| Neuromuscular junction(NMJ)is the key structure for transmitting a neural signal between nerves and muscles,and achieving the motor function of skeletal muscle.NMJs are widely distributed in skeletal muscle,its three-dimensional spatial distribution is closely related to motor function.As everyone knows,the development of motor function is one of the most obvious features after birth,and infants are gradually able to lift their heads,crawl,stand and walk,which usually shows heterochrony of motor development in different body parts(heterochrony broadly refers to differences in the rate or timing of development).However,it remains unclear whether the NMJ development is heterochronic.The basic reason is that most of the current studies on the structural development of NMJ have focused on the observation of fine morphological changes of individual NMJ from local areas in the cervical muscles.Thus,few studies have targeted the structural development of NMJ at the overall level of skeletal muscle.In addition,myofiber is the basic structure of skeletal muscle,and its total number change after birth remains inconclusive.The lack of information on these overall structures limits the interpretation of the relationship between the nerve and muscle structure of skeletal muscle and the development of the motor function.This work is focused on the problems related to the structural development of nerve and muscle in mouse skeletal muscle.Based on the spatial distribution characteristics of NMJs in mouse skeletal muscle from different body parts after birth,we further investigated the developmental changes of innervating nerves and myofibers,and the relationships between structural changes and motor function development.The main contents are as follows:(1)A 3D imaging method of NMJs in mouse skeletal muscle: The fluorescent labeling of NMJs at the overall level of the muscle was achieved by the transgenic labeling and specific ligand-receptor binding-based method.Then,several existing clearing methods were evaluated with clearing efficiency,morphological retention,and compatibility with fluorescent labeling,and FDISCO was selected as the suitable clearing method for skeletal muscle.Finally,the 3D structural information of NMJs and innervating nerves in different skeletal muscles(biceps brachii,triceps brachii,gastrocnemius,tibials anterior,quadriceps)of mice were obtained by using the light-sheet microscopy.The obtained 3D distribution of NMJs was applied to the study on retrograde labeling of spinal motor neurons to provide a precise location for tracer injection,which improves the transport efficiency of retrograde tracer and labeling efficiency for spinal motor neurons.(2)The development of spatial distributions of NMJs and innervating nerves in skeletal muscles: Based on the 3D imaging method for NMJs,the spatial distributions of NMJs and innervating nerves of different muscles(cervical cleidomastoid,forelimb biceps brachii,and hindlimb gastrocnemius)in mice during postnatal development(P0,P3,P6,P9,and adult)were obtained.And 3D structural features and muscle-derived collagen XXV m RNA expression were quantified.The results showed that the innervation pattern and innervation zone in these three muscles were consistent during postnatal development,respectively,but the development of NMJs and innervating nerves from different body parts is heterochronic.Axonal arborization and NMJ formation in forelimb biceps brachii and hindlimb gastrocnemius still occur until 3 and 9 days after birth,respectively,whereas they are completed before birth in cervical cleidomastoid.(3)The relationships among the development of NMJ spatial distribution,myofiber,and motor function: The development of myofibers in different muscles(cervical cleidomastoid,forelimb biceps brachii,and hindlimb gastrocnemius)during postnatal development was evaluated with the myonuclei location,the proportion of proliferating cells,and myofiber number.The results demonstrated that the development of myofibers in muscles from different body parts is heterochronic.The myofiber number in cervical cleidomastoid remained unchanged after birth,while the myofiber numbers of the forelimb biceps brachii and hindlimb gastrocnemius continued to increase after birth and it tended to stabilize earlier in the forelimb biceps brachii.The developmental characteristics of motor function during postnatal development were also evaluated using behavior testing and correlated with the structural development of NMJ and myofiber.The motor development of different body parts is heterochronic and corresponds to the sequence of structural development.And the improvement of motor function is related to the formation of myofibers and NMJs.In summary,combining tissue optical clearing and optical imaging enables us to obtain spatial distributions of NMJs in various skeletal muscles,and to find that the development of nerve and muscle structure in skeletal muscles in different body parts is heterochronic,which corresponds to the heterochrony of motor function development.This study provides important evidence to reveal the relationship between the structural and functional development of skeletal muscle.And the established 3D imaging method for skeletal muscle based on tissue clearing provides new perspectives for the research of the structure and function of skeletal muscle. |
