We inactivated the mouse metaflothionein (MT) -I and MT-Il genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal laboratory conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This proves that these widely expressed MTs are not essential for development but that they do protect against cadmium toxicity. These mice provide a means for testing other proposed functions of MT in vivo.Metallothioneins (MTs) were identified by Margoshes and Vallee (1); since then MTs have been described in most vertebrates and in a wide variety of invertebrate species (for review, see refs. 2 and 3). MTs are characterized by their low molecular weight, high cysteine content (=30%

MT-III, a brain-specific member of the metallothionein gene family, binds zinc and may facilitate the storage of zinc in neurons. The distribution of MT-III mRNA within the adult brain was determined by solution and in situ hybridization and compared to that of MT-I mRNA. MT-III mRNA is particularly abundant within the cerebral cortex, hippocampus, amygdala, and nuclei at base of the cerebellum. Transgenic mice generated using 11.5 kb of the mouse MT-III 5′ flanking region fused to the E. coli lacZ gene express beta-galactosidase in many of the same regions identified by in situ hybridization. MT-III mRNA was present in readily identifiable neurons within the olfactory bulb, hippocampus, and cerebellum, and beta-galactosidase activity was localized to neurons throughout the brain, but not to glia, as determined by costaining with X-Gal and neural- and glia-specific antibodies. There is marked correspondence between the neurons that are rich in MT-III mRNA and those neurons that store zinc in their terminal vesicles. MT-III is found complexed with zinc in vivo and its expression in cultured cells leads to the intracellular accumulation of zinc and enhanced histochemical detection of zinc. These results are discussed in light of the possibility that MT-III may participate in the utilization of zinc as a neuromodulator.

Competition and cooperation between type II and type III receptor protein tyrosine phosphatases (RPTPs) regulate axon extension and pathfinding in Drosophila. The first step to investigate whether RPTPs influence axon growth in the more complex vertebrate nervous system is to identify which neurons express a particular RPTP. We studied the expression of mouse PTPRO, a type III RPTP with an extracellular region containing eight fibronectin type III domains, during embryogenesis and after birth. Mouse PTPRO mRNA is expressed exclusively in two cell types: neurons and kidney podocytes. Maximal expression in the brain was coincident with mid to late gestation and axonogenesis in the brain. We cloned two cDNAs, including a splice variant without sequence coding of 28 amino acids within the juxtamembrane domain that was found mostly in kidney. In situ hybridization detected mPTPRO mRNA in the cerebral cortex, olfactory bulb and nucleus, hippocampus, motor neurons, and the spinal cord midline. In addition, mPTPRO mRNA was found throughout dorsal root, cranial, and sympathetic ganglia and within kidney glomeruli. Mouse PTPRO mRNA was observed in neuron populations expressing TrkA, the high-affinity nerve growth factor receptor, or TrkC, the neurotrophin-3 receptor, and immunoreactive mPTPRO and TrkC colocalized in large dorsal root ganglia proprioceptive neurons. Our results suggest that mPTPRO is involved in the differentiation and axonogenesis of central and peripheral nervous system neurons, where it is in a position to modulate intracellular responses to neurotrophin-3 and/or nerve growth factor.

Leukemia inhibitory factor (LIF) is a cytokine growth factor that induces rat sympathetic neurons to switch their neurotransmitter phenotype from noradrenergic to cholinergic in vitro. To test whether LIF can influence neuronal differentiation in vivo, we generated transgenic mice that expressed LIF in pancreatic islets under the control of the insulin promoter and evaluated the neurotransmitter phenotype of the pancreatic sympathetic innervation. We also used the insulin promoter to coexpress nerve growth factor in the islets, which greatly increased the density of sympathetic innervation and facilitated analysis of the effects of LWF. Our data demonstrate that tyrosine hydroxylase and catecholamines declined and choline acetyltranserase increased in response to LWF. We conclude that LIF can induce neurotransmitter switching of sympathetic neurons in vivo.

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of 125I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t1/2 of dissociation of 26 minutes. Cross-linking of 125I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the beta subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [3H]norepinephrine uptake. Insulin inhibition of [3H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in the Km, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.