G-protein coupled receptor 124 (GPR124) is an orphan receptor in the adhesion family of GPCRs and previous global or endothelial-specific disruption of
Gpr124
in mice led to defective CNS ...angiogenesis and blood brain barriergenesis. Similar developmental defects were observed following dual deletion of
Wnt7a/Wnt7b
or deletion of β-catenin in endothelial cells, suggesting a possible relationship between GPR124 and canonical WNT signaling. Here, we show using in vitro reporter assays, mutation analysis and genetic interaction studies in vivo, that GPR124 functions as a WNT7A/WNT7B specific co-stimulator of β-catenin signaling in brain endothelium. WNT7-stimulated β-catenin signaling was dependent upon GPR124’s intracellular PDZ binding motif and a set of leucine rich repeats in its extracellular domain. This study reveals a vital role for GPR124 in potentiation of WNT7 induced canonical β-catenin signaling with important implications for understanding and manipulating CNS-specific angiogenesis and blood brain barriergenesis.
Provirus insertion in the last intron of the Tpl-2 gene in retrovirus-induced rat T-cell lymphomas results in the enhanced expression of a carboxy-terminally truncated Tpl-2 kinase. Here we show that ...the truncated protein exhibits an approximately sevenfold higher catalytic activity and is two- to threefold more efficient in activating the MAPK and SAPK pathways relative to the wild-type protein. The truncated Tpl-2 protein and a GST fusion of the Tpl-2 carboxy-terminal tail interact when coexpressed in Sf9 cells. Their interaction down-regulates the kinase activity of the truncated protein suggesting that tail-directed intramolecular interactions regulate the Tpl-2 kinase. Tpl-2 transgenic mice expressing the wild-type protein from the proximal Lck promoter fail to show a biological phenotype, whereas mice expressing the truncated protein develop large-cell lymphoblastic lymphomas of T-cell origin. These results show that Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation.
Half of all familial breast cancers are due to mutation in the BRCA1 gene. However, despite its importance, attempts to model BRCA1‐induced disease in the mouse have been disappointing. Heterozygous ...Brca1 knockout mice do not develop mammary tumors and homozygous knockout mice die during embryogenesis from ill‐defined causes. Sequence analysis has shown that the coding region, genomic organization, and regulatory sequences of the human and mouse genes are not well conserved. This has raised the question of whether the mouse can serve as an effective model for functional analysis of the human BRCA1 gene. To address this question we have introduced a bacterial artificial chromosome containing the human BRCA1 gene into the germline of Brca1 knockout mice. Surprisingly, we have found that the embryonic lethality of Brca1 knockout mice is rescued by the human transgene. We also show that expression of human BRCA1 transgene mirrors the endogenous murine gene. Our “humanized” transgenic mice can serve as a model system for functional analyses of the human BRCA1 gene. genesis 29:72–77, 2001. Published 2001 Wiley‐Liss, Inc.
The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the ...middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human beta-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a16H allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a16H phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein.
Transgenic mouse lines have been developed that express the tv-a receptor under the control of the chicken β -actin promoter. These mice express the tv-a receptor in most or all tissues and in the ...early embryo. An avian leukosis virus (ALV)-based retroviral vector system was used for the efficient delivery of genes into preimplantation mouse embryos from these transgenic lines. Experimental animals could be generated quickly and easily by infecting susceptible blastocysts with ALV-based retroviral vectors. Expression of the delivered genes was controlled by either the constitutive viral promoter contained in the long terminal repeat or an internal nonviral tissue-specific promoter. Mating the infected founder chimeric animals produced animals that carry the ALV provirus as a transgene. A subset of the integrated proviruses expressed the chloramphenicol acetyltransferase reporter gene from either the promoter in the long terminal repeat or an internal promoter, which we believe indicates that many of the sites that are accessible to viral DNA insertion in preimplantation embryos are incompatible with expression in older animals. This approach should prove useful for studies on murine cell lineage and development, providing models for studying oncogenesis, and testing gene therapy strategies.
Federspiel et al used an avian leukosis virus (ALV)-based retroviral vector system to efficently deliver genes into preimplanted mouse embryos from transgenic mouse lines expressing the tv-a receptor ...under the control of the chicken Beta-actin promoter.
The murine dilute suppressor gene (dsu) suppresses the coat-color phenotype of three pigment mutations, dilute (d), ashen (ash) and leaden (ln), that each produce adendritic melanocytes. Suppression ...is due to the ability of dsu to partially restore (ash and ln), or almost completely restore (d), normal melanocyte morphology. While the ash and ln gene products have yet to be identified, the d gene encodes a novel myosin heavy chain (myosin 12), which is speculated to be necessary for the elaboration, maintenance, and/or function of melanocyte cell processes. To begin to discriminate between different models of dsu action, we have produced aggregation chimeras between mice homozygous for dsu and mice homozygous for d to determine if dsu acts cell autonomously or cell nonautonomously. In addition, we have further refined the map location of dsu in order to examine a number of possible dsu candidate genes mapping in the region and to provide a genetic basis for the positional cloning of dsu.
The mouse pancreatic amylase Amy-2.2 gene was fused to the structural gene for SV40 T antigen, and 51 independent transgenic founder mice carrying the fusion gene were generated. The majority of the ...founders and 100% of their offspring in the derived transgenic lines developed pancreatic acinar cell carcinomas and stomach carcinomas. Transgenic animals also had a high incidence of metastatic carcinomas in other tissues. The development of stomach carcinomas was unexpected because the Amy-2.2 promoter was not previously known to be expressed in stomach. Northern blot analyses and ribonuclease protection assays showed that Amy-2.2 is expressed in stomach, at approximately 0.05% of the level in pancreas. Expression of the fusion gene in stomach, therefore, appears to represent a previously unrecognized activity of the Amy-2.2 promoter. Examination of young transgenic mice demonstrated that preneoplastic lesions were present in pancreas and stomach before the development of neoplastic lesions in either tissue, consistent with the notion that stomach neoplasms are primary neoplasms and not metastases from the pancreas. Ribonuclease protection assays demonstrated that properly initiated large T and small t antigen transcripts were present in pancreas and stomach during tumorigenesis. T antigen protein was also detected in pancreas and stomach by immunohistochemistry. A time course for tumorigenesis was established for several transgenic mouse lines in which distinct types of lesions appeared at predictable times. This study provides the basis for future analysis of the role of SV40 T antigen in the progression and maintenance of pancreatic and stomach carcinomas.
BDNF signaling in hypothalamic circuitries regulates mammalian food intake. However, whether BDNF exerts metabolic effects on peripheral organs is currently unknown. Here, we show that the BDNF ...receptor TrkB.T1 is expressed by pancreatic β-cells where it regulates insulin release. Mice lacking TrkB.T1 show impaired glucose tolerance and insulin secretion. β-cell BDNF-TrkB.T1 signaling triggers calcium release from intracellular stores, increasing glucose-induced insulin secretion. Additionally, BDNF is secreted by skeletal muscle and muscle-specific BDNF knockout phenocopies the β-cell TrkB.T1 deletion metabolic impairments. The finding that BDNF is also secreted by differentiated human muscle cells and induces insulin secretion in human islets via TrkB.T1 identifies a new regulatory function of BDNF on metabolism that is independent of CNS activity. Our data suggest that muscle-derived BDNF may be a key factor mediating increased glucose metabolism in response to exercise, with implications for the treatment of diabetes and related metabolic diseases.
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