Using Zebrafish To Study The Pathogenesis Of Several Skeletal Genetic Diseases

Skeletal genetic diseases are a group of inherited skeletal system diseases characterized by skeletal growth and development disorders.Clinically,the main manifestations of various types of bone tissue growth,development abnormalities,such as short stature,skull limbs deformity,scoliosis,abnormal bone density.Currently,according to the 2015 revision of the international classification standards for hereditary bone diseases,436 species and 42 categories are classified according to their known molecular genetic basis,etiology and phenotype.Such diseases often lead to various birth defects,the total incidence of the population is about 1/1500,which serious harm to the human health.At present,with the deepening of research,the pathogenic genes of many skeletal genetic diseases have been identified,but the pathogenic genes of some skeletal genetic diseases are still unclear,and the pathogenic mechanism of about half of the skeletal genetic diseases has not been fully clarified.Therefore,elucidating the mechanism of bone development and the molecular mechanism of bone genetic diseases will provide more theoretical basis for early diagnosis and effective prevention of human bone diseases.Patients are the most direct research objects of these skeletal genetic diseases.However,these genetic resources are difficult to obtain and relatively small pedigree size.Considering the constraints of social ethics and morality,it is impossible to use patients with genetic diseases as experimental objects in many scientific experiments.Therefore,model animals with stable genetic background are ideal candidates for genetic disease research.Zebrafish,as a new type of vertebrate model,has been widely used in human disease models.Compared with traditional animal models such as chicken and mouse,zebrafish has the characteristics of small individual,fast-developing process,large offspring numbers,external development,transparency and the availability of multiple genetic manipulations.The similarity of zebrafish genome and human genome is up to 87%.The structure of zebrafish skeleton,especially skull and spine,which is similar to that of mammals.As a result,zebrafish has become an ideal animal model for studying bone development and skeletal diseases.Our study selected zebrafish as the animal model,using ENU large-scale mutation screening and CRISPR/Cas9 gene knockout technology,focusing on three different skeletal genetic diseases.1.Congenital scoliosis(CS)is a complex genetic disorder characterized by vertebral malformations at embryonic stage.The detailed genetic etiology and underlying mechanisms of CS remain elusive.Through large-scale mutagenesis screening,new genes causing CS in zebrafish could be found and its pathogenesis further clarified.2.Cartilage-hair hypoplasia(CHH)is an autosomal recessive syndrome characterized by short stature,hypotrichosis and immunodeficiency.It is caused by mutations of the long non-coding RNA RMRP.As there is no viable CHH animal model,the pathogenesis of CHH causing skeletal dysplasia has not been clarified.Using CRISPR/Cas9 system to generate a zebrafish model of CHH by knocking-out the RMRP ortholog in zebrafish,rmrp,and its pathogenesis could be further studied.3.CATSHL syndrome is an autosomal dominant or recessive syndrome characterized by tall stature,camptodactyly and hearing loss.It is caused by heterozygous or homozygous mutations of FGFR3 gene.The cellular and molecular mechanisms by which CATSHL syndrome leads to phenotypes such as bone development and hearing abnormalities have not been fully elucidated.The generation of a fgfr3 knockout zebrafish model by CRISPR/Cas9 technique to simulate CATSHL syndrome can further study its pathogenesis.Methods:Part I Screening of congenital scoliosis mutants in zebrafish and functional mechanism study of the mutants1.ENU-induced large-scale genetic screening of zebrafish: ENU treated F3 generation embryos,gross phenotype was observed by stereoscopic microscope,and vertebral mineralization was observed by living Calcein staining.2.Smt mutant position cloning: generate a zebrafish strain with single-strand fragment length polymorphism group and the mutant gene was position cloning by Bulk Segregant Analysis(BSA),and mRNA was injected for recover experiment.3.The gene expression pattern of dstyk was detected by whole mount in situ hybridization,and the mutant phenotype was verified by CRISPR/Cas9 knockout of dstyk gene.4.Using transgenic fish Tg(β-actin: ras-GFP)to label cell membrane,and transgenic fish Tg(col2a1a:EGFP)to label notochord cells to analyze the notochord phenotype of the mutant.5.Tracker in vivo staining,immunofluorescence,and whole mount in situ hybridization were used to analyze the causes of notochord vacuole biogenesis defect in the mutant.6.Transmission electron microscope was used to detect the ultrastructural changes of notochord and notochord sheath in mutant.7.Transgenic fish Tg(col2a1a: EGFP);Tg(osterix:mCherry)and living Calcein staining were used to analyze the causes of scoliosis in the mutant.8.Immunofluorescence and transgenic fish lines were used to detect the subcellular localization of DSTYK,and the impact of lysosome biogenesis after DSTYK siRNA knockdown was analyzed.9.Immunofluorescence,WB detection and analysis of the molecular mechanism of Dstyk knockout inhibiting the formation of lysosomes and notochord vacuole,and recover experiments at the level of cells in vitro and zebrafish in vivo to verify the phenotype changes.Part II The generation of zebrafish model of cartilage-hair hypoplasia and its pathogenesis study1.Multiple sequence alignment was used to compare the conservation of transcription and promoter sequences of zebrafish rmrp.The expression pattern of rmrp in zebrafish was analyzed by whole mount in situ hybridization.2.Using CRISPR/Cas9 technology to knock out zebrafish rmrp and analyze its general phenotype.3.Alcian Blue staining,chondrocyte labeled transgenic fish Tg(col2a1a:EGFP)and whole mount in situ hybridization were used to analyze the chondrogenic phenotype of rmrp mutants.4.The osteogenic development phenotype of rmrp mutants was analyzed using Alizarin red staining and transgenic fish line Tg(osterix:EGFP)labeled with osteoblasts.5.Transgenic fish lines and HE staining were used to analyze the development of intestinal and cranial blood vessels in rmrp mutants.6.Cell proliferation and apoptosis in rmrp mutants were analyzed by EdU and TUNEL assay.7.WB,quantitative PCR,immunofluorescence,in situ hybridization were used to analyze the mechanism of rmrp mutant phenotype and carry out rescue experiments to observe whether the mutation phenotype could be alleviated.Part Ш The generation of zebrafish model of CATSHL syndrome and its pathogenesis study1.Multiple sequence alignment was used to compare the conservation of zebrafish fgfr3 coding sequences and amino acid sequences.The expression pattern of fgfr3 in zebrafish was analyzed by whole mount in situ hybridization.2.Using CRISPR/Cas9 technology to knock out zebrafish fgfr3,and its skeletal phenotype was analyzed by X-ray,micro-CT and Alcian blue and Alizarin red Whole skeleton staining.3.Using living Alizarin red staining and transgenic fish line Tg(osterix:EGFP)which labeled with osteoblasts to analyze the phenotype of fgfr3 mutants at different time points in cranial osteogenesis,spine development and fish scales development.4.Chondrocyte-labeled transgenic fish Tg(col2a1a:EGFP)was used to analyze the chondrogenic phenotype of fgfr3 mutants at different time of development,and histological staining was used to analyze the growth plate phenotype of fgfr3 mutants.5.Using EdU assay,WB,in situ hybridization,transgenic fish lines to analyze the cell and molecular mechanisms of fgfr3 mutant phenotypes.Results:Part I Screening of congenital scoliosis mutants in zebrafish and functional mechanism study of the mutants1.A shortened somites(smt)mutant was screened by ENU,and the smt mutant had severe scoliosis and kyphosis.2.Position cloning and recover of pathogenic genes revealed that the phenotype of smt mutants was caused by dstyk mutation.3.Dstyk is widely expressed in early embryonic development and then dynamically expressed in the notochord.CRISPR/Cas9 knockout of the dstyk gene also found phenotypes of shortened somites and spinal deformity.4.Dstyk gene mutation leads to incomplete vacuole inflation of notochord cells,leading to abnormal notochord development.5.Dstyk gene mutation leads to abnormal vesicle transport,which leads to impaired formation of notochord vacuole,decreased expression of shh,cmn,col8a1 a,col9a1b and col11a2 in the notochord,and abnormal notochord function.6.Ultrastructure showed that the formation of vacuoles was defect,arrangement and structure of notochord sheath were disordered in dstyk mutant.7.The dstyk mutation leads to abnormal axial skeleton segmentation,vertebral body formation defects,and eventually leading to scoliosis.8.In vivo and in vitro studies have shown that DSTYK is located in the late endocytosis/lysosomes and is involved in the formation of mammalian cell lysosomes.9.Dstyk knockdown activates the mTORC1 signaling pathway and inhibits TFEB phosphorylation,thereby inhibiting the nuclear translocation of transcription factor TFEB and thereby inhibiting the formation of lysosomes and notochord vacuoles.Treatment with mTORC1 inhibitor Torin1 can partially rescue TFEB nuclear translocation defect and lysosome biogenesis defect after dstyk knockout.Torin1 treatment of dstyk mutants partially recovers the phenotype of scoliosis.Part II The generation of zebrafish model of cartilage-hair hypoplasia and its pathogenesis study1.Multiple sequence alignment of RMRP transcription and promoter regions in human,mouse and zebrafish showed that RMRP was highly conserved among multiple species.Rmrp is expressed in a variety of tissues including the cartilage.2.CRISPR/Cas9 knockout of the rmrp gene leads to abnormal mandibular development,pericardial edema,inability to form swim bladder,and small eyes and head.3.Rmrp gene knockout leads to hypoplasia of pharyngeal arch cartilage and delayed expression of chondrogenic development-related molecules sox9 a and col2a1 a.4.The intramembranous ossification of rmrp mutant was significantly inhibited and the endochondral ossification was delayed secondary to chondrodysplasia,but the mineralization of the vertebral body accelerated,and the number of mineralized vertebral bodies increased significantly.5.Rmrp mutant intestinal epithelial cells were reduced,intestinal villi were absent,and cranial blood vessels were malformed.6.Rmrp mutation inhibits cell proliferation by affecting the expression of cell cycle related genes,and up-regulates the expression of apoptosis-related genes to promote apoptosis.7.Rmrp mutation up-regulated the canonical Wnt/beta-catenin signaling pathway,and treatment with the beta-catenin inhibitor XAV939 partially alleviated chondrodysplasia and the increased vertebral mineralization in rmrp mutants.Part Ш The generation of zebrafish model of CATSHL syndrome and its pathogenesis study1.Multiple sequence alignment of FGFR3 coding and amino acid sequence in humans,mice,and zebrafish indicate that fgfr3 is highly conserved across species.Fgfr3 is expressed in a variety of tissues including cartilage.2.Fgfr3 knockout by CRISPR/Cas9 resulted in malformation of craniofacial bone,severely inhibited ossification of the mandible and skull,and abnormal development of swim bladder.3.In fgfr3 mutants,intramembranous ossification and endochondral ossification were significantly inhibited,scale development and formation were also blocked,and some auditory and sensory organs were abnormal.4.Zebrafish knockout of fgfr3 resulted in abnormal hypertrophy of chondrocytes and disordered arrangement of chondrocytes,which disrupted the patterning and shaping of pharyngeal arch,and resulted in chondroma-like lesions in some cartilage.5.Zebrafish fgfr3 mutation enhance IHH signaling and promote chondrocytes proliferation.6.Fgfr3 mutation resulted in an increase in Wnt/ beta-catenin pathway,and inhibition of Wnt signal partially alleviated chondrogenic dysplasia caused by fgfr3 mutation.Conclusion:1.Dstyk mutation affects the development of notochord vacuoles through mTORC1/TFEB pathway,leading to abnormal axial skeleton segmentation,vertebral body formation defects and eventually leading to scoliosis.2.Rmrp mutation disrupts chondrogenesis and bone ossification in zebrafish model of cartilage-hair hypoplasia via enhanced Wnt/β-catenin signaling3.Zebrafish fgfr3 mutations result in abnormal hypertrophy of chondrocytes and disordered arrangement of chondrocytes,which can lead to mechanical changes of mandibular cartilage and craniofacial deformity.