Regulatory Roles Of Bone Morphogenetic Protein Family Genes During Intermuscular Bone Development In Blunt Snout Bream

Blunt snout bream(Megalobrama amblycephala),belonging to Cypriniformes,Cyprinid,Cultrinae,Megalobrama genus,is also known as Wuchang fish,which has the advantages of good taste,small head and high meat content.It has been widely cultured as an excellent herbivorous fish in China.M.amblycephala,like most other freshwater fishes such as carassius carassius,grass carp(Ctenopharyngodon idella),and carp(Hypophthalmichthys molitrix),has a large number of intermuscular bones with complex shapes.With the improvement of people's living standards,people's consuming attitudes have changed.The existence of intermuscular bones has severely hindered the breeding and further processing of these carp species.Bone morphogenetic proteins(BMPs)are a class of multifunctional growth factors that participate in various physiological processes and play an important role in bone and cartilage formation.In this study,the cDNA sequences of nine BMP genes were obtained for M.amblycephala.To gain insight into the role of BMPs in IB development,a spatiotemporal expression of bmp2 a,bmp2b,bmp3,bmp4,bmp5,bmp7 b,bmp8a,bmp14 and bmp16 was described during different developmental stages of IBs.In addition,homozygous bmp3-knockout zebrafish was produced by using CRISPR/Cas9 genome-editing technology.The main research contents are as below:(1)First of all,nine BMP genes(bmp2a,bmp2 b,bmp3,bmp4,bmp5,bmp7 b,bmp8a,bmp14 and bmp16)from M.amblycephala were cloned and characterized.The phylogenetic tree was constructed by comparing the amino acid sequence of BMP family members with the homologues of seven fish species and 8 out-group species from the Ensembl and GenBank Database.Phylogenetic analysis demonstrated that the BMP genes from M.amblycephala cluster with closely related to zebrafish,then form a clade with other teleost,and have a distant genetic relationship with mammals.This result is consistent with the traditional taxonomic relationship of these species,indicating that the function of each BMP gene has been conserved throughout evolution.These results provide a theoretical basis for the role of BMPs in the development of intermuscular bones.(2)Semi-quantitative PCR was used to detect the expression of BMPs in eight tissues of 1-year-old and 2-year-old M.amblycephala.In the light of the results from semi-quantitative PCR,high expression of most BMP genes was observed in calcified tissues-IB,rib and fin,consistent with the idea that BMP genes play a vital role in bone induction.Spatial tissue distribution showed that most BMP genes were observed at the highest level in developing IBs at one year old fish,thus suggesting a key role for these BMP genes in IBs growth and maintenance.Notably,the expression pattern of most BMP genes in the two-year-old fish was dramatically different from that in the one-year-old fish but bmp3.(3)Quantitative Real-time PCR technology was applied to analyze the of temporal expression patterns of bmps during four stages of IB development.The results showed that during the ossification process from stage ?(the IBs haven't emerged)to stage ?(all of the IBs ossified in the tail with the mature morphology),the expression profiles revealed that bmp16 was the most abundant transcript while bmp4 had the lowest abundance.The mRNA levels of bmp3,bmp4,bmp5 and bmp8 a increased significantly at stage ?,suggesting their roles in stimulating IB formation;The expression of bmp7 b reached the highest level at stage ?(the rapid period of IB development);With the exception of bmp7 b and bmp16,most BMP genes appeared a significant increase at IB maturation phase(stage IV).(4)Two lines of bmp3-knockout zebrafish were obtained by using CRISPR/Cas9 genome-editing technology.The results of Alizarin Red staining showed that there was no detectable difference in skeletal development,number and types of intermuscular bones between the mutants and wild-type zebrafish.Quantitative results showed that the expression levels of runx2 a,osterix,smad4 and smad8 were significantly higher in mutants than wild-type zebrafish,consistant with that bmp3 act as negative role in bone formation and the expression levels of runx2 b and ocn were significantly lower in mutants.