The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2alpha phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2alpha phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic beta cells. The results demonstrate that regulation of translation through eIF2alpha phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.

Sodium channel ␤-subunits modulate channel gating, assembly, and cell surface expression in heterologous cell systems. We generated ␤2 ؊/؊ mice to investigate the role of ␤2 in control of sodium channel density, localization, and function in neurons in vivo. Measurements of [ 3 H]saxitoxin (STX) binding showed a significant reduction in the level of plasma membrane sodium channels in ␤2 ؊/؊ neurons. The loss of ␤2 resulted in negative shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons. The integral of the compound action potential in optic nerve was significantly reduced, and the threshold for action potential generation was increased, indicating a reduction in the level of functional plasma membrane sodium channels. In contrast, the conduction velocity, the number and size of axons in the optic nerve, and the specific localization of Na v1.6 channels in the nodes of Ranvier were unchanged. ␤2 ؊/؊ mice displayed increased susceptibility to seizures, as indicated by reduced latency and threshold for pilocarpine-induced seizures, but seemed normal in other neurological tests. Our observations show that ␤2-subunits play an important role in the regulation of sodium channel density and function in neurons in vivo and are required for normal action potential generation and control of excitability.auxiliary subunits ͉ gene targeting ͉ epilepsy ͉ action potential conduction

To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H+-K+-adenosinetriphosphatase (H+-K+-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H+-K+-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.

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A CCK-deficient mouse mutant generated by gene targeting in embryonic stem cells was analyzed to determine the importance of CCK for growth and function of the exocrine pancreas and for pancreatic adaptation to dietary changes. RIAs confirmed the absence of CCK in mutant mice and demonstrated that tissue concentrations of the related peptide gastrin were normal. CCK-deficient mice are viable and fertile and exhibit normal body weight. Pancreas weight and cellular morphology appeared normal, although pancreatic amylase content was elevated in CCK-deficient mice. We found that a high-protein diet increased pancreatic weight, protein, DNA, and chymotrypsinogen content similarly in CCK-deficient and wild-type mice. This result demonstrates that CCK is not required for protein-induced pancreatic hypertrophy and increased proteolytic enzyme content. This is a novel finding, since CCK has been considered the primary mediator of dietary protein-induced changes in the pancreas. Altered somatostatin concentrations in brain and duodenum of CCK-deficient mice suggest that other regulatory pathways are modified to compensate for the CCK deficiency.

Mutations in Prophet of PIT1 (Prop1), one of several homeodomain transcription factors that are required for the development of the anterior pituitary gland, are the predominant cause of MPHD (multiple pituitary hormone deficiency) in humans. We show that deletion of Prop1 in mice causes severe pituitary hypoplasia with failure of the entire Pit1 lineage and delayed gonadotrope development. The pituitary hormone deficiencies cause secondary endocrine problems and a high rate of perinatal mortality due to respiratory distress. Lung atelectasis in mutants correlates with reduced levels of NKX2.1 and surfactant. Lethality of mice homozygous for either the null allele or a spontaneous hypomorphic allele is strongly influenced by genetic background. Prop1-null mice are an excellent model for MPHD and may be useful for testing the efficacy of pharmaceutical intervention for neonatal respiratory distress.

The fucose ␣(132) galactose ␤ structure is expressed by uterine epithelial cells in the mouse and has been implicated in blastocyst adhesion events thought to be required for murine implantation.

Cholecystokinin (CCK) is a regulatory peptide that is primarily expressed in two adult cell types: endocrine cells of the intestine and neurons of the central nervous system. To determine the ontogeny of CCK expression during intestinal organogenesis, we created a mouse strain in which the CCK gene was replaced by a lacZ reporter cassette using homologous recombination in embryonic stem cells. Initially, CCK expression in the developing intestine was limited to the myenteric plexus of the enteric nervous system. This expression pattern was widespread, extending from the proximal stomach into the colon, yet transient, being detected soon after gut tube closure [embryonic day 10.5 (E10.5)] through E15.5. Since enteric neurons are derived from the neural crest, we examined earlier (E8.5-9.5) embryos and concluded that lacZ was expressed in subpopulations of neural tube and neural crest cells. Endocrine cell expression in the intestinal epithelium occurred later, beginning at E15.5 as enteric neuronal expression was dwindling. This expression persisted to yield the adult pattern of scattered single endocrine cells in the upper small intestine. The data show that CCK is a very early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that CCK receptor transcripts were detected in embryos as early as E10.5, suggesting that CCK signaling is established early in mouse development.