ABSTRACT
One of the earliest events in bone morphogenesis is the condensation of embryonic mesenchymal cells into chondroblasts and their subsequent proliferation and differentiation into chondrocytes. During this time, certain signaling cascades operate to establish proper patterning and differentiation of the cartilaginous skeleton. Characterization of the signaling pathways involved in these processes remains to be accomplished. We have identified a novel murine cytosolic tyrosine phosphatase termed PTPPBS gamma (+/-) which is a member of the PTP PC12,Br7,Sl (PTPPBS) family. Spatio-temporal expression analysis of the members of this tyrosine phosphatase family demonstrates significant expression of the gamma (-) splice variant in the cartilaginous skeleton. Using an embryonic mandibular explant culture system to serve as a model for cartilage formation, we examined the potential roles of the PTPPBS gamma phosphatase by loss-of-function studies achieved with antisense oligodeoxynucleotides. These studies demonstrated that loss of expression of the PTPPBS gamma (-) isoform resulted in abnormal patterning of Meckel's cartilage and an increase in the size of the chondrogenic regions. In gamma antisense-treated explants, bromodeoxyuridine-pulse labeling studies revealed increased proliferation of chondroblasts bordering along precartilaginous condensations and bordering populations of maturing chondrocytes. These studies provide evidence that in early skeletal development, PTPPBS gamma may regulate the rate of chondroblast proliferation in the cartilaginous skeleton.
Subject(s)
Chondrogenesis/physiology , Protein Tyrosine Phosphatases/physiology , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Body Patterning/genetics , Body Patterning/physiology , Cartilage/embryology , Chondrogenesis/genetics , DNA/genetics , DNA Primers/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , In Vitro Techniques , Isoenzymes/genetics , Isoenzymes/physiology , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , Protein Tyrosine Phosphatases/geneticsABSTRACT
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.
Subject(s)
Gene Expression Regulation, Enzymologic , Nerve Tissue Proteins/genetics , Protein Tyrosine Phosphatases/genetics , Adult , Alternative Splicing/physiology , Amino Acid Sequence , Animals , Base Sequence , Brain/enzymology , Female , Humans , In Situ Hybridization , Intestines/enzymology , Intracellular Signaling Peptides and Proteins , Mice , Molecular Sequence Data , Multigene Family/genetics , Organ Specificity , Placenta/enzymology , Protein Biosynthesis , RNA, Messenger/analysis , Receptor-Like Protein Tyrosine Phosphatases, Class 7 , Sequence Analysis, DNA , Uterus/enzymologyABSTRACT
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.
Subject(s)
Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/genetics , Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Animals , Base Sequence , DNA Primers/genetics , Diabetes Mellitus, Type 2/immunology , Disease Models, Animal , Gene Expression , Humans , Insulin Resistance , Islets of Langerhans/pathology , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/genetics , Liver/pathology , Male , Mice , Mice, Transgenic , Phenotype , Proto-Oncogene Proteins , Rats , Testis/pathology , Tumor Necrosis Factor-alpha/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
Obesity is a heath problem affecting a significant fraction of adult Americans and is on the rise globally. It is of importance to find treatments that achieve medically significant weight loss and successful long-term maintenance of a desired weight. Recent transgenic mouse studies and genetic characterization of spontaneous rodent obesity mutants, together with gene linkage analysis in humans, have led to an increased understanding of the physiologic and molecular mechanisms underlying obesity. However, much remains to be studied in this complex field of research. In this review, we discuss the physiology and genetics underlying obesity and how studies in rodents and humans are converging, producing a greater understanding of the mechanisms underlying this health problem.
Subject(s)
Disease Models, Animal , Mice, Mutant Strains , Mice, Obese , Obesity/physiopathology , Adult , Animals , Humans , Mice , Obesity/geneticsSubject(s)
Anatomy/trends , Biotechnology/trends , Career Choice , Economics, Pharmaceutical/trends , HumansABSTRACT
Antisense oligonucleotide targeting of engrailed-1 (En-1) in early somite mouse embryos resulted in reduced En protein levels and produced abnormalities of the brain, face, and heart and shortening of the embryonic axis (caudal dysgenesis). Defects of the brain and limbs were consistent with abnormalities observed in null mutant mice described by other investigators. Abnormalities of the face and heart may be related to alterations in neural crest cells. Caudal dysgenesis suggested a role for En-1 in axis formation and this hypothesis was supported by results showing that En-1 protein and mRNA are present in the primitive streak. Thus, in addition to participating in the signaling pathway for brain and limb development, En-1 appears to play a role in patterning the embryonic axis.
Subject(s)
Embryo, Mammalian/abnormalities , Embryonic and Fetal Development , Homeodomain Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Animals , Base Sequence , Blotting, Southern , Brain/abnormalities , Brain/embryology , Culture Techniques , Dose-Response Relationship, Drug , Extremities/embryology , Female , Genes, Homeobox , Homeodomain Proteins/physiology , Limb Deformities, Congenital , Mice , Mice, Inbred ICR , Molecular Sequence Data , Morphogenesis , Nervous System/embryology , Nervous System Malformations , Phenotype , Pregnancy , Thionucleotides/pharmacologyABSTRACT
The roles of engrailed-1 (En-1) and engrailed-2 (En-2) have been investigated during gastrulation and neurulation in mouse embryos. Using antisense oligonucleotides and murine whole embryo culture, early somite embryos were injected with antisense phosphorothioated oligonucleotides directed against En-1 or En-2 transcripts and then grown in vitro for 48 hr. Inhibition of En-1 by antisense targeting during this period resulted in embryos with craniofacial abnormalities, specifically loss of mid- and hindbrain tissue and hypoplasia in associated neural crest derived areas such as the face and first and second pharyngeal arches. In addition, En-1 appeared to be essential in early patterning of the neural tube. Embryos removed from culture as early as 8 hr following injection exhibited undulations in the presumptive spinal cord. Histological analysis of the affected neural tubes at 48 hr showed disrupted cytoarchitecture and in some cases, apparent dorsal-ventral duplication of the neural tube and underlying notochord. Using S100 beta as a notochord marker, embryos removed from culture at 8 or 12 hr following injection exhibited loss of S100 beta expression in the notochord region subjacent to affected neural tube segments, suggesting that S100 beta, or other notochordal genes, may be downstream components of the En-1 regulatory cascade. Furthermore, antisense inhibition of En-1 induced caudal dysgenesis, suggesting disruption in primitive streak function. Antisense targeting of En-2 expression of early somite stages produced few alterations in development, although approximately one third of the embryos exhibited a series of lateral folds in the spinal cord at the level of the forelimb-bud.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Embryonic and Fetal Development , Facial Bones/embryology , Homeodomain Proteins/genetics , Nerve Tissue Proteins/genetics , Nervous System/embryology , Oligonucleotides, Antisense/pharmacology , Skull/embryology , Animals , Base Sequence , Culture Techniques , Embryo, Mammalian/abnormalities , Female , Gastrula/physiology , Genes, Homeobox , Homeodomain Proteins/physiology , Mice , Mice, Inbred ICR , Molecular Sequence Data , Nerve Tissue Proteins/physiology , Nervous System Malformations , Phenotype , Pregnancy , Thionucleotides/pharmacologyABSTRACT
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.