Regulatory Mechanism Of Col22a1 In The Development Of The Myodural Bridge In Xenopus Laevis

Objective:The myodural bridge(MDB)has been defined as a normal physiological structure located in the suboccipital region.It is a dense fibrous connective tissue bridge between the suboccipital muscles and the dura mater.These interconnected fibers,together with the muscles where they origin and the dura mater,form a functional unit called the myodural bridge complex(MDBC).Previous studies have confirmed that MDB is a highly conserved structure existing in mammals,birds,and reptiles by plastination section observation,gross dissection,histological staining,and electron microscopy.In order to further investigate the extent of MDB in vertebrates,this study selected the amphibian model animal the African clawed frog,i.e.,Xenopus laevis,as the experimental object,utilizing morphological experimental methods such as gross dissection,paraffin section staining,and sweeping electron microscopy to investigate the forms and characteristics of MDB in the African clawed frog.In recent years,imaging and functional studies have shown that MDB has important physiological functions and may affect local or even overall cerebrospinal fluid circulation by pulling the dura mater.Clinical studies have also shown that hypertrophy of the rectus capitis posterior minor muscle is associated with chronic headache,and the mechanisms of pain may also be associated with MDB.Therefore,exploring the key molecular signals during the development or injury of MDB may be important for a deeper understanding of their role in cerebrospinal fluid circulation and headache development.Multiple staining methods have suggested that MDB is mainly composed of type I collagen fibers,which are extracellular matrix components synthesized and secreted by multiple cells.However,the origin of the cells that secrete MDB collagen fibers is unclear,and the developmental origin of these cells is also unclear.Considering that in the functional unit of MDBC,muscle is the power provider and the main starting point of MDB,this study focused on the junction of muscle and collagen fibers.By specifically knocked down COL22A1,a protein that regulates the development of myotendinous junction,we aimed to investigate the relationship between MDB development and muscle,and to further verify the functional and clinical significance of MDB from a developmental perspective.Materials and methods:In the present study,12 cases of sexual matured Xenopus laevis were selected(10cases of morphological experiments were males,and the remaining 2 cases were one male and one female for breeding),and were subjected to gross anatomy,histological section staining,scanning electron microscopy observation.According to Nieuwkoop and Faber(NF)staging criteria,NF54,NF58,NF61,and NF66 stage Xenopus laevis tadpoles were selected.Four tadpoles of each stage were selected and stained for histological sections.16 tadpoles of each NF58,NF61 and NF66 stages were selected and divided into experimental and control groups,and Morpholino knockdown modeling was performed on the experimental group at NF56 stage.Then q PCR and histological experiments were performed to observe the effect of low expression of COL22A1 on MDB development from the RNA and histological levels,respectively.Results:In adult Xenopus laevis,gross anatomical findings showed that there were string fibers connecting the dorsal longest muscle to the dura mater at the atlanto-occipital space.This string fiber connection were considered as the MDB in Xenopus laevis.Histologic staining showed that MDB was the connection from the thickened perimysium of the longissimus dorsi muscle(LGD)and the interarcualis muscle to the dorsal atlanto-occipital membrane(DAOM)and dura mater at the atlanto-occipital space.These fibrous connections were mainly composed of type I and type III collagen fibers with scarcely any elastic fibers,which clearly distinguished from the ligamentum flavum(LF)structure at the rest intervertebral spaces.Scanning electron microscopy showed that the perimysium was connected ventrally to DAOM and there were also dense fibrous connections between DAOM and dura mater.These results mutually corroborate the existence of MDB structure in Xenopus laevis.For Xenopus laevis tadpoles,the histological staining results showed that there was no muscle coverage at the atlanto-occipital space in stage NF54,the basic structural unit of MDB did not exist.In stage NF58 tadpoles,muscle coverage could be seen at the atlanto-occipital space,the exoccipital bone and the posterior part of atlas had appeared.Collagen fibers among the muscle fibers were connected to DAOM and dura mater ventrally but relatively weak because the muscle was not mature.In stage NF61 tadpoles,the neck was further developed and MDB were more obvious than in stage NF58.In stage NF66 tadpoles,the metamorphosis was completed and they were in the form of juvenile frogs,but the amount of MDB fibers were obvious less than that in the adult ones.These results suggest that MDB of Xenopus laevis tadpoles began to form at NF58 and were fully formed at NF66.After local injection of Vivo-Morpholino to establish an animal model of COL22A1 knockdown,the q PCR results of the experimental group relative to the control group showed that at the m RNA level,the expression of muscle,tendon,and matrix-related genes were altered as follows: the expression of all genes was increased in NF58 phase,except for the expression of Myo D,which was decreased;the expression of all genes was increased in NF61 phase,except for the expression of Myo D.The expression trend of each gene was higher and then lower,except for Myo D expression,which was lower and then higher,and the expression of other genes was higher and then lower.The histological results showed that the tadpoles in the NF58 and NF61 stages showed delayed development of MDB and the muscle near the median sagittal position of the neck was not completely covered.However,the neck muscles in the NF66 stage experimental group were enhanced compared with the control group.Conclusion:1、In adult Xenopus laevis,there existed MDB,which was in form of a connection from perimysium to DAOM and dura mater in the atlanto-occipital space.The MDB in the adults were characterized by collagen fibers,including type I and type III collagen fibers,and almost no elastic fibers;the fibers between the perimysium,DAOM and dura mater were fused with each other in a woven manner.2、The development of MDB in Xenopus laevis was consistent with the process of metamorphosis,which starts to form at the NF58 stage(pre-metamorphosis peak)and is completely formed at the NF66 stage(end of metamorphosis).3、COL22A1 plays a regulatory role in the development of the myocardial bridge in the African clawed toad over a specific time frame,and it may affect genes related to muscle and tendon development or matrix integration,and probably perform a developmental regulation through the TGFβ signaling pathway.