Cloning and characterization of developmental endothelial locus-1: An embryonic endothelial cell protein that binds the αvβ3 integrin receptor

We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the αvβ3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the αvβ3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.     

Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.     

Abnormal craniofacial growth and early mandibular osteoarthritis in mice harbouring a mutant type II collagen transgene

Skull morphology and histology in the heterozygous offspring of a transgenic founder mouse Del1, harbouring 6 copies of deletion mutation in Col2a1 gene, were compared with those in normal siblings. On visual observation and roentgenocephalometric examination the heads of heterozygous Del1 mice were smaller than normal. Histologically the sizes of cartilaginous structures of the cranial base were reduced. Severe defects were seen in the temporomandibular joint as progressive osteoarthritic lesions. These observations elucidate the relationship between the genotype and phenotype and demonstrate that heterozygous Del1 mice are a useful model for studies on a genetic disturbance where 'clinical' manifestations are not evident until adult age.     

Peroxiredoxin 6 is a potent cytoprotective enzyme in the epidermis

Peroxiredoxin 6 is an enzyme that detoxifies hydrogen peroxide and various organic peroxides. In previous studies we found strongly increased expression of peroxiredoxin 6 in the hyperproliferative epidermis of wounded and psoriatic skin, suggesting a role of this enzyme in epidermal homeostasis. To address this question, we generated transgenic mice overexpressing peroxiredoxin 6 in the epidermis. Cultured keratinocytes from transgenic mice showed enhanced resistance to the toxicity of various agents that induce oxidative stress. However, overexpression of peroxiredoxin 6 did not affect skin morphogenesis or homeostasis. On skin injury, enhancement of wound closure was observed in aged animals. Most importantly, peroxiredoxin 6 overexpression strongly reduced the number of apoptotic cells after UVA or UVB irradiation. These findings demonstrate that peroxiredoxin 6 protects keratinocytes from cell death induced by reactive oxygen species in vitro and in vivo, suggesting that activation of this enzyme could be a novel strategy for skin protection under stress conditions.     

Modulation of activin/bone morphogenetic protein signaling by follistatin is required for the morphogenesis of mouse molar teeth.

Teeth form as ectodermal appendages, and their morphogenesis is regulated by conserved signaling pathways. The shape of the tooth crown results from growth and folding of inner dental epithelium, and the cusp patterning is regulated by transient signaling centers, the enamel knots. Several signal proteins in the transforming growth factor-beta (TGF beta) superfamily are required for tooth development. Follistatin is an extracellular inhibitor of TGF beta signaling. To investigate the roles of follistatin during tooth development, we analyzed in detail the expression patterns of follistatin, activin beta A, as well as Bmp2, Bmp4, and Bmp7 during tooth morphogenesis. We also examined the tooth phenotypes of follistatin knockout mice and of transgenic mice overexpressing follistatin in the epithelium under the keratin 14 (K14) promoter. The folding of the dental epithelium was aberrant in the molars of follistatin knockout mice, and the cusps were shallow with reduced cell proliferation and lack of anteroposterior polarization. The functions of both primary and secondary enamel knots were apparently disturbed. In K14-follistatin transgenic mice, the molar cusp pattern was also seriously affected (although different from the follistatin knockouts) and the occlusal surfaces of the molars were whorled. Their enamel was prematurely worn. In addition, all of the third molars were missing. Our results indicate that follistatin regulates morphogenesis and shaping of the tooth crown. We propose that finely tuned antagonistic effects between follistatin and TGF beta superfamily signals are critical for enamel knot formation and function, as well as for patterning of tooth cusps.     

Bone formation in the context of growth retardation induced by hIGFBP-1 overexpression in transgenic mice

In humans, intrauterine growth retardation (hIUGR) is correlated with an overexpression of insulin-like growth factor binding protein 1 (IGFBP-1). The affected children also present a delay in bone mineralization. In this study, transgenic 12-day-old mutant mice overexpressing human IGFBP-1 hepatospecifically showed a severe growth retardation. Alcian blue and alizarin red S staining of the skeleton revealed mineralization defects at the posterior level of the skull (delayed suture closure) and in appendicular and axial skeleton. Furthermore, microradiographic analysis showed a reduced bone density in the same areas. Thus, overexpressing of hIGFBP-1 demonstrates early postnatal life growth retardation and a delay in mineralization in transgenic mutant mice. These data show the involvement of the IGF/IGFBP system and more particularly IGFBP-1 in the biomineralization process.     

Bone Formation in the Context of Growth Retardation Induced by hIGFBP-1 Overexpression in Transgenic Mice

In humans, intrauterine growth retardation (hIUGR) is correlated with an overexpression of insulin-like growthfactor binding protein 1 (IGFBP-1). The affected children also present a delay in bone mineralization. In this study, transgenic 12-day-old mutant mice overexpressing human IGFBP-1 hepatospecifically showed a severe growth retardation. Alcian blue and alizarin red S staining of the skeleton revealed mineralization defects at the posterior level of the skull (delayed suture closure) and in appendicular and axial skeleton. Furthermore, microradiographic analysis showed a reduced bone density in the same areas. Thus, overexpressing of hIGFBP-1 demonstrates early postnatal life growth retardation and a delay in mineralization in transgenic mutant mice. These data show the involvement of the IGF/IGFBP system and more particularly IGFBP-1 in the biomineralization process.     

GDNF-induced seminomatous tumours in mouse--an experimental model for human seminomas?

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-alpha1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/- mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

COMMENTS

Glial-cell-line-derived neurotrophic factor (GDNF) is a distant member of the transforming growth factor superfamily. It binds to and activates a receptor complex consisting of GFR-α1 and Ret receptor tyrosine kinase. In testis, GDNF is expressed by Sertoli cells. We have shown by transgenic loss- and gain-of-function mouse models that GDNF regulates the cell fate decision of undifferentiated spermatogonia. In the GDNF +/− mice, the spermatogonia differentiate in excess leading to the depletion of germ cells. In the mice overexpressing GDNF in testes, undifferentiated spermatogonia accumulate in the tubules, no sperm is produced, and the mice are infertile. After a year, the GDNF overexpressing mice frequently (89%) develop testicular tumours, and most of them are bilateral (56%). All these tumours show the same histological pattern. They are composed of round spermatogonial/gonocytic cells with only a scant cytoplasm. The tumours are locally invasive but do not metastasise. They express germ line markers, are positive for alkaline phosphatase, and aneuploid with a triploid peak. Thus, by several histological, molecular, and histochemical characteristics, the GDNF-induced tumours mimic classical seminomas in men, but the precursor lesions are apparently different in mouse and man.     

Characterization of transgenic mice that overexpress both copper zinc superoxide dismutase and catalase

Transgenic mice overexpressing both Cu/ZnSOD and catalase [Tg(SOD1/CAT) +/o] were used to evaluate the effects of overexpression of both genes against oxidative stress. Characterization of these transgenic mice revealed that catalase or Cu/ZnSOD activities were two- to fourfold higher in the tissues of transgenic mice compared to wild-type mice, and the activities of the other major antioxidant enzymes were not altered in the tissues of the transgenic mice. The murine embryonic fibroblasts (MEFs) from the Tg(SOD1/CAT) +/o and MEFs overexpressing Cu/ZnSOD were more resistant to paraquat cytotoxicity, relative to wild-type MEFs. The MEFs from Tg(SOD1/CAT) +/o tended to be more resistant (up to 2.25-fold) to paraquat cytotoxicity than MEFs overexpressing either Cu/ZnSOD or catalase alone. MEFs from Tg(CAT) +/o and Tg(SOD1/CAT) +/o were equally as resistant to hydrogen peroxide cytotoxicity. However, there were no significant differences in whole animal survival against either paraquat or gamma-radiation.     

Loss of tolerance of anti-dsDNA B cells in mice overexpressing CD19

Mice transgenic for the R4A-Cmu heavy chain of an anti-dsDNA antibody, maintain tolerance by anergy and deletion. In C57BL/6 mice overexpressing CD19, a molecule, which lowers the threshold for B cell activation, elevated levels of serum autoantibodies have been observed. In the present study, we wished to determine whether CD19 overexpression could alter the induction of tolerance in R4A-Cmu mice and lead to the secretion of transgenic anti-dsDNA antibodies. We, therefore, bred R4A-Cmu transgenic mice-to-mice transgenic for human CD19 (hCD19) and generated R4A-Cmu mice heterozygous and homozygous for hCD19. We, now report the spontaneous secretion of transgenic IgM anti-dsDNA antibody in the sera of R4A-Cmu mice overexpressing CD19, indicative of a loss of B cell tolerance. We observe that transgenic B cells secreting anti-dsDNA antibody in these mice are T independent and display a marginal zone like phenotype althought they do not reside in the MZ. In addition, they appear to be derived from the conventional B2 subset rather than the B1 subset. Interestingly, a subset of the anti-dsDNA B cells in these mice still display the phenotype and functional characteristics of anergic B cells. These B cells cannot be activated to secrete antibody following BCR crosslinking, however, they are hyper-responsive to activation by innate signaling mechanisms. This suggests that CD19 overexpression may promote anergic B cells to escape tolerance by converging with BCR independent pathways, thereby rendering these B cells hyper-responsive to innate signaling.     

四环素调控的小鼠LAIR-1/CD305转基因小鼠的初步建立

目的：建立四环素调控的小鼠LAIR-1/CD305转基因小鼠,为进一步研究mLAIR-1分子的体内功能奠定基础.方法：构建pBI-5-mLAIR-1载体,显微注入B6D1F1受精卵,PCR检测新生小鼠基因组DNA中LAIR-1与荧光素酶（Luciferase）基因.将mLAIR-1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含盐酸强力霉素（Dox）的培养基进行培养,检测细胞裂解液中荧光素酶活性.将荧光素酶表达依赖Dox小鼠与C57BL/6交配,采用PCR对子代鼠进行检测.结果：共获得9只首建鼠,其目的基因表达高度依赖Dox,并得到其中5只首建鼠的F1代小鼠.结论：获得了四环素调控的小鼠LAIR-1转基因小鼠,可用于该分子体内功能的研究.     

Generation and phenotypic characterization of a galanin overexpressing mouse

In most parts of the peripheral nervous system galanin is expressed at very low levels. To further understand the functional role of galanin, a mouse overexpressing galanin under the platelet-derived growth factor-B was generated, and high levels of galanin expression were observed in several peripheral tissues and spinal cord. Thus, a large proportion of neurons in autonomic and sensory ganglia were galanin-positive, as were most spinal motor neurons. Strong galanin-like immunoreactivity was also seen in nerve terminals in the corresponding target tissues, including skin, blood vessels, sweat and salivary glands, motor end-plates and the gray matter of the spinal cord. In transgenic superior cervical ganglia around half of all neuron profiles expressed galanin mRNA but axotomy did not cause a further increase, even if mRNA levels were increased in individual neurons. In transgenic dorsal root ganglia galanin mRNA was detected in around two thirds of all neuron profiles, including large ones, and after axotomy the percentage of galanin neuron profiles was similar in overexpressing and wild type mice. Axotomy reduced the total number of DRG neurons less in overexpressing than in wild type mice, indicating a modest rescue effect. Aging by itself increased galanin expression in the superior cervical ganglion in wild type and transgenic mice, and in the latter also in preganglionic cholinergic neurons projecting to the superior cervical ganglion. Galanin overexpressing mice showed an attenuated plasma extravasation, an increased pain response in the formalin test, and changes in muscle physiology, but did not differ from wild type mice in sudomotor function. These findings suggest that overexpressed galanin in some tissues of these mice can be released and via a receptor-mediated action influence pathophysiological processes.     

Retarded skeletal development in transgenic mice with a type II collagen mutation.

Transgenic mice harboring specific mutations provide a tool for systematic studies on the effects of dominant mutations on embryonic development and growth. In the present study, several different techniques were employed to follow the development of growth abnormalities and other phenotypic consequences in the mouse litters from matings of heterozygous Del1 mice carrying six copies of a mouse type II collagen transgene with a small engineered deletion mutation. Skeletal staining of complete litters with alcian blue/alizarin red S revealed that the onset of chondrogenesis and subsequent endochondral ossification were delayed in heterozygous and homozygous Del1 embryos. The lengths, widths, and shapes of long bones were all affected. Histological and in situ hybridization analyses revealed several types of abnormalities in the epiphyseal growth plates of homozygous Del1 embryos including disorganization of growth plate architecture, abnormal appositional growth activity, an increase in the number of hypertrophic chondrocytes, a deficiency in the formation of metaphyseal cancellous bone, and development of necrotic areas in the epiphyseal heads. Many of these findings parallel those seen in human chondrodysplasias. The basic information obtained on the effects of a specific deletion mutation in the type II collagen gene on the development and growth of the transgenic embryos provides a background for testing the efficacy of novel therapeutic strategies for prevention of such growth abnormalities.     

EGFR ligands exert diverging effects on male reproductive organs

While the EGFR and most of its ligands are expressed in the male reproductive tract, their functions in male reproduction are poorly understood. Interestingly, male transgenic mice overexpressing EGF are sterile, and transgenic mice overexpressing TGFA, another EGFR ligand, show an enlarged coagulation gland (anterior prostate) due to severe hyperplasia with focal dysplasia. We studied the male reproductive tract of transgenic mice overexpressing betacellulin (BTC-tg) under the control of a promoter conferring widespread transgene expression. Despite strong overexpression of BTC in different parts of the male reproductive tract, the gross appearance and histology of the reproductive organs of BTC-tg males were normal and the same were true for sperm parameters and the in vitro fertilization rate. Collectively, our findings demonstrate that excess of BTC exerts no deleterious effects on the structure or function of the male reproductive tract in mice and indicates unique, non-overlapping functions of specific EGFR ligands in male reproduction.     

Overexpression of neurofilament subunit NF-L and NF-H extends survival of a mouse model for amyotrophic lateral sclerosis

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in a subset of patients. Neurofilaments (NFs), the most abundant protein in motoneurons, may play a role in motoneuron degeneration. To investigate this role, we crossed transgenic mice expressing SOD1 mutant G93A (G93A mice) with mice overexpressing mouse neurofilament subunit H (H mice) or L (L mice). G93A mice overexpressing either NF-L or NF-H developed ALS later and survived longer than the G93A mice on a wild type background. These results illustrate a beneficial role of neurofilaments in ALS and call into question of several hypotheses regarding the role of neurofilaments in the development of ALS.     

Intact pericellular matrix of articular cartilage is required for unactivated discoidin domain receptor 2 in the mouse model

Increased expression of the discoidin domain receptor 2 (DDR2) results from its interaction with collagen type II. This induces expression of matrix metalloproteinase (MMP)-13, leading to osteoarthritis (OA). To investigate the impact of the pericellular matrix of chondrocytes on DDR2, we generated a mouse model with inducible overexpression of DDR2 in cartilage. Conditional overexpression of DDR2 in mature mouse articular cartilage was controlled via the cartilage oligomeric matrix protein promoter using the Tet-Off-inducible system. Doxycycline was withdrawn at 1 month of age, and knee joints were examined at 2, 3, and 4 months of age. Microsurgery was performed on 3-month-old transgenic mice overexpressing DDR2 to destabilize the medial meniscus, and serial paraffin sections were examined at 2, 4, 8, and 12 weeks after surgery. DDR2 expression increased in the knee joints of transgenic mice. However, the increased DDR2 did not induce MMP-13 expression. No OA-like changes were observed in the transgenic mice at the age of 4 months. When transgenic mice were subjected to destabilizing of the medial meniscus, we observed accelerated progression to OA, which was associated with DDR2 activation. Therefore, conditionally overexpressing DDR2 in the mature articular cartilage of mouse knee joints requires activation to induce OA, and altered biomechanical stress can accelerate the onset of cartilage loss and progression to OA in transgenic mice.     

A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease

This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.