Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.

At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation.

A new isoleucine substitution of Val-20 in transthyretin tetramers selectively impairs dimer-dimer contacts and causes systemic amyloidosis.

The most frequent form of inherited amyloidoses is associated with mutations in the transthyretin (TTR) gene coding for 127-amino acid residues of four identical, noncovalently linked subunits that form a pair of dimers in the plasma protein complex. Amyloid fibrils containing the variant and to a lesser extent the wild-type form of the TTR molecule are deposited in various organs, including peripheral nerves and the myocardium, with polyneuropathy and cardiomyopathy as major clinical manifestations. So far, more than 40 distinct amino acid substitutions distributed throughout the TTR sequence over 30 positions have been found to be correlated with an increased amyloidogenicity of TTR. Most of these amyloidogenic amino acid substitutions are suspected to alter the conformation and stability of the monomer. Here we identify and characterize by protein and DNA analysis a novel amyloidogenic Val-20 to Ile mutation in a German three-generation family. The index patient suffered from severe amyloid cardiomyopathy at the age of 60. Conformational stability and unfolding behavior of the Ile-20 monomer in urea gradients was found to be almost indistinguishable from that of wild-type TTR. In contrast, tetramer stability was significantly reduced in agreement with the expected change in the interactions between the two opposing dimers via the side chain of Ile-20. Our observations provide strong evidence for the view that amyloidogenic amino acid substitutions in TTR facilitate the conversion of tetrameric TTR complexes into those conformational intermediates of the TTR folding pathway that have an intrinsic amyloidogenic potential.

Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.

Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.

Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis.

The CBFA2 (AML1) gene encodes a DNA-binding subunit of the heterodimeric core-binding factor. The CBFA2 gene is disrupted by the (8;21), (3;21), and (12;21) chromosomal translocations associated with leukemias and myelodysplasias in humans. Mice lacking a CBF alpha 2 protein capable of binding DNA die between embryonic days 11.5 and 12.5 due to hemorrhaging in the central nervous system (CNS), at the nerve/CNS interfaces of cranial and spinal nerves, and in somitic/intersomitic regions along the presumptive spinal cord. Hemorrhaging is preceded by symmetric, bilateral necrosis in these regions. Definitive erythropoiesis and myelopoiesis do not occur in Cbfa2-deficient embryos, and disruption of one copy of the Cbfa2 gene significantly reduces the number of progenitors for erythroid and myeloid cells.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color.

Mutations in the gene encoding the endothelin receptor type B (EDNRB) produce congenital aganglionic megacolon and pigment abnormalities in mice and humans. Here we report a naturally occurring null mutation of the EDNRB gene in spotting lethal (sl) rats, which exhibit aganglionic megacolon associated with white coat color. We found a 301-bp deletion spanning the exon 1-intron 1 junction of the EDNRB gene in sl rats. A restriction fragment length polymorphism caused by this deletion perfectly cosegregates with the sl phenotype. The deletion leads to production of an aberrantly spliced EDNRB mRNA that lacks the coding sequence for the first and second putative transmembrane domains of the G-protein-coupled receptor. Radioligand binding assays revealed undetectable levels of functional EDNRB in tissues from homozygous sl/sl rats. We conclude that EDNRB plays an essential role in the normal development of two neural crest-derived cell lineages, epidermal melanocytes and enteric neurons, in three mammalian species--humans, mice, and rats. The EDNRB-deficient rat may also prove valuable in defining the postnatal physiologic role of this receptor.

Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice.

Translational control is a major form of regulating gene expression during gametogenesis and early development in many organisms. We sought to determine whether the translational repression of the protamine 1 (Prm1) mRNA is necessary for normal spermatid differentiation in mice. To accomplish this we generated transgenic animals that carry a Prm1 transgene lacking its normal 3' untranslated region. Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein. Premature accumulation of Prm1 within syncytial spermatids in mice hemizygous for the transgene caused dominant male sterility, which in some cases was accompanied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prm1 is necessary for the transition from nucleohistones to nucleoprotamines.

A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the beta or gamma subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of beta and gamma subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the beta subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Failure of programmed cell death and differentiation as causes of tumors: some simple mathematical models.

Most models of tumorigenesis assume that the tumor grows by increased cell division. In these models, it is generally supposed that daughter cells behave as do their parents, and cell numbers have clear potential for exponential growth. We have constructed simple mathematical models of tumorigenesis through failure of programmed cell death (PCD) or differentiation. These models do not assume that descendant cells behave as their parents do. The models predict that exponential growth in cell numbers does sometimes occur, usually when stem cells fail to die or differentiate. At other times, exponential growth does not occur: instead, the number of cells in the population reaches a new, higher equilibrium. This behavior is predicted when fully differentiated cells fail to undergo PCD. When cells of intermediate differentiation fail to die or to differentiate further, the values of growth parameters determine whether growth is exponential or leads to a new equilibrium. The predictions of the model are sensitive to small differences in growth parameters. Failure of PCD and differentiation, leading to a new equilibrium number of cells, may explain many aspects of tumor behavior--for example, early premalignant lesions such as cervical intraepithelial neoplasia, the fact that some tumors very rarely become malignant, the observation of plateaux in the growth of some solid tumors, and, finally, long lag phases of growth until mutations arise that eventually result in exponential growth.

Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts.

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.

Y chromosome short arm-Sxr recombination in XSxr/Y males causes deletion of Rbm and XY female sex reversal.

We earlier described three lines of sex-reversed XY female mice deleted for sequences believed close to the testes-determining gene (Sry) on the Y chromosome short arm (Yp). The original sex-reversed females appeared among the offspring of XY males that carried the Yp duplication Sxr on their X chromosome. Earlier cytogenetic observations had suggested that the deletions resulted from asymmetrical meiotic recombination between the Y and the homologous Sxr region, but no direct evidence for this hypothesis was available. We have now analyzed the offspring of XSxr/Y males carrying an evolutionarily divergent Mus musculus domesticus Y chromosome, which permits detection and characterization of such recombination events. This analysis has enabled the derivation of a recombination map of Yp and Sxr, also demonstrating the orientation of Yp with respect to the Y centromere. The mapping data have established that Rbm, the murine homologue of a gene family cloned from the human Y chromosome, lies between Sry and the centromere. Analysis of two additional XY female lines shows that asymmetrical Yp-Sxr recombination leading to XY female sex reversal results in deletion of Rbm sequences. The deletions bring Sry closer to Y centromere, consistent with the hypothesis that position-effect inactivation of Sry is the basis for the sex reversal.