Genetic Basis Of Characteristic Styliform Cartilage Development In Pteromyini

Among land-dominated placental mammals,flight and glide are two special forms of movement that they adapted to the environment.Among these,flying squirrels are the only group within the Sciuridae capable of glide,and with a remarkable species diversity and wide range of geographical distribution.It belongs to the Tribe Pteromyini in the Class Mammalia,Order Rodentia,Family Sciuridae in the subfamily Sciurinae.There are 52 Species within 15 Genera in the world,and 16 species of 7 genera in China,among which 3 species are endemic to China:Trogopterus xanthipes,Aeretes melanopterus and Hylopetes phayrei.The flying squirrel have a skin membrane connecting the front and rear legs,which stretched out like a gliding wing,and have a special cartilage structure compare to other gliding species:styliform cartilage,to increase the membrane area.Until then,most researches mainly focused on the kinematic dynamics of flying squirrel gliding,as well as morphology and anatomy of the specially developed styliform cartilage.However,few information was reported on the molecular development of this structure.In order to explore the molecular mechanism of development of this cartilage structure,using Trogopterus xanthipes as representative,we collected forelimb and styliform cartilage tissues of the embryos of Trogopterus xanihipes at 6 different developmental stages(TXS4-TXS9),as well as forelimb tissues from Mus musculus of the corresponding periods(E10.5-E17.5)as control.We first applied bone double staining experiments at various embryonic stages,observed the development of styliform cartilage,and secondly conducted transcriptome sequencing of each stage(6GB data for each organism on average),and analyzed differentially expressed genes.Results of bone double staining experiments showed that the styliform cartilage was occurred at TXS6.Based on the third generation genome assembled by assistance of HI-C(genome size 3.17 GB,coverage 71X,contig N50=4.1Mb,scaffold N50=7.04 Mb,and annotated 25,726 genes)recently sequenced in our lab,we performed differentially transcriptomic analyses with reference genome.Using forlimbe as control,the results turned to be congruence with bone double staining experiments,that the differentially-expressed genes which related to development of styliform cartilage were significantly down-regulated during TXS6.We found that the signaling pathways regulating pluripotency of stem cells may highly relevant to the development of styliform cartilage,and the family of BMP signaling pathways,TGFbeta family factor,LIF together mediates to control this pathway.In the key stage of styliform cartilage,the Bambi,Bmp5,Gdf5,Dkk1,and Frat1 genes,which inhibit the expression of downstream genes,are down-regulated,so that the downstream genes are activated,and the pathway is finally accessed to achieve osteoblast differentiation,and finally result in the appearance of styliform cartilage.In the subsequent stages,Bmprla,Bmpr2,Bmp8a,Bmp6,Bmp5,and are up-regulated,which promotes iron metabolism,osteoblast differentiation,and induces differentiation of mesoderm and endoderm.The iron element can affect the metabolism of calcium,and such indirectly affecting the activity of osteoblasts may contribute to sustained development of styliform cartilage.This study revealed for the first time the genetic basis of the styliform cartilage development and the molecular mechanism of flying squirrel adaptation to gliding movement from the perspective of evolutionary development,providing a new understanding for the complex character of gliding adaptation.