immunoprecipitation assays to determine which genes are directly regulated by FOXO1. 2) Determine when Foxo1 is required for GH production. This aim will analyze GH production at early stages of development in mouse embryos that have the Foxo1 gene excised from the pituitary. Our expertise in both forkhead transcription factor function and pituitary development places us in a unique situation to address this fundamental question. The knowledge generated by these studies will further this field by exploring the mechanism of action of a candidate gene for pituitary hormone deficiency and by adding to our knowledge of pituitary organogenesis.