Genetic control of hypothalamo-pituitary axis development and function in mice.

Congenital dysfunction of the hypothalamic-pituitary (HP) axis occurs in approximately one birth per 2,200 and is associated with a broad range of common disease states including impaired growth (short stature), infertility, hypogonadism poor responses to stress and slow metabolism (Pescovitz and Eugster, 2004). Although, a number of genes have been linked to diseases of the HP axis, the genetic cause in many patients remains unknown. This thesis examines two aspects of HP axis development and function. The first aim was to identify Sox3 targets by examining gene expression differences between three mouse lines: Sox3-null (mice lacking Sox3; loss of function), Extra-Sox3 (mice overexpressing Sox3; gain of function) and wild-type, by genome wide profiling using the Illumina BeadChip microarray platform. The second aim was to characterize the downstream effects relative to HP development in a novel recessive dwarf mouse model with pituitary hypoplasia and growth hormone (GH) deficiency, generated by N-ethyl-Nnitrosourea (ENU) mutagenesis that produces a point mutation in the gene for the enzyme tryptophanyl-tRNA synthetase (WARS). The first project (project 1) examined Sox3, the causative gene associated with Xlinked hypopituitarism (XH), in wild-type and transgenic mice. SOX3 is a member of the SOX (SRY-related HMG box) gene family of transcription factors that is expressed in progenitor cells of the mouse embryonic central nervous system (CNS) including the developing and postnatal hypothalamus (Rizzoti et al., 2004). It is the only member of the SOXB1 subfamily positioned on the X chromosome (Collignon et al., 1996; Stevanovic, 2003). Appropriate dose- and time-dependent expression of Sox3 in the developing hypothalamus is required for normal neuroendocrine function, particularly related to growth and growth hormone (GH). Changes associated with a loss-of-function and/or gain-of-function of Sox3 may contribute to a better understanding of other important genes, currently not known, involved in XH and/or X-linked mental retardation. At this point, however, the mechanisms linking SOX3 to its direct targets and their interplay within other downstream signaling cascades regulating HP axis development remain unknown. In order to identify Sox3-dependent genes, in mice, I performed microarray analysis of RNA extracted from embryonic mouse heads at 10.5 days post coitum (dpc) and compared RNA from wild-type, loss-of-function (Sox3-null) and gain-of-function (Extra-Sox3) mice. Several emergent candidate genes were further tested by quantitative mRNA expression analysis (qPCR). One of these was Neurogenin-3 (Ngn3), which showed a 2.5- fold decrease (P<0.001) in expression by microarray in Sox3-null (n=6), compared with wild-type (WT; n=6) mice and 1.8-fold decrease (P<0.001) by qPCR between Sox3-null (n=6) and WT (n=6) mice. To evaluate the relationship between Ngn3 and Sox3 at a cellular level immunohistochemistry was performed on 10.5 dpc and 12.5 dpc brains. In WT mice at 10.5 dpc and 12.5 dpc Ngn3 and Sox3 expression overlapped in a subset of cells across the ventral-midline of the developing hypothalamus. In addition and in contrast to WT mice, in Sox3-null mice, there were few Ngn3 positive cells, localized to the arcuate hypothalamic nucleus. Neurogenin-3 (Ngn3) is a member of the Neurogenin gene family of proneural basic helix-loop-helix proteins. Although previous data show the importance of Ngn3 during pancreatic development, there is no information on the mechanisms and actions of Ngn3 or a relationship between NGN3 action and SOX3 during hypothalamic development. These results suggest Ngn3 is a downstream target of Sox3 that is contributing to appropriate development of the hypothalamic-pituitary axis. The second study (project 2) aimed to characterize and further examine a novel recessive ENU mouse mutant, called Tukku¹ , exhibiting HP axis dysfunction resulting in dwarfism, pituitary hypoplasia and GH deficiency. Adult Tukku mice are 30-40% smaller than their WT littermates. The primary focus was to characterize the dwarfism phenotype in relation to the somatotropic axis and to identify the causative gene. The mutation was identified as a leucine to proline substitution in tryptophanyl-tRNA synthetase (WARS), a member of the aminoacyl-tRNA synthetase (AARS) enzyme family that link amino acids to their specific tRNAs. For proper function of this enzyme the specific recognition of substrates is critical for the fidelity of protein synthesis. The Wars mutation is contained within the N-terminal WHEP domain, from residue 16-69, and likely causes the disruption of the alpha helical structure. The N-terminal WHEP domain has only been found in eukaryote Wars enzyme. Importantly, AARS have been linked to regulating the noncanonical activity of angiogenesis (Otani et al., 2002; Wakasugi, 2010; Wakasugi and Schimmel, 1999; Wakasugi et al., 2002b). Along with pituitary hypoplasia, Tukku mice show a significant reduction in pituitary GH and serum levels of IGF-1, suggesting the defect leading to pituitary hypoplasia involves brain regions implicated in growth of the anterior pituitary. The reduction in pituitary GH levels may also involve delivery of GHreleasing hormone (GHRH) to GH-secreting cells since preliminary data also indicate that WARS is expressed within blood vessels of the pituitary and hypothalamus. To assess this, quantitative mRNA expression analysis (qPCR) of GHRH and somatostatin (Sst) was performed. qPCR revealed a decrease in both GHRH and Sst (fold change >2) indicating that the defect is likely to be within the hypothalamic hypophysial vasculature that extends and makes a connection with the pituitary. To evaluate the relationship between Wars and pituitary vasculature, immunohistochemistry was performed on pituitaries at 8-weeks postnatal. Pituitary sections were co-stained with antibodies against platelet endothelial cell adhesion molecule (PECAM) + Wars or vascular-endothelial cadherin (VE-Cadherin; an endothelial specific, transmembrane protein, which clusters at adheren junctions where it promotes homotypic cell-cell adhesion) + Wars. Wars immunostaining was expressed within the endothelial cells of the pituitary vasculature, both in the anterior and posterior pituitary. Both PECAM and Wars appeared co-expressed within the vascular wall. VECadherin was expressed in vessels together with Wars. Overall, the data gathered from these projects highlight important insights into the identification of Ngn3 as a likely Sox3 target gene (project 1) and have identified a novel dwarf mouse model with a genetic determinant of HP axis function (project 2). These results have application to the study of HP axis development, to the study of vascular development during embryology and postnatally, and to possible avenues of genetic screen testing and development of new treatments related to GH deficiencies. ¹ Tukku, meaning ‘small’ in Kaurna Aboriginal language.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201