Iain C. A. F. Robinson

Ribosomal Protein S6 Kinase 1 Signaling Regulates Mammalian Life Span

Mimicking Caloric Restriction The extended life span and resistance to age-related diseases in animals exposed to caloric restriction has focused attention on the biochemical mechanisms that produce these effects. Selman et al. (p. 140; see the Perspective by Kaeberlein and Kapahi) explored the role of the mammalian ribosomal protein S6 kinase 1 (S6K1), which regulates protein translation and cellular energy metabolism. Female knockout mice lacking expression of S6K1 showed characteristics of animals exposed to caloric restriction, including improved health and increased longevity. The beneficial effects included reduced fat mass in spite of increased food intake. Thus, inhibition of signaling pathways activated by S6K1 might prove beneficial in protecting against age-related disease. A signaling pathway in mice mediates the effects of caloric restriction that protect against age-related diseases. Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse

During early mouse development the homeobox gene Hesx1 is expressed in prospective forebrain tissue, but later becomes restricted to Rathkes pouch, the primordium of the anterior pituitary gland. Mice lacking Hesx1 exhibit variable anterior CNS defects and pituitary dysplasia. Mutants have a reduced prosencephalon, anopthalmia or micropthalmia, defective olfactory development and bifurcations in Rathkes pouch. Neonates exhibit abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum. A comparable and equally variable phenotype in humans is septo-optic dysplasia (SOD). We have cloned human HESX1 and screened for mutations in affected individuals. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain which destroyed its ability to bind target DNA. These data suggest an important role for Hesx1/HESX1 in forebrain, midline and pituitary development in mouse and human.

Genetic Regulation of Pituitary Gland Development in Human and Mouse

Normal hypothalamopituitary development is closely related to that of the forebrain and is dependent upon a complex genetic cascade of transcription factors and signaling molecules that may be either intrinsic or extrinsic to the developing Rathkes pouch. These factors dictate organ commitment, cell differentiation, and cell proliferation within the anterior pituitary. Abnormalities in these processes are associated with congenital hypopituitarism, a spectrum of disorders that includes syndromic disorders such as septo-optic dysplasia, combined pituitary hormone deficiencies, and isolated hormone deficiencies, of which the commonest is GH deficiency. The highly variable clinical phenotypes can now in part be explained due to research performed over the last 20 yr, based mainly on naturally occurring and transgenic animal models. Mutations in genes encoding both signaling molecules and transcription factors have been implicated in the etiology of hypopituitarism, with or without other syndromic features, in mice and humans. To date, mutations in known genes account for a small proportion of cases of hypopituitarism in humans. However, these mutations have led to a greater understanding of the genetic interactions that lead to normal pituitary development. This review attempts to describe the complexity of pituitary development in the rodent, with particular emphasis on those factors that, when mutated, are associated with hypopituitarism in humans.

Mutations within Sox2/SOX2 are associated with abnormalities in the hypothalamo-pituitary-gonadal axis in mice and humans

The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice.

SOX3 is required during the formation of the hypothalamo-pituitary axis

The pituitary develops from the interaction of the infundibulum, a region of the ventral diencephalon, and Rathkes pouch, a derivative of oral ectoderm. Postnatally, its secretory functions are controlled by hypothalamic neurons, which also derive from the ventral diencephalon. In humans, mutations affecting the X-linked transcription factor SOX3 are associated with hypopituitarism and mental retardation, but nothing is known of their etiology. We find that deletion of Sox3 in mice leads to defects of pituitary function and of specific central nervous system (CNS) midline structures. Cells in the ventral diencephalon, where Sox3 is usually highly expressed, have altered properties in mutant embryos, leading to abnormal development of Rathkes pouch, which does not express the gene. Pituitary and hypothalamic defects persist postnatally, and SOX3 may also function in a subset of hypothalamic neurons. This study shows how sensitive the pituitary is to subtle developmental defects and how one gene can act at several levels in the hypothalamic-pituitary axis.

SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland

The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (<0.05%) population of progenitor cells in the adult pituitary gland. We show that these cells express SOX2, a marker of several early embryonic progenitor and stem cell types, and form “pituispheres” in culture, which can grow, form secondary spheres, and differentiate to all of the pituitary endocrine cell types, as well as folliculostellate cells. Differentiation of cells in the pituispheres was associated with the expression of nestin, SOX9, and S100. Cells expressing SOX2 and E-cadherin are found throughout Rathkes pouch (RP) in embryos but persist in the adult gland, mostly in a narrow zone lining the pituitary cleft, but also are scattered throughout the pituitary. However, unlike in embryonic RP, most of these SOX2+ cells in the adult gland also express SOX9 and S100. We suggest that this SOX2+/SOX9+ population represents transit-amplifying cells, whereas the SOX2+/SOX9− cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.

Over- and Underdosage of SOX3 Is Associated with Infundibular Hypoplasia and Hypopituitarism

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

Differential Clearance of Neurophysin and Neurohypophysial Peptides from the Cerebrospinal Fluid in Conscious Guinea Pigs

The clearance of neurohypophysial peptides from cerebrospinal fluid (CSF) in conscious unrestrained guinea pigs. 125I-labelled peptides were detectable in the cisterna magna within 2 min of their intracerebroventricular injection, reaching peak concentrations 10-15 min post-injection and declining exponentially over the next hour. 125I-oxytocin (125I-OT) and 125I-vasopressin (125(I-AVP) were cleared at similar rates, whereas 125I-labelled neurophysin (125I-NP) disappeared significantly more slowly; mean half-times of clearance (t1/2) from cisternal CSF were 28, 24 and 46 min, respectively. 125I-NP was cleared at the same rate as 3H-inulin (t1/2 40 min), as was an antibody to OT (anti-OT, t1/2 37 min). Intracerebroventricular infusions of iodinated peptides produced constant levels in CSF within 3 h. 125I-AVP reached lower plateau levels and disappeared twice as fast as 125I-NP, although the apparent equilibrium distribution space was the same for both peptides. Although NP was cleared half as fast as OT or AVP, this difference was not sufficient to account for the large molar excess of NP over the nonapeptides in guinea pig CSF. There is an effective blood/CSF barrier to neurohypophysial peptides in the guinea pig; intravenous infusions of OT or porcine NP did not raise the CSF levels of these peptides (measured by specific radioimmunoassays) except when very high concentrations were maintained in peripheral plasma. However, single intravenous injections of anti-OT produced low but significant titres in CSF, persisting for several days.

Effects of adrenalectomy and glucocorticoids on the peptides CRF‐41, AVP and oxytocin in rat hypophysial portal blood.

1. The effects of adrenalectomy (3 weeks) and dexamethasone (3 h) treatment on the release of corticotrophin‐releasing factor‐41 (CRF‐41), arginine vasopressin (AVP), oxytocin (OT), adrenocorticotrophin (ACTH) and corticosterone were studied in adult female Wistar rats. 2. The animals were anaesthetized with sodium pentobarbitone which, as assessed by the effects on the circadian rhythm of plasma ACTH and corticosterone, appeared to be a better anaesthetic than either urethane or alphaxalone for studies on the hypothalamic‐pituitary‐adrenal system. 3. Adrenalectomy increased the concentrations of ACTH in peripheral plasma and the output of CRF‐41 and AVP into hypophysial portal plasma. 4. Dexamethasone administered to adrenalectomized rats significantly reduced the concentration of ACTH in peripheral plasma and the amount of AVP released into portal plasma. However, dexamethasone did not affect the output of CRF‐41 into portal blood. 5. The output of OT into portal plasma was unaffected by either adrenalectomy or dexamethasone treatment. 6. Dexamethasone administered to adrenalectomized rats reduced significantly the ACTH response to CRF‐41. 7. These results show that the feed‐back action of glucocorticoids is mediated by two mechanisms. The increased release of ACTH which follows adrenolectomy [corrected] is produced predominantly by an increased release of both CRF‐41 and AVP into hypophysial portal blood. The intermediate negative feed‐back of glucocorticoids is produced by a reduction in the output of AVP but not CRF‐41 into portal blood and, as well, by a significant reduction in the responsiveness of the anterior pituitary gland to CRF‐41.

SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

CONTEXT Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. OBJECTIVE We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. RESULTS All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathkes pouch, the developing anterior pituitary, and the eye. CONCLUSIONS Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.