细胞极化对多种细胞的发育和功能的正确行使都至关重要。极化的目的在于打破细胞形态和功能上的对称性，从而更好地响应细胞内外的信息流。神经元是生物体内极化程度最高的细胞类型之一，神经元极化的形成和维持，是调控神经发生、再生以及塑型的基础，因此对于神经系统的发育至关重要。在整个神经极化过程中，任一阶段受损都可能会导致神经发育障碍或神经退行等神经系统疾病的发生。 轴突是神经元极性的主要标志之一，同时也是神经元形态和功能的基础。轴突的发育过程一般可以分为三个阶段，包括轴突发生，轴突生长和导向，以及轴突延伸和分支等。神经元将电信号沿轴突传播到突触，通过释放神经递质将信号传播到另一神经元或效应细胞，例如肌肉或腺体，所以轴突不仅介导了不同神经元之间的信息传递，还能介导神经元与效应细胞的信息传递，因此轴突发育的异常除了可能导致阿尔茨海默症、帕金森综合征等神经退行性疾病，还会导致神经肌肉疾病的发生。 神经肌肉疾病(NMDs)是一类肌肉和周围神经系统病变的疾病，主要影响细胞体位于脊髓前角的运动神经元和细胞体位于背根神经节以接受传入纤维的感觉神经元，以及肌肉本身或者神经肌肉接头的功能。斑马鱼是一种新兴的模式动物，将其作为研究神经肌肉疾病的动物模型具有很多显而易见的优点。首先斑马鱼脊髓运动神经元轴突发育是一个非常严谨的过程。基于它们何时分化并支配它们的靶肌肉组织，可分为初级运动神经元(PMN)和次级运动神经元(SMN)。PMN较大，在原肠胚形成过程中出现，并在斑马鱼发育的第一天出现轴突发生。通过它们细胞体的位置，轴突轨迹和膜电位特性可以在每个脊髓半球中鉴定出三到四个单独的PMN，分别是：骶管初级运动神经元(CaP MN)，主要支配腹主干肌肉组织；中间初级运动神经元(MiP MN)，主要支配背部躯干肌肉组织；延髓初级运动神经元(RoP MN)，主要支配前两者之间的肌纤维。第二，斑马鱼的基因组和蛋白组和人类相比具有较高的同源性和功能的保守性。第三，斑马鱼胚胎体外发育，早期发育过程胚胎透明可见，并且发育周期短，繁殖能力强。最后，目前斑马鱼研究的技术方法正在迅速的丰富和发展，比如高分辨率的活体成像系统，更多样的运动行为学测试以及更高效的大批量动物化学药物筛选等技术方法正在获得广泛应用。这些优势不仅有助于探究神经肌肉疾病的起源和机理，还有助于更有效的获得用于治疗人类疾病的药物。 先天性肌无力综合征(CMS)和遗传性痉挛性截瘫(HSP)是两种典型的神经肌肉疾病，因此我们的工作分为两个部分，分别以斑马鱼为模式动物，研究了GMPPB和ENC1两个基因对脊髓运动神经元轴突发育的影响，进而探索了这两类疾病CMS和HSP的发病机理以及可能的治疗方案。CMS是由一些对维持神经肌肉信号传导至关重要的基因发生突变，从而引起的一类异质遗传性疾病，与之类似的还有先天性肌营养不良症(CMD)和肢带肌营养不良症(LGMD2T)等。CMD是骨骼肌的遗传性疾病，其特征为出生的头两年内出现肌肉张力减退和虚弱，大肌肉群活动能力获得延迟，同时在骨骼肌组织活检中出现营养不良特征。这类疾病表型多样，且都具有遗传异质性。LGMD2T是由FKRP基因突变引起的常染色体隐性肌营养不良症。FKRP编码一个可能在高尔基体上驻留的糖基转移酶fukutin相关蛋白，参与β-葡糖糖基化过程。CMS， CMD以及LGMD2T之间并没有明确的界限，在致病基因，临床表现和组织病理学特征上部有重叠，而GMPPB恰好是这三者的重叠致病基因。 GMPPB全称是鸟苷二磷酸甘露糖焦磷酸化酶B，它是一种参与生物体内糖基化作用的重要的酶，能够催化三磷酸鸟苷(GTP)和甘露糖-1-磷酸形成鸟苷二磷酸甘露糖(GDP-甘露糖)。GDP-甘露糖是多重糖基化反应的结构单元，是包括α-肌营养不良蛋白聚糖(α-DG)在内的蛋白质的O-甘露糖基化所必需的，并且它是胞浆内甘露糖基转移酶的底物。在斑马鱼中抑制gmppb的表达会引起肌肉和中枢神经系统异常，并且α-DG的糖基化减少。 目前关于GMPPB突变导致的CMS，CMD以及LGMD2T的报道有很多，但是大都集中在新的突变位点的发现以及临床表型的描述上，对其在轴突发育过程中的作用以及突变致病机理和相应的治疗方案尚无深入研究。我们的工作首先鉴定了GMPPB两个新的突变位点，并且发现在斑马鱼中抑制gmppb的表达会导致其脊髓运动神经元轴突发育的异常，主要表现为轴突生长受到抑制。进一步的工作表明GMPPB基因不同位点的突变会导致其活性不同程度的降低，并且活性的下降程度与病人的症状严重程度相关，在斑马鱼中过表达突变体的mRNA不能恢复抑制gmppb表达所造成的轴突生长抑制和肌肉纤维紊乱，而直接补充其催化代谢产物GDP-甘露糖，能够恢复其神经肌肉发育障碍。 HSP是一种描述遗传性疾病的综合征，主要症状为下肢无力和痉挛。目前关于HSP已经鉴定出了超过50种遗传类型。在之前的一些研究中发现，病人发病后均会出现皮质脊髓束轴突的变性和纤维束纤维变性，因此HSP可能涉及了运动感觉神经元轴突变性。由HSP基因编码的蛋白质具有非常多样的功能，主要包括：轴突运输、内质网(ER)形态建成、线粒体功能、髓磷脂形成、蛋白质折叠和ER应激反应、皮质脊髓束和其他神经发育，脂肪酸和磷脂代谢以及胞内体膜运输和囊泡形成等。尽管有大量不同关基因编码各种蛋白，但是有超过50％的HSP患者中存在以下3个基因中的一个具有致病性突变：spastin，atlastin-1，REEP1。 Atlastin-1是发动蛋白相关GTP酶家族中的一员，主要在真核生物管状内质网中发挥功能，它能够介导内质网微管的同型融合从而形成三路连接。在斑马鱼中抑制atlastin1的表达会导致斑马鱼运动能力的严重下降，脊髓运动神经元轴突结构异常，并且与BMP信号通路的显著上调相关。Ectoderm-Neural Cortex-1(encl)是一个在神经系统特异表达的基因，主要包括三个结构域，即BTB／POZ结构域，BACK结构域和Kelch结构域。之前的研究表明ENCl在PC12细胞中能够促进细胞分化以及轴突形成，而在胶质母细胞瘤和星形细胞瘤中的表达量都显著上升，最近的研究还表明，ENCl可能还与神经细胞凋亡以及内质网应激相关。 尽管有越来越多的证据表明ENCl在神经系统中发挥重要功能，但是关于ENCl和Atlastin1之间的关系尚无报道。前期的工作中我们发现斑马鱼encl和atlastin1之间存在相互作用，但是两者如何相互作用，并且怎样共同调控斑马鱼脊髓运动神经元轴突分支形成尚不清楚，现在我们发现，在斑马鱼中抑制encl的表达同样导致脊髓运动神经元轴突分支增多，斑马鱼运动能力下降，并且过表达encl能够恢复抑制atlastinl造成的表型。同时我们将两者相互作用的结合区域范围缩小到了50个氨基酸，atlastin1控制管状内质网的结构和高尔基体的形态建成，并且在斑马鱼神经元中和Rab7共定位，encl则可能影响了高尔基体的形态和Rab7的分布，在调控脊髓运动神经元轴突发育的过程中encl在atlastin1下游，两者可能在细胞内经过Rab7标记的晚期胞内体短暂的相互作用，共同介导了细胞内由内质网到高尔基体的囊泡运输过程。 主要结果： 1.GMPPB突变导致的先天性肌无力综合征在斑马鱼中的机制研究与治疗 1.1 病人体内鉴定的GMPPB突变及其临床表型 我们通过全外显子测序技术，在病人身上鉴定出了两个新的GMPPB突变640G>A和332T>G，病人的主要临床表现为小脑发育不全，四肢抽搐等。 1.2 Gmppb在斑马鱼中的时空表达 我们发现Gmppb能够在斑马鱼胚胎早期发育阶段各个时期中表达，而且在神经和肌肉系统特异性表达，因此gmppb在斑马鱼早期发育过程中可能发挥重要作用。 1.3 在斑马鱼中抑制gmppb表达造成脊髓运动神经元轴突变短 我们发现在斑马鱼中通过注射Morpholino(MO)的方式抑制gmppb的表达，能够导致其脊髓运动神经元轴突长度缩短，并且脊髓位置HuC:GFP阳性的细胞数显著下降，因此gmppb影响了斑马鱼神经系统的发育，这和我们在病人身上观察到的临床表现是一致的。 1.4 抑制gmppb表达导致斑马鱼肌肉纤维紊乱，运动能力下降 我们发现在斑马鱼中抑制gmppb的表达不仅会影响其脊髓运动神经元轴突的正常形成，而且会导致其肌肉组织形态异常，并最终影响其运动能力，这和我们在临床病人身上发现的各项病征一致，因此斑马鱼对于我们研究gmppb突变导致的先天性肌无力综合征是一种理想的动物模型。 1.5 GMPPB突变体蛋白的活性显著下降 我们的合作研究者通过酶活性检测实验发现，在V111G和G214S这两个突变中，V111G活性显著下降。在进一步比较多个文献中报道的突变体活性之后我们发现，GMPPB活性下降越严重的突变，其对应的临床症状也越严重。因此GMPPB突变导致的蛋白活性下降是疾病发生直接原因。 1.6 在斑马鱼中补充GMPPB V111G不能恢复MO造成的表型 我们在体外转录了GMPPB野生型及其突变体的mRNA并且与gmppb的MO共注射，结果不管是神经元轴突还是肌肉纤维，过表达V111G突变体mRNA均不能恢复MO造成的表型。这表明蛋白活性的下降确实影响了gmppb正常的生理功能。 1.7 补充GDP-甘露糖能够恢复gmppb MO造成的表型 将GDP-甘露糖溶解在水中孵育注射了MO的斑马鱼不能使其恢复，而直接注射GDP-甘露糖才能够达到恢复的效果，证明了通过补充GMPPB催化代谢产物GDP-甘露糖，能够弥补GMPPB的功能缺失，同时由于GDP-甘露糖不能直接被斑马鱼吸收，因此需要开发更加直接有效的给药方式以达到对疾病治疗的效果。 2.Encl和Atlastin1相互作用共同调控斑马鱼运动神经元轴突分支形成 2.1 斑马鱼encl影响脊髓运动神经元轴突发育 通过在斑马鱼中注射MO抑制encl的表达，我们发现encl影响了斑马鱼脊髓运动神经元轴突的发育，表现为神经元轴突分支增多。并且encl的BTB／POZ结构域以及Kelch结构域等均参与了轴突发育调控， encl结构的完整性是运动神经元轴突正常发育的基础。 2.2 抑制encl的表达导致斑马鱼运动能力下降 通过“接触-反应”实验评估抑制encl表达后斑马鱼运动能力。结果发现其移动距离较对照组显著缩短。因此，抑制encl的表达导致斑马鱼运动能力的下降。 2.3 Encl在atlastin1下游参与调控运动神经元轴突发育 通过向Hb9:GFP转基因斑马鱼中注射MO分别抑制encl和atlastin1的表达，我们发现两者都会导致斑马鱼脊髓运动神经元轴突的分支增多，其中抑制atlastin1还会导致斑马鱼体轴的弯曲，而过表达encl mRNA则能够恢复atlastin1 MO造成的表型。因此，ericl可能在atlastin1的下游参与了对脊髓运动神经元轴突分支的调控。 2.4 Encl和atlastin1存在蛋白质之间相互作用 通过IP实验我们发现encl能够特异性的和atlastin1相互作用。目前我们将两者的结合区域分别缩小到了encl蛋白的BACK功能域的前50个氨基酸以及atlastin1的GTP酶功能域。 2.5 Encl和atlastin1相互作用的细胞生物学机制 我们在细胞内特异性表达了encl蛋白，免疫荧光染色的结果表明，细胞内encl的弥散分布与actin共定位，而囊泡样分布在部分情况下和高尔基体标志Giantin存在共定位，也能和晚期胞内体Rab7共定位， Encl和atlastin1之间仅存在小部分的共定位。在斑马鱼脊髓运动神经元中，抑制encl的表达导致Rab7在轴突起始位置的堆积。 关键词：GMPPB ENCl Atlastinl 轴突 先天性肌无力综合征 遗传性痉挛性截瘫

Cell polarization is critical to the proper functioning of many cell types. Neurons are one of the cell types with the highest degree of polarization in vivo. The purpose of polarization is to break the symmetry of cell morphology and function, so as to better respond to intra- and extra-cellular information flow. The formation and maintenance of neuronal polarization is the basis for regulation of neurogenesis, regeneration, and plasticity, and is therefore essential for the development of the nervous system. During the entire process of neuropolarization, damage at any stage may cause neurological disorders such as neurodevelopmental disorders or neurodegeneration. Axon is one of the main signs of neuronal polarity, and it is also the basis of neuronal morphology and function. Axon development can generally be divided into three stages, including axonogenesis, axonal growth and guidance, and axon elongation and branching. Neurons propagate electrical signals along the axons and extend them to synapses. By releasing neurotransmitters to transmit signals to another neuron or effector cells, such as muscles or glands. Axons not only mediate the transmission of information between different neurons, but also mediate the transmission of information between neurons and effector cells such as muscles or glands. Therefore, abnormal development of axons may not only lead to neurodegenerative diseases such as Alzheimer's disease and Parkinson's syndrome, but also cause neuromuscular diseases. Neuromuscular disorders (NMDs) are a class of diseases of the muscle and peripheral nervous system that mainly affect motoneurons whose cell bodies are located in the anterior horn of the spinal cord. The cell bodies are located in the dorsal root ganglion to accept sensory neurons of the afferent fibers, and the muscle itself or the function of the neuromuscular junction. The use of zebrafish as a model for the study of the pathogenesis of neuromuscular diseases has many obvious advantages. Firstly, axonal development of zebrafish spinal motor neurons is a very rigorous process. Based on when they differentiate and control their target muscle tissue, they can be divided into primary motor neurons (PMNs) and secondary motor neurons (SMNs). . Primary motor neurons are larger and appear during gastrulation and axons occur on the first day of development. By their cell body location, axon loci and membrane potential characteristics can identify three to four separate PMNs in each spinal hemisphere, namely: primary motor neurons of the fistula (CaP MN), which dominate the abdominal trunk muscle tissue; Intermediate primary motoneurons (MiP MNs) innervate the back trunk muscle tissue; medulla oblongata motor neurons (RoP MN) dominate the muscle fibers between the formers. Second, the zebrafish genome and Proteome have high homology and functional conservation compared to humans. Third, zebrafish embryos develop in vitro. The embryos in the early stage of development are transparent and have short development cycle and strong reproductive capacity. Finally, the current zebrafish research methods are rapidly developing and enriching, such as high resolution in vivo imaging systems, kinesiology tests, and convenience for screening and testing of large numbers of animals for chemical drugs.These not only help to explore the origin and mechanism of neuromuscular diseases, but also help to obtain effective drugs for the treatment of human diseases through screening. Congenital myasthenic syndrome (CMS) and hereditary spastic paraplegia (HSP) are two typical neuromuscular diseases. Therefore, our work is divided into two parts. Developmental disorders of axons in zebrafish motor neurons are as a starting point, the mechanism of this two type neuromuscular disease caused by GMPPB mutations and Atlastinl/ENCl mutations, respectively, was studied in depth. CMS is a type of heterogeneous genetic disease caused by mutations in a number of genes encoding proteins essential for maintenance of neuromuscular signaling, Similar to this is congenital muscular dystrophy (CMD) and limb muscular dystrophy (LGMD2T). CMD is a hereditary disease of skeletal muscle and is characterized by decreased muscle tone and weakness during the first two years of life, delays in the acquisition of mobility of large muscle groups, and malnutrition in skeletal muscle tissue biopsy. These diseases have multiple phenotypes and all have genetic heterogeneity. LGMD2T is an autosomal recessive muscular dystrophy caused by mutations in the FKRP gene. FKRP encodes a glycosyltransferase fukutin-rclated protein that presumably resides on the Golgi apparatus and is involved in beta-glucosylation. There are no clear boundaries between CMS, CMD, and LGMD2T. There are overlaps in disease genes, clinical manifestations, and histopathological features, and GMPPB happens to be an overlapping gene for these three genes. The full name of GMPPB is guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B, which is an important enzyme involved in glycosylation in vivo, and it can catalyze the formation of GTP and mannose-1-phosphate to form GDP- Mannose. GDP-mannose is a structural unit of a multiplexed glycosylation reaction and is required for O-mannosylation of a protein including α-DG, and it is a substrate of intracytoplasmic mannosyltransferase. Inhibition of gmppb expression in zebrafish causes abnormalities in the muscles and central nervous system, and glycosylation of α-dystrophin is reduced. At present, there are many reports on CMS, CMD and LGMD2T caused by GMPPB mutations, but mainly focused on the discovery of new mutation sites and the description of clinical phenotypes. There is no in-depth study on its pathogenic mechanism and corresponding treatment options. In our work, we not only detected two new mutation sites in clinical patients, but also found that inhibition of gmppb expression in zebrafish leads to abnormal development of axons in spinal cord motor neurons and inhibition of axonal growth. . We also found that mutations at different sites of the GMPPB gene resulted in different degrees of reduction in activity, and the degree of decline in activity was related to the severity of the patient's symptoms. Overexpression of mutant mRNA in zebrafish could not restore the inhibition of gmppb expression caused axon growth inhibition and muscle fiber disruption, and finally through the direct recruitment of its catalytic metabolite GDP-mannose, can restore inhibition of gmppb-induced neuromuscular developmental disorders in zebrafish. Hereditary spastic paraplegia (HSP) is a syndrome that describes a hereditary disease, in which lower limb weakness and delirium are the main symptoms. More than 50 genetic types of HSP have been identified so far. In previous studies, it was found that all patients had degeneration of the corticospinal tract axons and fiber bundle fibrosis. Therefore, HSP syndrome involves axonal degeneration of motor sensory neurons that mainly affect the distal axon of the central nervous system. The proteins encoded by HSP genes have a variety of functions, including: axonal transport, endoplasmic reticulum (ER) morphogenesis, mitochondrial function, myelin formation, protein folding and ER stress response, corticospinal tract and other neurodevelopment. Metabolism of fatty acids and phospholipids, and intracellular membrane trafficking and vesicle formation. Although there are a large number of different genes encoding related proteins, in more than 50% of HSP patients there is a pathogenic mutation in one of the following three genes: spastin, atlastin-1, and REEP1. The Atlastin-1 protein is a member of the activator-associated GTPase and functions mainly in the eukaryotic tubular endoplasmic reticulum. It can mediate isotypic fusion of endoplasmic reticulum microtubules and thus form a three-way linkage. Inhibition of atlastinl expression in zebrafish results in severely reduced zebrafish locomotor capacity and axonal abnormalities in spinal motor neurons and is associated with a significant upregulation of the BMP signaling pathway. In our previous work, we initially found a gene that is specifically expressed in the nervous system, Ectoderm-Neural Cortex-1 (encl), which mainly includes three domains, ie, the BTB/POZ domain, the BACK domain, and the Kelch domain. Previous studies have shown that ENC1 can promote cell differentiation and axon formation in PC 12 cells, and the expression levels in both glioblastoma and astrocytoma are significantly increased, indicating that there is a link between ENC1 and brain tumors. Recent studies have shown that ENC1 may also be associated with neuronal apoptosis and endoplasmic reticulum stress. Although more and more evidence shows that ENC1 plays an important role in the nervous system, there is no report about the relationship between ENC1 and Atlastinl. In the previous work, we found that there is an interaction between end and atlastinl, but how the two interact, and how to jointly regulate axonal branches of zebrafish spinal motor neurons is unclear. We have now found that inhibiting the expression of end and atlastinl in zebrafish both results in abnormal development of axons in spinal motor neurons, increased axon branching, and reduced zebrafish locomotor capacity, and overexpression of end can restore inhibition of the atlastinl phenotype. At the same time, we reduced the scope of the interaction between the two proteins to 50 amino acids. Atlastinl controls the structure of tubular endoplasmic reticulum and morphogenesis of the Golgi apparatus, and colocalizes with Rab7 in zebrafish neurons. Encl may affect the morphology of Golgi and the distribution of Rab7, end is still downstream of atlastinl, both of which may be intracellularly transiently interacted by Rab7-labeled late endosomes and mediate intracellular trafficking of vesicles from the endoplasmic reticulum to the Golgi apparatus. Results 1.The mechanism and treatment of congenital myasthenic syndrome caused by GMPPB mutation in zebrafish 1.1GMPPB mutations found in patients and their clinical phenoty pes. We identified two new GMPPB mutations, 640G﹥A and 332T﹥G, in the patient through full exon sequencing. The main clinical manifestations of the patient were cerebellar hypoplasia and limb twitching. 1.2Spatio-temporal expression of Gmppb in zebrafish We found that Gmppb can be expressed in various stages of the early development stage of zebrafish embryos, and is specifically expressed in the nervous and muscle systems. Therefore, gmppb may play an important role in the early development of zebrafish, so we will use zebrafish as a follow-up study animal. 1.3Inhibition of gmppb expression in zebrafish causes short axons in motor neurons We found that inhibiting the expression of gmppb in zebrafish by injection of Morpholino can lead to abnormal axon development in spinal motor neurons, shortening of axon length, and a significant decrease in the number of HuC: GFP positive cells at the spinal cord site. Therefore, gmppb does affects the development of the zebrafish nervous system, which is consistent with the clinical picture we have observed in patients. 1.4Inhibition of gmppb expression leads to zebrafish muscle fiber disturbances and decreased motor capacity We have found that inhibiting the expression of gmppb in zebrafish not only affects the normal formation of axons in spinal cord motor neurons, but also results in abnormal muscle tissue morphology and eventually affects their motor ability, which we found in clinical patients. All indications are consistent, so for us to cany out the study of congenital myasthenic syndrome caused by gmppb mutation, zebrafish is a very ideal animal model. 1.5The activity of the GMPPB mutant protein is significantly reduced Our co-investigators have discovered through enzyme activity assays that V111G activity is significantly reduced in the two mutations, V111G and G214S. After further comparing the mutant activity reported in several literatures, we found that the more severe the decline in GMPPB activity, the corresponding patient's symptoms are more severe. Therefore, the decrease of protein activity caused by GMPPB mutation is the direct cause of the disease. 1.6Supplementation of GMPPB V111G in zebrafish does not restore the phenotypes caused by MO We transfected the mRNA of the GMPPB wild-type and its mutants in vitro and co-injected with the gmppb MO. As a result, none of the V111G mutants could restore the phenotype caused by MO regardless of neuronal axons or muscle fibers. This shows that the decline in activity does affect the normal physiological function of gmppb. 1.7Supplementing GDP-mannose can restore the phenotype caused by gmppb MO The GDP-mannose was dissolved in water to incubate the zebrafish injected with MO and could not be recovered. The direct injection of GDP-mannose could achieve the recovery effect. It was proved that by supplementing GMPPB catalytic metabolite GDP-mannose, the lack of GMPPB function couble be restore, and because GDP-mannose can not be directly absorbed by the zebrafish, it is necessary to develop more direct and effective drug delivery methods to achieve the effect of disease treatment. 2.Encl and atlastinl regulate axonal branching in zebrafish motor neurons 2.1Zebrafish end affects axon development of spinal motor neurons By inhibiting the expression of end in zebrafish injected with Morpholino, we found that end does affect the development of axons of spinal motoneurons in zebrafish. Inhibition of end expression leads to an increase in axonal branches of neurons. And end BTB/POZ domain and Kelch domain are involved in the regulation of axon development. The integrity of end structure is the basis for the normal development of motor neuron axons. 2.2Inhibition of the expression of end leads to decreased zebrafish motility The zebrafish motility was assessed by "contact-response" experiments after inhibition of end expression. As a result, it was found that the moving distance was significantly shorter than that of the control group. Therefore, inhibition of the expression of end can lead to a decrease in zebrafish motility. 2.3Encl participates in the regulation of motor neuron axon development downstream of atlastinl By injecting Morpholino into the Hb9:GFP transgenic zebrafish to inhibit the expression of end and atlastinl, respectively, we found that both resulted in increased branching of axons in spinal motoneurons of zebrafish, and inhibition of atlastinl also caused bending of the zebrafish body axis. Overexpression of end mRNA restored the phenotype of atlastinl MO. We further confirmed this result by in situ hybridization experiments. Therefore, both may be involved in the regulation of axon branching of spinal cord motor neurons, and end may be downstream of atlastinl. 2.4Protein interaction between Encl and atlastinl Through IP experiments we found that end can specifically interact with atlastinl. Through further experiments, we have now reduced the binding region of the two to the first 50 amino acids of the BACK domain of end protein and the GTPase domain of atlastin 1. 2.5Cytobiological mechanism of interaction between Encl and atlastinl We specifically expressed end protein in cells. The results of immunofluorescence staining showed that the diffuse distribution of intracellular end was co-localized with actin, whereas the vesicle-like distribution was partially colocalized with the Golgi marker Giantin in some cases. It also colocalizes with the late endosome Rab7, and we have also validated it in different cell lines. There is only a small fraction of colocalizations between Encl and atlastinl. In zebrafish spinal motor neurons, inhibition of end expression results in the accumulation of Rab7 at the axon initiation site. Keywords: GMPPB ENC1 Atlastinl axon CMS HSP