David L. Hurley

CENTRAL HYPOTHYROIDISM REVEALS COMPOUND HETEROZYGOUS MUTATIONS IN THE PIT-1 GENE

Mutations in the gene encoding the Pit-1 transcriptional activator interfere with the embryologic determination and ultimate functions of anterior pituitary cells that produce growth hormone (GH), prolactin (Prl) and thyroid-stimulating hormone (TSH). Central hypothyroidism is often the presenting feature of combined pituitary hormone deficiency (CPHD), but it is not detected in screening programs that rely upon elevation of TSH. We report a child whose hypothyroidism was recognized clinically at age 6 weeks, and subsequently found to have GH and Prl as well as TSH deficiency. With thyroxine and GH replacement he has reached the 70th percentile for height and has normal intelligence. Molecular analysis of genomic DNA for Pit-1 revealed the presence of compound heterozygous recessive mutations: a nonsense mutation in codon 172 and a novel missense mutation substituting glycine for glutamate at codon 174. This case is the first demonstration of CPHD due to compound heterozygous Pit-1 point mutations, as most reported cases of the CPHD phenotype involve either the dominant negative R271W allele or homozygosity for recessive Pit-1 mutations. Therefore, in cases of CPHD, the possibilities of compound heterozygosity for two different Pit-1 mutations, or homozygosity for mutations in the epigenetic gene, Prop-1, should be considered.

The opiate system in invertebrates

The presence in diverse species of a similar mode of communication, that of a soluble messenger binding to a receptor, raises the question as to whether the specific components of this system are equally widespread. Do invertebrates use the same hormones and receptors as vertebrates do? Invertebrates ranging from unicellular organisms to insects have been shown to contain opiate-like peptides and binding sites, and they exhibit biological responses to opiates. However, critical genetic data are lacking. It is not known how signal systems arise phylogenetically, but it is conceivable that signal molecules that are already present cause the formation of their own receptors from membrane proteins.

Growth Hormone-Releasing Hormone-Producing and Dopaminergic Neurones in the Mouse Arcuate Nucleus Are Independently Regulated Populations

Differentiation of hypophysiotropic neurones that regulate the secretion of growth hormone (GH) and prolactin is influenced by GH and prolactin. Genetic GH and prolactin deficiency in mutant rodent models such as the Ames dwarf (df/df) mouse results in an increase in the number of GH‐stimulatory GH‐releasing hormone (GHRH) neurones and a reduction of prolactin‐inhibitory tuberoinfundibular dopaminergic (TIDA) neurones in the arcuate nucleus during postnatal development. The present study tested the hypothesis that these concomitant changes in numbers of tyrosine hydroxylase (TH)‐ and GHRH‐immunoreactive neurones in df/df hypothalamus might represent a neuronal population of fixed number that undergoes a partial change in phenotype during postnatal development. To evaluate this possibility, the postnatal reduction of the df/df TIDA population was prevented by administering prolactin neonatally to preserve TH phenotype; dwarf and normal sibling mice were treated with daily injections of ovine prolactin or vehicle starting at postnatal day 12 and continuing for 30 days. Following this treatment, numbers of arcuate neurones containing GHRH or TH, or both, were quantified using immunocytochemistry. It was hypothesized that prolactin preservation of TH‐immunoreactive cell number would be accompanied by either a decrease in the GHRH‐producing population or an increase in numbers of cells producing both TH and GHRH. In prolactin‐treated normal (DF/df) mice, numbers of arcuate TH‐immunoreactive neurones were similar to those in vehicle‐treated normals. Numbers of TH‐positive neurones in prolactin‐treated dwarfs were higher than in vehicle‐treated dwarfs, and did not differ from numbers in DF/df. Numbers of GHRH‐immunoreactive cells in vehicle‐treated df/df were higher than in vehicle‐treated DF/df, and were not different in prolactin‐treated groups of either dwarf or normal mice. Neurones containing both TH and GHRH constituted 15% of the TH population, and 76% of the GHRH population, in control normal mice; in control dwarfs, double‐labelled cells were 9.3% of TH and 9.9% of GHRH. Numbers of cells immunoreactive for both TH and GHRH were not affected by prolactin treatment in either mouse type. These results demonstrate that the increase in number of GHRH‐expressing neurones in the df/df arcuate nucleus does not occur at the expense of the TH phenotype, and that this increase is not influenced by prolactin feedback. Although coexpression of TH and GHRH in a subpopulation indicates that TIDA and GHRH populations are not exclusive, they appear to be influenced independently by prolactin and GH signals during development.

Prolactin replacement must be continuous and initiated prior to 21 d of age to maintain hypothalamic dopaminergic neurons in hypopituitary mice

The prolactin (PRL) deficit in mice homozygous for the spontaneous Ames dwarf (df) mutation coincides with a marked reduction in the number of PRL-regulating tuberoinfundibular dopaminergic (TIDA) neurons. The TIDA deficit develops after 14–21 d postnatally and may be prevented by PRL replacement initiated at 12, but not at 60, d of age. The present study was designed to define further the developmental period during which PRL can prevent the deficit in the number of TIDA neurons in df/df mice, as well as to evaluate whether exposure to PRL neonatally affects the response to PRL by TIDA neurons in later development. To address the first aim, litters of df/df and normal (DF/df) mice were treated daily with ovine PRL (50 µg intraperitoneally), starting at 12, 21, or 30 d of age. To address the second aim, DF/df and df/df animals treated with PRL for 30 d starting at 12 d of age were subjected to PRL withdrawal (15 d of saline vehicle treatment), followed by PRL retreatment. All brains were evaluated using both catecholamine histofluorescence and tyrosine hydroxylase (TH) immunocytochemistry. Total numbers of TH-immunostained cells were counted in area A12 (TIDA neurons) and in A13 (medial zona incerta). Qualitatively, catecholamine fluorescence in A12 perikarya and terminals in df/df mice was enhanced by PRL treatment initiated at 12 or 21, but not at 30, d of age. TH immunostaining intensity was enhanced in all df/df PRL-treated groups, compared with saline treatment. However, total numbers of TH-positive TIDA neurons were reduced significantly in df/df mice treated with PRL beginning at 21 or 30 d, as well as with saline at 12 d, compared with similarly treated DF/df groups and with df/df animals treated with PRL beginning at 12 d (p<0.01 for all comparisons). Among dwarf mice treated with PRL beginning at 12 d, followed by PRL withdrawal, the numbers of TH-positive TIDA neurons were greater than those of saline-treated dwarfs, but less than those in DF/df mice (p<0.05 for both comparisons). In dwarfs retreated with PRL after withdrawal, the TIDA population was also smaller than that in normal animals (p<0.05), although it was larger than in vehicle-treated dwarfs of the same age (p<0.05). No effect of PRL treatment on TIDA cell numbers in normal mice, or of treatment or mouse phenotype on the number of TH-positive cells in zona incerta, occurred in either experiment. These results indicate that the effect of PRL on preventing the reduction in the TIDA population in df/df mice is limited to a developmental period prior to 21 d postnatally. In addition, this study provides evidence that continuous PRL feedback is required to maintain normal numbers of TIDA neurons. These findings extend the evidence for a critical role of PRL feedback in the differentiation and preservation of phenotype in TIDA neurons.

Mouse growth hormone transcription factor Zn-16: unique bipartite structure containing tandemly repeated zinc finger domains not reported in rat Zn-15.

Rat Zn-15 is a transcription factor activating GH gene expression by synergistic interactions with Pit-1, named for 15 DNA-binding zinc fingers, including fingers IX, X, and XI that are responsible for GH promoter binding. In this study, a mouse cDNA for Zn-15 was characterized. The predicted 2192-amino acid mouse protein is 89% identical to rat (r) Zn-15 overall, and is 97% similar in the C-terminal domain necessary for binding the GH promoter. However, the mouse cDNA encodes 16 zinc fingers, and sequences of rZn-15 pituitary cDNAs were the same as the mouse (m) Zn-16; the rat sequence in GenBank has a one nucleotide offset of a 17-bp segment in the finger V region. The mouse and corrected rat sequences contain four tandemly repeated fingers in the N-terminus, each separated by seven amino acids, typical of zinc finger proteins of the transcription factor IIIA-type. Analysis of mZn-16 expression by RT-PCR showed that the mRNA is, produced at similar levels in normal and GH-deficient Ames dwarf (Prop-1 ) mouse pituitaries at postnatal day 1. Mouse Zn-16 mRNA also was detected by ribonuclease protection assay in the pre-somatotrophic mouse cell line GHFT1-5. The Zn-16 protein is bipartite in that the N-terminal half displays tandem spacing typical of most zinc finger proteins, while the C-terminal portion contains long linkers between fingers that cooperatively bind to a DNA response element. Expression in early postnatal pituitary and in pre-somatotrophic cells suggests that Zn-16 could play a role in pituitary development prior to somatotroph differentiation.

Altered Growth Hormone-Releasing Hormone mRNA Expression in Transgenic Mice with Excess or Deficient Endogenous Growth Hormone

Hypothalamic expression of growth hormone-releasing hormone (GHRH) mRNA was examined in two transgenic mouse models displaying excess or deficient endogenous GH. Transgenic dwarf mice bore a gene construct consisting of the rat growth hormone (GH) promoter fused to a diphtheria toxin A chain structural gene (DT-A); the GH promoter restricted DT-A expression to endogenous GH-producing cells, which were destroyed. GH was undetectable in either the pituitary or the peripheral circulation (Behringer et al., Genes Devel. 2: 453-461, 1988). Transgenic giant mice carried a construction joining the metallothionein promoter to the human GHRH structural gene, which stimulated endogenous pituitary GH production (Hammer et al., Nature 315: 413-417, 1985). In situ hybridization to GHRH mRNA in transgenic dwarf, giant, and nontransgenic controls was performed using single-stranded RNA probes generated from cloned mouse GHRH cDNA. Hybridization to GHRH mRNA was limited to the neurons of the hypothalamic arcuate nucleus (ARC). Autoradiographic densities on X-ray films were quantified by computerized image analysis. There was an increase in GHRH signal intensity in the dwarfs (282 +/- 20 units; mean +/- SEM) relative to that measured in control animals (107 +/- 8 units; P < 0.001), while giant mice had decreased signal (42 +/- 7 units; P < 0.001) in the ARC. The present studies demonstrate that increase in GHRH mRNA expression accompanies GH deficiency, while a decrease in GHRH mRNA accompanies GH excess, suggesting both positive and negative feedback upon steady-state mRNA levels in hypophysiotropic neurons by target pituitary hormone.

Transcript abundance in mouse pituitaries with altered growth hormone expression quantified by reverse transcriptase polymerase chain reaction implicates transcription factor Zn-16 in gene regulation in vivo.

The correlation of growth hormone (GH) mRNA abundance and expression of specific transcription factors was studied in pituitaries of panhypopituitary (Ames df/df and Snell dwJ/dwJ dwarf), isolated GH-deficient (lit/lit), and GH-overproducing (growth hormone-releasing hormone [GHRH] transgenic) mice compared with normal littermates. A fluorescence-based reverse transcriptase polymerase chain reaction assay was developed for seven target mRNAs: GH, prolactin (PRL), proopiomelanocortin (POMC), α-subunit of the glycoprotein hormones (αSU), Pit-1, Prop-1, and Zn-16. Amplification parameters for each of these primer pairs were determined in order to calculate initial mRNA transcript number. The reproducibility of the assay was found to be ±10% for either Pit-1 or Zn-16 mRNAs measured in characterized murine GHFT1-5 somatotroph precursor cells. The cell extracts also showed an increased abundance of both Zn-16 and Pit-1 mRNAs when compared with whole pituitary extracts. Measurement of copy number in normal pituitaries showed that for every 106 GH or PRL mRNAs, there were 3 × 105 POMC, 4 × 104 αSU, 2 × 103 Pit-1, and only 70 Zn-16 or Prop-1 transcripts. Transcript abundance in GH-altered mice as a percentage of copy number per normal gland showed that POMC was significantly reduced in dwJ/dwJ (p<0.01) and df/df (p<0.05) mice. αSU mRNA was reduced in df/df (p<0.05), dwJ/dwJ (p<0.05), and lit/lit (p<0.05) mice, but not in GHRH-excess mice. PRL mRNA was not detected in dwarf mice, reduced to 52% of normal in lit/lit (p<0.05), and unchanged in GHRH-excess animals. GH mRNA was not detected in dwarf mice, reduced to 1.3% in lit/lit (p<0.005), and increased to 242% in GHRH-excess mice (p<0.05). Pit-1 mRNA was not detected in dwarf mice, was 2.9% of normal in lit/lit (p<0.005) mice, and increased to 200% in GHRH-excess mice (p<0.05). Prop-1 was not present in dwarf mice, was decreased to 1.4% in lit/lit (p<0.01), and increased to 223% in GHRH-excess mice (p<0.05). Zn-16 abundance in df/df mice was significantly reduced (p<0.05) to 4.8% of normals, to 6.3% of normals in dwJ/dwJ (p<0.005), to 6.1% of normals in lit/lit (p<0.005) mice, and significantly elevated in GHRH-excess mice to 197% (p<0.05). Altered pituitary mRNA abundance was found for several products not previously measured, or thought not to be affected by these mutations. Correlation of GH mRNA abundance with transcription factor copy number showed a significant correlation for Pit-1, Prop-1, and Zn-16. These quantitative analyses provide the first in vivo evidence that Zn-16 mRNA abundance correlates with GH expression.

Increased Hypothalamic Somatostatin Expression in Mice Transgenic for Bovine or Human GH

Acute studies of GH removal by hypophysectomy or GH replacement in adult rats have shown that GH has a positive influence on its hypothalamic inhibitory hormone somatostatin (SRIH). The present study was undertaken to assess the effect of lifelong exposure to elevated GH on the development and differentiation of SRIH‐producing hypothalamic neurons, including comparison of differing GH levels and heterologous species of GH. Expression of somatostatin peptide and mRNA was evaluated using respective immunocytochemistry and in situ hybridization in brains of transgenic mice bearing constructs of either human (hGH) or bovine (bGH) linked to metallothionein (MT) promoter or bGH linked to phosphoenolpyruvate carboxykinase (PEPCK) promoter. Nontransgenic littermates served as controls. All transgenic constructs resulted in high levels of circulating heterologous GH and significantly elevated body weights. Both bGH levels and body weights were higher in PEPCK‐bGH than in MT‐bGH mice; mean weights were not different between MT‐bGH and MT‐hGH mice. Numbers of SRIH‐immunoreactive neurons in the hypophysiotropic periventricular nucleus (PeN) of transgenic mice showed a two‐fold increase (P<0.01) relative to control animals; the number of SRIH‐positive cells in the medial basal hypothalamus (MBH) was comparable for transgenic and control mice. Total SRIH mRNA in situ hybridization intensity also showed a two‐fold increase (P<0.05) in the PeN of all transgenic mice compared with controls, and was not elevated in the MBH. The higher levels of GH produced in PEPCK‐bGH transgenic mice led to greater weight gain, but not to greater SRIH expression than in other GH‐transgenic mice, suggesting that the increased SRIH cell number and mRNA in the PeN of MT‐GH‐transgenic mice may represent a plateau of maximal feedback stimulation. The results indicate that lifelong elevated heterologous GH in mice stimulates hypothalamic SRIH expression markedly. It is not known whether this mechanism is direct or indirect via a mediator of GH such as IGF, but the heterologous GH appears to be specific to these hypophysiotropic neurons.

Discordant molecular and morphological evolution in buffalofishes (Actinopterygii: Catostomidae)

Buffalofishes (Genus Ictiobus) are large, robust-bodied suckers adapted to large rivers and lakes of North America. Currently recognized species are readily diagnosed by morphological characters, and the group is known from fossils dating back to the Miocene. However, sympatrically occurring species in the Mississippi River Basin are known to hybridize in nature and in the laboratory. Here we describe patterns of morphological (morphometric) and DNA sequence variation (mitochondrial and nuclear genes) across the geographic ranges of extant species of genus Ictiobus. We show that Ictiobus species form more of less discrete entities based on body morphometry, consistent with current taxonomy. However, except for I. labiosus, there is extensive sharing of alleles of nuclear and mitochondrial genes among species, and the species do not form reciprocally monophyletic groups in nuclear or mitochondrial gene trees. Moreover, the pattern is not confined to the broad area of sympatry in the Mississippi River Basin. We attribute this to a long history of introgressive hybridization and gene flow among species inhabiting the present-day Mississippi River Basin, and recent colonization of the Great Lakes, Hudson Bay drainage and gulf coastal rivers east and west of the Mississippi River by introgressed Mississippi River Basin stocks.

Growth Hormone Releasing Hormone Expression During Postnatal Development in Growth Hormone-Deficient Ames Dwarf Mice:mRNA in situ Hybridization

Several genetic mutations in mice and rats that produce lifelong growth hormone (GH) deficiency result in overexpression of GH-releasing hormone (GHRH) mRNA in hypothalamic arcuate nucleus neurons. In order to examine the development of this condition, GHRH mRNA expression was quantified in Ames dwarf (df/df) and normal (DF/?) mice at 1 (day of birth), 3, 7, 14, 21 and 60 postnatal days (d) following in situ hybridization. Total mRNA was assessed using computer-assisted densitometry after X-ray film autoradiography, and mRNA expression per neuron was quantified by counts of grains per cell after emulsion autoradiography. Total GHRH mRNA was the same in dwarf and normal mice at 1, 3 and 7d. GHRH mRNA in dwarfs increased at 14d to 240% of that in DF/? (p < 0.005); the percentage overexpression in dwarf mice remained ≧200% through 60d, although total GHRH mRNA increased in both dwarfs and normals during this period. GHRH mRNA per neuron was the same in normal and dwarf mice at 1d, then increased in dwarfs to 190% of that in normals at 3d (p < 0.05), and rose to 300% of normal levels by 7d and beyond (p < 0.005). There was no sexual dimorphism in expression by either measure in normal or dwarf mice. These results indicate that an increase in GHRH mRNA in Ames dwarf mice is first detectable at 3d, a period of approximately 7d after the failure to initiate GH production, which occurs normally at embryonic day 17.5. The onset of GHRH overexpression occurs earlier than the decline of either hypophysiotropic somatostatin or dopamine in Ames dwarf mice. This difference may be due to the stimulatory action of GHRH, as opposed to the inhibitory effects of factors examined previously.