斑马鱼模型,骨骼发育和疾病 z-bo 2020-11-02 414

　　Abstract: In the past two decades, research on zebrafish as a model organism has been greatly increased. Zebrafish has many advantages, such as high fecundity, optical transparency, in vitro development and genetic ease, which is very suitable for studying the development process and the influence of genetic mutations. Recently, the zebrafish model has been used to study autophagy. In many cases, the homeostasis of cells and tissues requires this important protein degradation pathway. Correspondingly, its disorder is related to a variety of diseases, including bone diseases. In this review, we explored how zebrafish are used to study autophagy in bone development and diseases, and how these fields intersect each other to help identify potential therapeutic targets for bone diseases.

　　Autophagy: Autophagy is a catabolic process that decomposes cytosolic components into basic biomolecular components through lysosome degradation, so that they can be recycled. This is an indispensable process in the process of cell differentiation. It helps to maintain cell homeostasis. Its main function is to mobilize nutrients under pressure to maintain important cell functions. In order to understand the autophagy pathway and its molecular control, extensive research has been conducted. Although these studies have established the importance of autophagy in cell differentiation and survival, they have also emphasized the important role of autophagy in many diseases, and how autophagy dysregulation leads to the pathology of many diseases, including common Bone diseases such as arthritis and osteoporosis. Autophagy can be divided into three main forms. Chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy; each is described by the method of transporting cargo to the lysosome. This review will focus on macro-autophagy (hereinafter referred to as autophagy), because this is considered the main form of autophagy and is still the most widely studied. Autophagosomes are unique double-membrane structures that capture proteins, organelles and other cell debris before fusing with lysosomes to form degraded autolysosomes. This isolation of cytoplasmic components can be performed as a non-selective or selective process. The former is considered to be a large number of non-specific degradation processes, while the latter requires specific receptor proteins to identify and isolate target proteins, molecules, organelles or Invading pathogens.

　　Autophagy mechanism: The autophagy pathway is mainly mediated by the highly conserved autophagy-related (ATG) protein family, which was first discovered and identified in budding yeast by Yoshinori Ohsumi and colleagues. So far, more than 40 ATGs have been identified in yeast, most of which are preserved in higher eukaryotes. Roughly speaking, the pathway can be divided into three main steps: initiation and formation of phagosomes; elongation of phagosomes; and finally lysosome fusion. In each step, specialized ATG proteins and complexes are recruited and activated at different phagosome assembly points, called autophagosome initiation points. When new autophagosomes are expanded, blocked, and transported to lysosomes, proteins from other cell membrane transport pathways play an important role. We will focus on the mammalian biology of the core proteins related to the zebrafish autophagy model so far. These include ULK1 complex composed of ULK1, ATG13, FIP200 and ATG101, and Phosphatidylinositol 3-kinase complex I (PI3KC3) containing VPS34, Beclin1 (BECN1), ATG14, AMBRA1 and p115, which are involved in autophagy Initiation and the formation of phagocytic function. Next, two binding systems: ATG5-ATG12-ATG16L and MAP1LC3-ATG3 cooperatively recruit MAP1LC3 and couple it to the lipid phosphatidylethanolamine (PE) present on the fluorophore membrane to form lipidated MAP1LC3- II. Before this step, ATG4 and ATG7 are responsible for processing MAP1LC3 into MAP1LC3-I for lipid coupling. These steps are essential for the detection and analysis of autophagy, because fluorescently labeled MAP1LC3 is the most commonly used marker for monitoring autophagy activity in cells and whole organisms. Finally, in the case of selective autophagy, receptor proteins (such as p62/SQSTM-1, optineurin, and NDP52) can specifically recognize and target polyubiquitinated substances to autophagosomes.

　　The core proteins involved in the autophagy pathway of zebrafish and their regulation. Knockout (KO) zebrafish strains are highlighted in red, and the box shows commonly used drugs that can activate (green) or inhibit (red) autophagy activity

　　In addition to these proteins, there are many other signaling pathways involved in initiating the autophagy response. At its core are the mechanistic targets of the rapamycin complex 1 (mTORC1) pathway and the cAMP-dependent protein kinase A (PKA) pathway. It is obvious from various studies on different organisms that the correct regulation of autophagy initiation is essential for maintaining cell homeostasis. Many studies have shown that autophagy disorders are related to the development of various neurological, cardiovascular, metabolic and recent bone diseases. At the same time, other studies have found the diversity of cell functions other than protein degradation mediated by autophagy. These processes include cell differentiation and proliferation, cell metabolism, endoplasmic reticulum stress relief, and non-cell autonomous nutrient mobilization. All these processes are essential for the development and survival of bone and cartilage cells. By understanding the unique functions of autophagy in specific cells and the key factors in their regulation, we can better understand the impact of autophagy in the development of bone diseases and how to manipulate autophagy pathways to achieve therapeutic goals.

　　Autophagy and bone development: A number of studies have shown that autophagy is related to the development and maintenance of the skeletal system. From the early stages of development, autophagy plays a vital role in the differentiation, transformation and functional activity of key skeletal cells, including osteoblasts (skeletal secretion cells), osteoclasts (skeletal absorption cells), Bone cells (bone maintenance cells) and chondrocytes (chondrocyte secreting cells). New evidence after development shows that once these cells have finally differentiated, they need basic autophagy at a constitutive level to ensure their normal function and survival in the hypoxic, nutrient-deficient and hypertonic environment.

　　Overview of the role of autophagy in bone and chondrocytes. Autophagy helps maintain the homeostasis, survival and function of osteoblasts, osteoclasts, osteocytes and chondrocytes

　　During embryonic development, vertebrate bones and their related bone, cartilage and connective tissue are formed by mesenchymal stem cells (MSCs). These cells were originally derived from three different embryonic cell lines and continue to form different areas of bone. In order to form bones, these cells first migrate to the appropriate area in the embryo for bone formation, then aggregate and proliferate to form mesenchyme, and then differentiate into osteoblasts or chondrocytes. The study emphasized the important role of autophagy in the differentiation process and differentiation ability of MSCs. Bone marrow mesenchymal stem cells have been shown to have a high level of basal autophagy. After treatment with 3-methyladenine (3-MA), a class III pi3 kinase inhibitor that prevents autophagy induction, it showed Its ability to differentiate into osteoblasts is reduced. In addition, studies on primary human bone marrow mesenchymal stem cells show that during the differentiation of bone marrow mesenchymal stem cells into osteoblasts, these cells accumulate a large number of autophagic vacuoles, which are then broken down to provide energy. Autophagy is needed to help balance energy supply during the differentiation process, so it is essential for the differentiation and function of MSC. In vitro studies using primary mouse osteoblasts also showed that by knocking out FIP200, an important component of the mammalian ULK1 complex, autophagy was blocked and osteoblast differentiation was inhibited, which further proved that autophagy is establishing osteoblast populations. The importance of. Bone is formed by osteoblasts and chondrocytes through two different mechanisms. In the process of intramembranous ossification, bone is directly secreted by osteoblasts, which are the main components of zebrafish craniofacial bones, spine and fins. As the organism develops, the bones will be lengthened and modeled until the final bones are formed, although the bones will continue to reshape in response to changes in mechanical loads or fractures throughout their lives. The process of bone modeling and remodeling is mediated by osteoblasts, osteoclasts and osteocytes. Osteoblasts are located on the surface of bones and are responsible for the synthesis, secretion and mineralization of bone matrix. At the same time, osteoclasts migrate to the active bone remodeling area to help degrade and absorb bone. The coordinated activity of these cells is essential to ensure bone homeostasis, because disturbance of this balance can lead to disease. Autophagy is a process that has been shown to be essential for maintaining this balance and regulating the differentiation, formation and function of bone and cartilage cells.

　　Autophagy in bone formation: Osteoblasts are the main cell type for bone formation, and their survival and function are regulated by autophagy. The differentiation of bone marrow mesenchymal stem cells into osteoblasts is regulated by the transcription factors RUNX2 and SP7 (also called Osterix). In this process, studies have shown that autophagy is up-regulated to help these cells survive in the hypoxic bone environment and fight oxidative stress, because controlling the level of autophagy activity is positively correlated with osteoblast survival. In addition to survival, autophagy activity is closely related to osteoblast mineralization. Osteoblasts form mineralized bone by depositing hydroxyapatite crystals into the collagen-based bone matrix. As the matrix matures, these crystals form a lattice structure in collagen to form bones. In addition, the absence of ATG5, ATG7 or Beclin1 is essential for the formation of autophagosomes, and they have all been shown to cause bone loss and mineralization. In the case of targeted ATG7 loss in mice, an increase in the number of fractures was recorded, suggesting that it was related to endoplasmic reticulum stress and a decrease in the number of osteoblasts. Another in vitro study showed that the loss of FIP200 in osteoblasts can disrupt their terminal differentiation, inhibit bone formation and lead to osteopenia.

　　The role of autophagy in bone maintenance: Osteocytes are terminally differentiated cells formed by osteoblasts, which are trapped in the bone matrix. They are essential for bone health and maintenance, and are responsible for regulating the bone remodeling process. By expanding the dendritic-like process in the bone matrix, bone cells connect to form a huge network, which detects and responds to hormonal and mechanical changes in the bone environment by guiding the recruitment of osteoblasts and osteoclasts to the local bone area. Just as autophagy plays a key role in the differentiation and function of osteoblasts, autophagy also plays an important role in the health and maintenance of bone cells. First of all, during the transformation of osteoblasts into bone cells, cells must undergo tremendous changes in cell morphology and composition, which requires active circulation of organelles. Second, a study using human and rat bone tissues showed that bone cells showed accumulation of MAP1LC3 punctate cells, and this expression was higher in bone cells than in osteoblasts.

　　The role of autophagy in cartilage formation and maintenance: In addition to bone cells, cartilage cells that form cartilage also play a vital role in bone development and function. Cartilage cells are responsible for the formation of the initial cartilage skeleton and the articular cartilage layer between the bones during the process of endochondral ossification, and the movement of joint fluid. Like other skeletal cell populations, autophagy has been shown to be an important process for chondrocyte differentiation, function and survival. In the process of intrachondral bone formation, chondrocytes secrete a matrix rich in collagen to form the cartilage of future bones. This process continues until the chondrocytes reach a non-proliferative, hypertrophic state, at which time the cells undergo apoptosis, triggering the absorption of cartilage and invading osteoblasts to mineralize them into bone. Some chondrocytes remain in the area near the end where the bone is formed, called growth plates. In this area, chondrocytes continue to proliferate and secrete cartilage matrix, so that they can grow longitudinally through ossification. Mice lacking ATG7 specifically for chondrocytes show impaired matrix secretion due to the retention of synthetic type II procollagen in the endoplasmic reticulum.

　　Zebrafish is used as a model to study autophagy in bone development and pathological processes: As mentioned above, autophagy plays an important role in the development and maintenance of bone and cartilage cells, and its activity is essential for maintaining the stability of the bone internal environment. In fact, due to the imbalance of bone or chondrocyte activity and the accompanying imbalance of autophagy activity, a variety of bone diseases are caused. Many of these diseases are chronic and debilitating, and currently available treatment options are limited. Therefore, expanding our understanding of the cellular and molecular processes that are essential for coordinating the development of bones and joints (such as autophagy) is essential to promote the development of new treatments for these diseases and to expand our understanding of the pathogenesis of these diseases. Through the use of animal models, I have learned a lot about the skeletal system and related diseases. In bone research, in vivo models have obvious advantages over in vitro cell models, because the complex, movable, three-dimensional structures of bones and joints cannot be fully reproduced in in vitro systems. Similarly, the effects of other cell and tissue types and their associated secretions on cartilage and bone cells cannot be recaptured in a single cell system. Although many different animals have been used as models for bone research, the mouse model is still the most widely used model because of its generally low cost of raising, short generation time, ease of handling and genetic tractability. Despite these advantages, rodent models do have some inherent limitations in bone and autophagy-based research.

　　Zebrafish is considered a useful alternative to rodent models for studying bones and autophagy. First of all, zebrafish have a strong reproductive ability. A pair of zebrafish can lay 300 eggs a week, and these eggs develop into translucent larvae on the outside. This allows the study of cell and general morphological changes during early development without the need for invasive experimental techniques or animal sacrifices. Secondly, zebrafish have strong genetic adaptability, because by using genetic tools such as TALEN and CRISPR/Cas9, embryos can be injected with constructs at the single-cell stage to produce transgenic or transgenic zebrafish lines. With the continuous improvement of these tools and access to complete sequenced genomes, it is possible for zebrafish to efficiently and specifically target multiple genes in a high-throughput manner. Through this method, many gene knockout and reporter gene lines have been developed and used to simulate specific diseases or visualize and track specific proteins or cell types, such as bone cells or autophagy-related proteins. As a vertebrate, zebrafish and humans show a high degree of genetic similarity, and all core proteins related to mammalian autophagy can be found in the zebrafish genome, and their overall amino acid identity with their human counterparts Between 40% and 96%. This high degree of conservation suggests that the autophagy pathway of zebrafish operates in a very similar manner compared to humans, and has contributed to the development of many mutant and transgenic autophagy zebrafish lines. Despite the obvious structural differences, the skeletal physiology of zebrafish is similar to that of mammals, including the same joint types and joint components, such as joint cavity, articular cartilage and synovium. It has been widely displayed in the larval jaws of zebrafish that have been extensively studied, and is still one of the main joint parts used to simulate joint development. In addition, it is emphasized that the overall molecular mechanisms of vertebrate bone segmentation, joint development and fin/limb development are very similar. Many human bone diseases can be simulated in zebrafish, and the phenotype of higher vertebrates can be reproduced. For example, similar disease models can be found in zebrafish, such as osteogenesis imperfecta, scoliosis, osteoporosis, Stickler syndrome, and osteoarthritis. In addition, because zebrafish naturally develop osteoarthritis during aging, the pathogenesis of the disease and its common symptoms (such as increased spinal deformity)

　　Plus, vertebral dislocation and fracture) and the formation of osteophytes are easy to explore. In summary, these data indicate that zebrafish is a representative, relevant and useful model that can be used to study bone and joint development, the pathology of bone diseases, and the genes involved in any process.

　　Currently available tools for studying zebrafish autophagy: genetically modified and mutant zebrafish strains: In view of the genetic and physiological similarities between humans and zebrafish, several transgenes targeting key autophagy and bone genes have been developed in zebrafish And mutant fish. Although these strains have been widely used in research in their respective fields, there are far fewer studies that combine autophagy strains with skeletal zebrafish strains. The first transgenic autophagy reporter genes produced in zebrafish were the GFP-Map1Lc3 and GFP-Gabarap transgenic lines, whose reporter genes were expressed under the control of the constitutive cytomegalovirus (CMV) promoter. Both Map1Lc3 and Gabarap are homologs of yeast Atg8, and each form a subfamily of proteins in mammals and fish. In mammalian cells, MAP1LC3 and GABARAP family members act synergistically to enable autophagosome formation and/or substance recognition. Therefore, measuring autophagy in vivo and in vitro is equally useful. In general, Map1Lc3 is the most widely used marker for identifying and visualizing autophagy activity.

　　The GFP-Map1Lc3 transgenic zebrafish strain has been used in many studies to explore the role of autophagy in bacterial clearance, blastocyst formation after fin removal, and liver homeostasis. We were able to determine that during development, zebrafish showed high expression of GFP-Map1Lc3 around the joints, and compared with surrounding cells, cells in the joint space specifically showed increased expression of GFP-Map1Lc3. Taking into account the optical clarity of zebrafish, these fish can be imaged in real time under anesthesia, and the expression of Map1lc3 can be tracked throughout the development of the same fish. In addition, by using a skeletal cell-specific transgenic line, the expression of GFP-Map1Lc3 can be correlated with specific cell types. For example, transgenic lines expressing Col10a1 or sp7 can be used to label osteoblasts at different stages of differentiation; transgenic GFP trap fish can be used to label osteoclasts; and type II collagen fluorescent labeling can be used to monitor the development and formation of chondrocytes and cartilage.

　　Bone repair and regeneration measurement: Since zebrafish's fins and scales can be obtained through optical means throughout adulthood, zebrafish provide a useful tool for dynamic observation of bone regeneration and bone repair related factors. Similar to other bony animals, zebrafish can regenerate certain parts of the body after injury. In the fins, this regeneration process is relatively fast. Within two weeks, all major tissues, including bones, joints, and nerves, are basically restored. Varga et al. used the fin regeneration test to explore the role of autophagy in this process and showed that the genetic and pharmacological inhibition of autophagy can impair fin regeneration. This highlights the important role of autophagy in tissue construction and renewal. Further research on this process may help determine how to use autophagy to promote the renewal and replacement of bone and cartilage cells, especially during aging.

　　Using the GFP-Map1Lc3 transgenic zebrafish strain to study the role of autophagy in fin fracture repair and bone regeneration-top, schematic depicting how to perform fracture repair and regeneration assays in zebrafish, and how to use Alizarin Red in adult zebrafish Perform live bone staining. At the bottom, a fluorescent stereo microscope image of a fracture repair assay performed in a transgenic CMV: EGFP-map1lc3b zebrafish 6 months after fertilization was stained with Alizarin Red (red) in vivo.

　　Zebrafish are also a useful model for fracture repair research because they show that the fracture healing response is accompanied by the formation of callus, which is very similar to mammals. Using the GFP-Map1Lc3 line and living bone staining Alizarin Red, we were able to analyze fin fractures, and the results showed that Map1Lc3 expression increased in the early stages of fracture repair. This indicates that autophagy activity may be up-regulated in the repair response, and suggests the role of autophagy in bone repair.

　　Future direction and conclusion: Existing studies have used zebrafish as a powerful model for studying vertebrate development and simulating genetic diseases (including various bone diseases). Zebrafish has only recently been used in autophagy research, but it is still growing. It takes advantage of the ease of use and flexibility of the real-time imaging options provided by zebrafish. Commonly used rodent autophagy models are simply incomparable. Preliminary studies using the zebrafish autophagy model have provided new perspectives on the role of autophagy in skeletal cell differentiation and function, bone repair and regeneration, and drug development.