Developmental Morphology And Ultrastructural Studies Of The Zebrafish Myodural Bridge-Like Structure

Objective:Traditionally,the main driving forces of cerebrospinal fluid(CSF)flow were thought to include respiration and pulsation of the ventricular choroid plexus.In recent years,it was shown that the myodural bridge(MDB)provide power for CSF circulation.It is noted that during head movements,the tension generated by the contraction of the suboccipital muscles pull the dura mater through MDB,causing changes in the volume of the subarachnoid space and generating negative pressure,thereby causes changes in CSF flow.The MDB is a fibrous connection between the suboccipital muscle and the dura mater.It is a prevalent and highly conserved structure in mammals,and has been found in reptiles and birds,further demonstrating the high degree of conservation of MDB.It has also been shown that MDB has important physiological functions,including preventing dura mater infolding,transmitting proprioception and being associated with cervicogenic headache etc.The previous studies have helped to clarify that the MDB is a highly conserved anatomical structure with important physiological functions.So is MDB ubiquitous in vertebrates? To elucidate this issue,this study was performed on fish among vertebrates,preferentially selecting the model organism zebrafish as the experimental subjects.As the oldest vertebrates,fish have not yet evolved cervical vertebrae and have only a cerebral(spinal)membrane.The gross anatomy revealed that the vertebral canal membrane in zebrafish and the mammalian dura mater is similar in the structural hierarchy.And the muscle lateralis,located on both sides of the spine,is connected to the vertebral canal membrane,similar to the MDB structure described in mammals,and exists in the form of a connection between the muscle lateralis and the vertebral canal membrane,a special presence of a myomembranous connection.During the contraction of the zebrafish lateralis muscle,the force generated pulled the vertebral canal membrane through the myo-membranous connection,causing changes in the volume of the intraspinal space,resulting in local negative pressures and thus changes in the flow of CSF,which may provide a basis for the hypothesis that MDB is one of the driving forces of CSF circulation.Recently,the development and morphological characteristics of MDB in mammals(human embryo,SD rats)have been reported in the literature,but there was no report focusing on the development of myodural bridge structure in fish and the cellular composition and morphological changes during development.Materials and Methods:In this study,wild-type zebrafish at different developmental stages were selected as experimental subjects.Histological staining(Masson,HE,immunohistochemical staining),transmission electron microscopy and scanning electron microscopy were used to observe the area conjunction to both sides of the spine of 6-60dpf(days post fertilization)zebrafish,to clarify whether the connections exist between the muscle lateralis and the vertebral canal membrane,and to elucidate their structural characteristics,ultrastructural features and developmental patterns.Results:Histological staining showed that there were myo-membrane connections in zebrafish.During the whole larval stage,the vertebral canal and spinal cord membrane were not separated and the vertebral canal membrane was dominated with fibroblasts and fibers components;until 23 dpf,the vertebral canal and spinal cord membrane were stratified;25dpf,the vertebral canal membrane remains fibroblasts;30dpf,the vertebral canal membrane was predominantly fibrous.Immunohistochemical results revealed that TNMD was expressed in the developing tendons of fish,and the vertebral membrane composition may be the same as myoseptum,consisting of primarily type I collagen fibres.Scanning electron microscopy found that the muscle lateralis was inserted obliquely into the vertebral canal membrane,and the myofibers were tightly attached to the membrane.Transmission electron microscopy clearly showed that the myofilaments were anchored obliquely to the vertebral canal membrane and that fibroblasts were present in them,further verifying the existence and cell type of the myo-membrane connections.Conclusion:The results of this study showed that zebrafish have myo-membrane connections similar to those in other vertebrates,it was termed MDB-like structure according to the results of this study.This research revealed the developmental process and ultrastructural features of MDB-like structure,and delineated that fibroblasts are closely related to the development of the MDB-like structure in zebrafish.The findings provide strong support for the idea that MDB is common in vertebrates and will contribute to future studies on the function and mechanism of MDB.