Using Conditional Knockout Technology Research Smad4 Gene In Endochondral Bone In The Process Of Function

TGF-β superfamily molecules play important roles in regulating cell proliferation,1ineage determination,motility, adhesion and apoptosis.Smad4 is the eentral mediator ofthe TGF-β superfamily signalings.In order to comprehensively study the funetion ofTGF-β superfalllily moleeules in the endoehondral ossification,we generated thechondrocyte specfic Smad4 gene knockout mice using the Cre-loxP system. Firstly,we investigated the temporal and spatial distribution of the Cre recombinase inchondrocyte specific Cre transgenic mousc using the ROSA26 reproter mice.The resultsshowed that the Cre recombinase began to express when the mesenehymal cellsdifferentiate into the chondroeytes.The aetivity of Cre recombinase can be detected in allthe cartilaginous tissues of vertebra,ribs,and limb bones et al,which were formedthrough the cndoehondral ossifieation.All these data indicated that the chondrocytespecifie Cre transgenic mice spceifically expressed the Cre recombinase in the cartilagetissues,and was a good tool for making chondrocyte specific gene knoekout mice. The chondrocyte specific Smad4 knockout mice were obtained by crossing thechondrocyte specific Cre transgenic mice with the Smad4 conditional knockout mice.Thechondrocyte specific Smad4 knockout mice cxhibited various phenotypes because thedifferent chondrocyte specific Cre transgenie founder mice were used.The mice eitherdied at the cmbryonic stages,or died right after birth or survive after birth.we found thatthe alveolus of the Smad4 knockout mice could not be distended.They died of lacking ofoxygen since the narrowed trachea prevented the air from entering the lung. We diselosed the roles of BMP and TGF-β signaling mediated by Smad4 molecule inthe endochondral ossification through studying the chondrocyte specific Smad4 knockoutmice that survived after birth.Mutant mice were smaller than their littermate countrols.The caleification of their long bones was delayed.At P4,the growth plates of the mutantmice became disorganized characterized by significantly expanded resting zone ofchondroeytes,reduced proliferating and hypertrophic zones of chondrocytes,andpremature hypertrophy of proliferating chondroeytes.BrdU ineorporation assay showedthat the Proliferation rate ofProliferating ehondroeytes was low inthemutantmiee.Inthegrowth Plates of P 1 6 and P22 mutant miee,the disorganization of ehondroeytes beeamemore aPParent.There were still areas of resting ehondroeytes in the bone marrow eavity.We further detected the moleeule markersofehondroeyteswithinsituhybridization.Theresults showed that there were some chondroeytes exPressing eollagen X loeating in theProliferating zone of ehondroeytes,indicating that some Proliferating ehondroeyteshyPertroPhied Prematurely.Together, these data suggested that Smad4 moleeule Promotedthe differentiation of the resting ehondroeytes,stimulated the Proliferation of theProliferating ehondroeytes,and inhibited the hyPertroPhie differentiation of ehondroeytes.In addition,these data also suggested that Smad4 mediated signals eould funetion as themorphogen to maintain the Polarity of arrangement and differentiation ofehondrocytes. We further studied the effeet of Smad4 gene knoekout on other signaling Pathwayswhieh regulate the bone develoPment.In situ hybridization and immunohistoehemistryanalysis revealed that the exPression of Ihh,Pte,PTHrP and PPR moleeules weredeereased in the mutant miee,suggesting thats"Zad4 deletion resulted in redueedlhl PTHrP signaling.Our results also showed thats Zad4 deletion eaused the increasedaetivity of FGF signaling.we found that PZI exPression was inereased,and the Stat-lwas aetivated as revealed by the nuelear loealization of Statl Protein in manyehondroeytes of the mutant miee.Through Von一Kossa staining,we found that the loeationof bone eollar of mutant miee was higher than that of eontrol miee.In the mutant miee,the bo-le eollar was abutted to thetlletllatexPressi()一1()f...