Wnt-1 and Wnt-3a proto-oncogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attenuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurally related to Wnt-1, targeted the forebrain and midbrain region, which were hypoplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nucleotides) make this antisense oligonucleotide/whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development.

Protein tyrosine phosphatases (PTPs) play important roles in modulating signals transduced by tyrosine kinases. Certain phosphatases have been implicated as having important roles in embryonic development as well as in adult physiology. Although both kinases and phosphatases are equally important in regulating signal transduction, phosphatases as a group have not been well characterized. Thus, characterization of sequence, expression, and biological function for additional phosphatases is informative. PTPBr7/PC12 and PTPSl are mouse receptor PTPs sharing similar amino acid sequences. Northern blot analysis demonstrated expression of these genes in adult rodent brain and revealed previously uncharacterized transcripts in the brain and other tissues. Our results demonstrate that PTPBr7/PC12 and PTPSl are members of a larger family of PTPs. We have identified two novel family members as well as several novel transcriptional splice variants from both human and mouse colon cDNA libraries. Expression analysis demonstrated that the various mRNA transcripts are differentially expressed, with the highest levels found in the brain, intestinal tract, uterus, and placenta. In situ hybridization analysis of mouse brain and intestinal tissues established that each isoform has a unique expression pattern in specific cell populations as well as in tissue regions. Furthermore, these restricted patterns suggest that the encoded family of phosphatases may play roles in modulating signal transduction pathways important for specific cell types and biological processes.

The axl tyrosine kinase receptor is aberrantly expressed on myeloid cells of many individuals afflicted with chronic myelogenous leukemia (CML) and other myeloid leukemias. Although previous studies demonstrated this kinase to have oncogenic potential, it is not known whether axl actively participates in the onset and/or progression of CML. We addressed this question by generating transgenic mice possessing constitutive ectopic expression of human axl throughout cells of the myeloid hematopoietic lineage through the use of the granulocyte colony-stimulating factor (GCSF) receptor promoter. The transgenics did not exhibit hematopoietic malignancies, but did exhibit phenotypic characteristics associated with noninsulin-dependent diabetes mellitus (NIDDM) including hyperglycemia and hyperinsulinemia, severe insulin resistance, progressive obesity, hepatic lipidosis, and pancreatic islet dysplasia. The obese-diabetes phenotype was similar to that observed in the agouti and melanocortin-4(-/-) mutants, however the axl transgenics were not hyperphagic. Axl transgenic animals expressed elevated serum tumor necrosis factor (TNF)-alpha levels that were further enhanced upon in vitro lipopolysaccharide (LPS) stimulation of peripheral blood. Administration of the axl ligand, gas6, to peripheral transgenic blood samples eliminated excessive TNF-alpha production in response to LPS stimulation. As a means to better understand axl-gas6 biology, transgenic animals were produced which systemically expressed the gas6-binding axl proteolytic cleavage product. A more severe NIDDM phenotype occurred in these mice. The observed phenotypes may be related to the axl receptor or proteolytic cleavage product competing with related axl family receptors for binding of the gas6 ligand. We conclude that axl expression in myeloid cells in itself does not lead to the onset or progression of leukemia and suggest that ectopic axl expression affects endogenous modulation of TNF-alpha production indirectly resulting in the NIDDM phenotype.

Wnt-1 and Wnt-3a have been postulated to share functional redundancy in spinal cord morphogenesis due to their homologies in protein structure and overlapping expression patterns. In this study, antisense oligonucleotides and a murine whole embryo culture system were used to examine functional interactions of Wnt-1 and Wnt-3a in late gastrulation and neurulation. Early somite mouse embryos were injected with combinations of Wnt-1 and Wnt-3a antisense oligonucleotides and then grown in vitro for up to 48 hr. Simultaneous inhibition of Wnt-1 and Wnt-3a expression resulted in pattern loss in the presumptive spinal cord, which was apparent within 4 hr following antisense treatment. The neural tube was wavy, there was a reduction in the number of nuclear layers in the walls of the neural tube, and evidence of decreased cell adhesion between neuroepithelial cells by 12 hr postinjection. In addition, notochord and primitive streak abnormalities accompanied neural tube abnormalities. The existence of regulatory interactions between Wnt-1, Wnt-3a, and engrailed genes was also examined in this study. Antisense inhibition of Wnt-1 or Wnt-3a expression resulted in reduction of engrailed protein levels in the brain, somites, and spinal cord. However, simultaneous inhibition of both Wnt genes resulted in more complete loss of engrailed protein in these regions. Herein, we present data suggesting functional redundancy of Wnt-1 and Wnt-3a in neural tube patterning and in regulation of engrailed expression.

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.