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Cardiac outflow tract defects in mice lacking LTBP_1L
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ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
b
Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, United States Dentin sialophosphoprotein (DSPP) is synthesized in both mesenchyme and epithelium at varying stages of tooth development. At the tooth cap stage, corresponding to day E-13.5 of mouse embryonic life, immunostaining of developing mouse tooth bud with anti-DMP2 antibody showed an intense staining in the oral ectoderm at the point where it had begun to invaginate into the mesenchyme, with no expression in dental follicle mesenchyme. At this stage, DPP was also expressed in the ureteric bud branches of embryonic metanephric kidney and the alveolar epithelial buds of developing lung while the remaining parenchyma did not show any anti-DMP2 reactivity. Interestingly, the staining showed a characteristic perinuclear pattern at the basal portion of the polarized epithelial cells. RT-PCR analysis verified the presence of DSPP mRNA with identical sequences in the tooth, lung and kidney. The DSPP−/− mouse with absence of DPP protein in tooth, kidney and lung exhibited aberrant organogenesis in lung and kidney, with malformed metanephric S-shaped bodies and increased mesenchymal apoptosis in kidney. Interestingly, inclusion of anti-DPP antibodies for 5 days in organ culture of metanephroi, harvested from day E-13.5 wild type mice, also resulted in altered morphogenesis of ureteric bud, and a decrease in the number of glomeruli, suggesting a role for DPP in epithelial-mesenchymal interactions in meristic tissues during embryonic development. doi:10.1016/j.matbio.2006.08.072
50 Cardiac outflow tract defects in mice lacking LTBP-1L V. Todorovic a, L. Freyer a, D. Gutstein a , D. Frendewey b, D. Rifkin a a
NYU School of Medicine, New York, NY 10016, United States Regeneron Pharmaceuticals, Inc., Tarrytown, NY, United States b
Latent TGF-β binding protein-1 (LTBP-1) is a member of the LTBP/fibrillin family of extracellular proteins. LTBP-1 binds latent TGF-β and regulates its bioavailability. Due to usage of different promoters, LTBP-1 exists in two forms: long (L) and short (S). To examine the function of LTBP-1L, we generated Ltbp-1L−/− mice. Homozygous mutants lack Ltbp-1L but retain expression of Ltbp-1S. 60% of expected Ltbp-1L mutants are recovered at birth. These mice have cardiovascular defects, namely persistent truncus arteriosus (PTA) and interruption of the aortic arch (IAA). Defects in septation of the cardiac outflow tract (OFT) and remodeling of pharyngeal arch arteries (PAA) have been related to aberrant biology of cardiac neural crest (CNC). Studies of Ltbp-1L expression in the pharynx and the developing heart showed that only those CNC that relocated to
the OFT express Ltbp-1L. Specification and migration of CNC to the cardiac tissue in Ltbp-1L mutants appears to be normal, as judged by fate mapping of CNC. Likewise, the number and the proliferation rate of CNC that invaded the OFT in Ltbp-1L mutants are normal. However, we detect fewer cells expressing CNC markers Plexin A2 and FoxC1 in Ltbp-1L mutant OFT from E11.5 onwards, suggesting an altered CNC function in the OFT.TGF-β signaling is crucial for normal development of the cardiac OFT and associated vasculature. Given the role of Ltbp1L in the regulation of TGF-β bioavailability, we believe that cardiac abnormalities of Ltbp-1L mutants result from abnormal TGF-β activity during heart development. doi:10.1016/j.matbio.2006.08.073
51 MIG-17(ADAMTS), fibulin-1 and nidogen interact in cell migration Y. Kubota, K. Nagata, R. Kuroki, K. Nishiwaki RIKEN CDB, Kobe 650-0047, Hyogo, Japan The gonadal distal tip cells (DTCs) migrate along the body wall basement membrane (BM) to form the U-shaped gonad of the C.elegans hermaphrodite. An ADAMTS family metalloprotease MIG-17 is secreted from the body wall muscles and localizes to the gonadal BM where it is required for directional migration of DTCs. To identify the molecules interacting with MIG-17, we isolated suppressors of DTC migration defects of a mig-17 (null) mutant. One of the suppressor genes encodes fibulin-1 spliced isoforms (FBL-1C and FBL-1D), calciumbinding extracellular matrix proteins. Suppressor mutations, fbl-1 (k201) and fbl-1(k206), are amino acid substitutions in the third EGF-like motif. Mutant FBL-1C but not FBL-1D can suppress mig-17 defects. FBL-1C is synthesized in the gut cells and localizes to the gonadal BM in a MIG-17-dependent manner (Kubota et al., 2004).We found that fbl-1(k201) also significantly suppresses the gonadal elongation defects of gon-1(e1254), a mutation in another ADAMTS (Hesselson et al., 2004). Since mammalian fibulin-1C binds strongly to nidogen-1, in vitro (Sasaki et al. 1995), we examined the requirement of nid-1 (nidogen-1) gene function in the mechanisms of suppression of mig-17 and gon-1 by fbl-1 mutations. Introduction a nid-1(null) mutation into fbl-1(k201); mig-17(null) double mutants abolished the suppression effect of fbl-1(k201). However, the suppression was only weakly affected by introducing nid-1(null) into fbl-1 (k201) gon-1(e1254) mutants. These results suggest that the FBL1(k201) mutant protein compensates loss of MIG-17 or GON-1 in gonad development strongly or partially in a wild-type NID-1 dependent manner, respectively. doi:10.1016/j.matbio.2006.08.074
ASMB Meeting Abstracts / Matrix Biology 25 (2006) S1–S94
b
Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, United States Dentin sialophosphoprotein (DSPP) is synthesized in both mesenchyme and epithelium at varying stages of tooth development. At the tooth cap stage, corresponding to day E-13.5 of mouse embryonic life, immunostaining of developing mouse tooth bud with anti-DMP2 antibody showed an intense staining in the oral ectoderm at the point where it had begun to invaginate into the mesenchyme, with no expression in dental follicle mesenchyme. At this stage, DPP was also expressed in the ureteric bud branches of embryonic metanephric kidney and the alveolar epithelial buds of developing lung while the remaining parenchyma did not show any anti-DMP2 reactivity. Interestingly, the staining showed a characteristic perinuclear pattern at the basal portion of the polarized epithelial cells. RT-PCR analysis verified the presence of DSPP mRNA with identical sequences in the tooth, lung and kidney. The DSPP−/− mouse with absence of DPP protein in tooth, kidney and lung exhibited aberrant organogenesis in lung and kidney, with malformed metanephric S-shaped bodies and increased mesenchymal apoptosis in kidney. Interestingly, inclusion of anti-DPP antibodies for 5 days in organ culture of metanephroi, harvested from day E-13.5 wild type mice, also resulted in altered morphogenesis of ureteric bud, and a decrease in the number of glomeruli, suggesting a role for DPP in epithelial-mesenchymal interactions in meristic tissues during embryonic development. doi:10.1016/j.matbio.2006.08.072
50 Cardiac outflow tract defects in mice lacking LTBP-1L V. Todorovic a, L. Freyer a, D. Gutstein a , D. Frendewey b, D. Rifkin a a
NYU School of Medicine, New York, NY 10016, United States Regeneron Pharmaceuticals, Inc., Tarrytown, NY, United States b
Latent TGF-β binding protein-1 (LTBP-1) is a member of the LTBP/fibrillin family of extracellular proteins. LTBP-1 binds latent TGF-β and regulates its bioavailability. Due to usage of different promoters, LTBP-1 exists in two forms: long (L) and short (S). To examine the function of LTBP-1L, we generated Ltbp-1L−/− mice. Homozygous mutants lack Ltbp-1L but retain expression of Ltbp-1S. 60% of expected Ltbp-1L mutants are recovered at birth. These mice have cardiovascular defects, namely persistent truncus arteriosus (PTA) and interruption of the aortic arch (IAA). Defects in septation of the cardiac outflow tract (OFT) and remodeling of pharyngeal arch arteries (PAA) have been related to aberrant biology of cardiac neural crest (CNC). Studies of Ltbp-1L expression in the pharynx and the developing heart showed that only those CNC that relocated to
the OFT express Ltbp-1L. Specification and migration of CNC to the cardiac tissue in Ltbp-1L mutants appears to be normal, as judged by fate mapping of CNC. Likewise, the number and the proliferation rate of CNC that invaded the OFT in Ltbp-1L mutants are normal. However, we detect fewer cells expressing CNC markers Plexin A2 and FoxC1 in Ltbp-1L mutant OFT from E11.5 onwards, suggesting an altered CNC function in the OFT.TGF-β signaling is crucial for normal development of the cardiac OFT and associated vasculature. Given the role of Ltbp1L in the regulation of TGF-β bioavailability, we believe that cardiac abnormalities of Ltbp-1L mutants result from abnormal TGF-β activity during heart development. doi:10.1016/j.matbio.2006.08.073
51 MIG-17(ADAMTS), fibulin-1 and nidogen interact in cell migration Y. Kubota, K. Nagata, R. Kuroki, K. Nishiwaki RIKEN CDB, Kobe 650-0047, Hyogo, Japan The gonadal distal tip cells (DTCs) migrate along the body wall basement membrane (BM) to form the U-shaped gonad of the C.elegans hermaphrodite. An ADAMTS family metalloprotease MIG-17 is secreted from the body wall muscles and localizes to the gonadal BM where it is required for directional migration of DTCs. To identify the molecules interacting with MIG-17, we isolated suppressors of DTC migration defects of a mig-17 (null) mutant. One of the suppressor genes encodes fibulin-1 spliced isoforms (FBL-1C and FBL-1D), calciumbinding extracellular matrix proteins. Suppressor mutations, fbl-1 (k201) and fbl-1(k206), are amino acid substitutions in the third EGF-like motif. Mutant FBL-1C but not FBL-1D can suppress mig-17 defects. FBL-1C is synthesized in the gut cells and localizes to the gonadal BM in a MIG-17-dependent manner (Kubota et al., 2004).We found that fbl-1(k201) also significantly suppresses the gonadal elongation defects of gon-1(e1254), a mutation in another ADAMTS (Hesselson et al., 2004). Since mammalian fibulin-1C binds strongly to nidogen-1, in vitro (Sasaki et al. 1995), we examined the requirement of nid-1 (nidogen-1) gene function in the mechanisms of suppression of mig-17 and gon-1 by fbl-1 mutations. Introduction a nid-1(null) mutation into fbl-1(k201); mig-17(null) double mutants abolished the suppression effect of fbl-1(k201). However, the suppression was only weakly affected by introducing nid-1(null) into fbl-1 (k201) gon-1(e1254) mutants. These results suggest that the FBL1(k201) mutant protein compensates loss of MIG-17 or GON-1 in gonad development strongly or partially in a wild-type NID-1 dependent manner, respectively. doi:10.1016/j.matbio.2006.08.074