Glenda J. Froelick

Disruption of the Metallothionein-III Gene in Mice: Analysis of Brain Zinc, Behavior, and Neuron Vulnerability to Metals, Aging, and Seizures

Metallothionein-III (MT-III), a brain-specific member of the metallothionein family of metal-binding proteins, is abundant in glutamatergic neurons that release zinc from their synaptic terminals, such as hippocampal pyramidal neurons and dentate granule cells. MT-III may be an important regulator of zinc in the nervous system, and its absence has been implicated in the development of Alzheimer’s disease. However, the roles of MT-III in brain physiology and pathophysiology have not been elucidated. Mice lacking MT-III because of targeted gene inactivation were generated to evaluate the neurobiological significance of MT-III. MT-III-deficient mice had decreased concentrations of zinc in several brain regions, including hippocampus, but the pool of histochemically reactive zinc was not disturbed. Mutant mice exhibited normal spatial learning in the Morris water maze and were not sensitive to systemic zinc or cadmium exposure. No neuropathology or behavioral deficits were detected in 2-year-old MT-III-deficient mice, but the age-related increase in glial fibrillary acidic protein expression was more pronounced in mutant brain. MT-III-deficient mice were more susceptible to seizures induced by kainic acid and subsequently exhibited greater neuron injury in the CA3 field of hippocampus. Conversely, transgenic mice containing elevated levels of MT-III were more resistant to CA3 neuron injury induced by seizures. These observations suggest a potential role for MT-III in zinc regulation during neural stimulation.

Ganglioneuromas and renal anomalies are induced by activated RETMEN2B in transgenic mice

Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant syndrome characterized by the development of medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies and mucosal ganglioneuromas. MEN2B is caused by a specific mutation (Met918→Thr) in the RET receptor tyrosine kinase. Different mutations of RET lead to other conditions including MEN2A, familial medullary thyroid carcinoma and intestinal aganglionosis (Hirschsprung disease). Transgenic mice were created using the dopamine β-hydroxylase promoter to direct expression of RETMEN2B in the developing sympathetic and enteric nervous systems and the adrenal medulla. DβH-RETMEN2B transgenic mice developed benign neuroglial tumors, histologically identical to human ganglioneuromas, in their sympathetic nervous systems and adrenal glands. The enteric nervous system was not affected. The neoplasms in DβH-RETMEN2B mice were similar to benign neuroglial tumors induced in transgenic mice by activated Ras expression under control of the same promoter. Levels of phoshorylated MAP kinase were not increased in the RETMEN2B-induced neurolgial proliferations, suggesting that alternative pathways may play a role in the pathogenesis of these lesions. Transgenic mice with the highest levels of DβH-RETMEN2B expression, unexpectedly developed renal malformations analogous to those reported with loss of function mutations in the Ret gene.

Oncogenesis and altered differentiation induced by activated Ras in neuroblasts of transgenic mice.

Sympathetic neurons, enteric neurons and adrenal chromaffin cells all derive from the neural crest. During development these cells migrate, proliferate, survive and differentiate in a highly controlled fashion influenced by local signals encountered during their migration. Aberrations of these processes are responsible for a variety of developmental defects and malignancies. Many of the environmental signals influencing these precursor cells activate receptor tyrosine kinases that can signal, at least in part, via Ras pathways. To assess the extent to which Ras can alter neuroblast cell number and fate in vivo, we expressed activated H-Ras in transgenic mice using the dopamine-β-hydroxylase promoter, which directs expression to these cells prior to and after their differentiation. Ganglioneuromas and occasional neuroblastomas formed in the adrenal gland and preaortic sympathetic ganglia. Curiously, neurons of the superior cervical ganglia and the gut were largely unaffected despite demonstrated expression of activated Ras. The sensitivity of preaortic sympathetic neurons and adrenal chromaffin cells to the effects of oncogenes such as Ras may explain the predilection of neuroblastomas in humans to these sites. The ability to analyse neuroblastoma development in these mice may shed light on the molecular basis of certain types of human neuroblastoma.

The d subunit of the vacuolar ATPase (Atp6d) is essential for embryonic development

In our attempt to inactivate the gene for agouti-related protein (Agrp), we also inadvertently removed the first exon of the Atp6d gene encoding the d subunit of the vacuolar V-ATPase. Mouse embryos deficient in both Agrp and Atp6d die shortly after implantation. Because mice lacking only Agrp are viable, these results indicate that the d subunit of the V-ATPase is essential for embryonic development. Figure 1 depicts the Agrp and Atp6d loci, which lie on mouse chromosome 11. The termination codon of the Agrp gene is ∼1000 base pairs upstream of the initiation codon of the Atp6d gene. In our attempt to inactivate the Agrp gene we replaced a 4.7 kb Pml1AvrII region of genomic DNA, that includes the coding exons of the Agrp gene along with the first exon of the Atp6d gene, with sequences that included a Pgk-Neo selectable gene and either uncoupling protein 1 (Ucp1) or enhanced green fluorescent protein (EGFP) genes (see Figure 1). Correctly targeted ES cells (129/SvJ) were identified by PCR and confirmed by Southern blot. They were injected into C57BL/6 blastocysts and transferred to foster mothers. Male chimeric offspring were mated with C57BL/6 dams and their agouti offspring were examined for the presence of the targeted allele. The targeting frequency was ∼1/100 and ES cell clones of each construct were transmitted through the germline. Agrp/Atp6d heterozygotes carrying either targeted allele manifested normal growth, adiposity, and longevity. To the extent examined, their

Visualization and ablation of phenylethanolamine N-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5′ flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli β-galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT8kb-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.