Hypothalamic growth hormone-releasing hormone (GHRH) deficiency: targeted ablation of GHRH neurons in mice using a viral ion channel transgene.

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

Physiological studies of transgenic mice overexpressing growth hormone (GH) secretagogue receptor 1A in GH-releasing hormone neurons.

The type 1A GH secretagogue (GHS) receptor (GHSR) has been proposed to mediate the effects of ghrelin on GH release, food intake, and body composition. We have overexpressed GHSR in GH-producing GC cells and GHRH neurons in an attempt to enhance signaling via this pathway selectively, in the GH axis. Constitutive overexpression of human GHSR in rat GC cell lines resulted in increased basal phosphoinositol turnover and rendered them responsive to GHS ligands. We then generated transgenic mice overexpressing human GHSR in GHRH neurons using a 38-kb rat GHRH cosmid promoter. GHRH-GHSR transgenic mice showed increased hypothalamic GHRH expression, pituitary GH contents, and postweaning growth rates. Body weights of the transgenic mice became similar in adulthood, whereas adipose mass was reduced, particularly so in female GHRH-GHSR mice. Organ and muscle weights of transgenic mice were increased despite chronic exposure to a high fat diet. These results suggest that constitutive overexpression of GHSR in GHRH neurons up-regulates basal activity in the GHRH-GH axis. However, GHRH-GHSR mice showed no evidence of increased sensitivity to acute or chronic treatment with exogenous GHS ligands. Food intake and adipose tissue responses to chronic high fat feeding and treatment with GHS ligands were unaffected, as were locomotor and anxiety behaviors, although GHRH-GHSR mice remained significantly leaner than wild-type littermates. Thus, constitutive overexpression of GHSR can up-regulate basal signaling activity in the GHRH/GH axis and reduce adiposity without affecting other GHSR-mediated signals.

Neural and mammary gland defects in ErbB4 knockout mice genetically rescued from embryonic lethality.

Mice lacking the epidermal growth factor receptor family member ErbB4 exhibit defects in cranial neural crest cell migration but die by embryonic day 11 because of defective heart development. To examine later phenotypes, we rescued the heart defects in ErbB4 mutant mice by expressing ErbB4 under a cardiac-specific myosin promoter. Rescued ErbB4 mutant mice reach adulthood and are fertile. However, during pregnancy, mammary lobuloalveoli fail to differentiate correctly and lactation is defective. Rescued mice also display aberrant cranial nerve architecture and increased numbers of large interneurons within the cerebellum.

Autosomal dominant growth hormone deficiency disrupts secretory vesicles in vitro and in vivo in transgenic mice.

Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.

Conditional ablation of T-cell development by a novel viral ion channel transgene.

A novel conditional-lethal transgene system is defined in which a mutated influenza A virus ion-channel protein, which is permeable to monovalent cations, is lethal to cells on heterotypic expression and whose activity can be blocked by an antiviral drug (amantadine), is used to reversibly disrupt T-cell development. In vivo expression of the M2 ion channel, as a transgene under control of the T-cell specific p56(Lck) proximal promoter, resulted in total ablation of T-cell development with the accumulation of three distinct populations of early progenitor cells (CD44(+) CD25(-); CD44(+) CD25(+); CD44(+) CD25(hi)) in the thymic rudiment. In vitro development of transgenic fetal thymic progenitors to single-positive T cells could be rescued by antiviral drug treatment. Moreover, there was a radical reduction in B-cell lymphopoiesis, evident at the pre-B-cell stage, with a twofold increase of lymphoid cells 'in cycle' in transgenic bone marrow, indicative of major changes in haematopoietic homeostasis. This system may provide a generic protocol for conditional, lineage-specific cell ablation with available tissue-specific promoters for any eukaryotic developmental system, and provide a window on early T-cell development.