RESUMO
Ryk is a member of the receptor tyrosine kinase (RTK) family of proteins that control and regulate cellular processes. It is distinguished by binding Wnt ligands and having no detectable intrinsic protein tyrosine kinase activity suggesting Ryk is a pseudokinase. Here, we show an essential role for Ryk in directing morphogenetic events required for normal cardiac development through the examination of Ryk-deficient mice. We employed vascular corrosion casting, vascular perfusion with contrast dye, and immunohistochemistry to characterize cardiovascular and pharyngeal defects in Ryk-/- embryos. Ryk-/- mice exhibit a variety of malformations of the heart and outflow tract that resemble human congenital heart defects. This included stenosis and interruption of the aortic arch, ventriculoarterial malalignment, ventricular septal defects and abnormal pharyngeal arch artery remodelling. This study therefore defines a key intersection between a subset of growth factor receptors involved in planar cell polarity signalling, the Wnt family and mammalian cardiovascular development.
Assuntos
Cardiopatias Congênitas/etiologia , Faringe/anormalidades , Receptores Proteína Tirosina Quinases/fisiologia , Proteínas Wnt/metabolismo , Animais , Aorta Torácica/anormalidades , Feminino , Camundongos , Morfogênese , GravidezRESUMO
Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a ß-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.
Assuntos
Polaridade Celular/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Células CHO , Cóclea/citologia , Cóclea/embriologia , Cricetinae , Cricetulus , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Células HEK293 , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Proteína Tirosina Quinases/genética , Proteínas Wnt/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The tumor suppressor gene p53 plays a major role in the maintenance of genomic integrity. The impact that variations in cellular turnover rates and sensitivity to DNA damage will have on the effectiveness of p53 in this role was examined by following the induction and persistence of mutations in the brain and small intestine of mice after exposure to ionising radiation (IR). The examination of mutagenesis was carried out using the pUR288 LacZ plasmid-based mouse model-consisting of mice containing a target gene for mutation analysis integrated into every cell. In addition the mice varied in their p53 status. The tissues were compared at post-irradiation time-points from 24h to 3 months. The mutation frequencies (MFs) in the p53 wildtype and heterozygous brains peaked at 24h post-irradiation, and then returned to background or close to background levels, respectively. The p53 nullizygous brain showed a more fluctuating MF pattern, but returned to background levels by 3 months, indicating that the effect of the loss of p53 did not result in lasting differences in the response to mutation induction in the brain. In the intestine, there was a different pattern; in the wildtype and heterozygous animals, the MFs increased from 24h to a peak at 4 weeks post-irradiation, before decreasing towards background levels at 3 months. The MFs in the intestine from the nullizygous animals did not decrease significantly between 4 weeks and 3 months, illustrating that the loss of p53 had a greater impact in this tissue than the brain. The variation in mutation frequencies and the type of mutations generated after DNA damage suggests that while p53 plays a significant role in the maintenance of genomic integrity, other mechanisms, such as the drive to replicate in progenitor cells, can reduce its effectiveness as the "guardian of the genome".
