Ramanoudjame Laetitia, Rocancourt Claire, Lainé Jeanne, Klein Arnaud, Joassard Lucette, Gartioux Corine, Fleury Marjory, Lyphout Laura, Kabashi Edor, Ciura Sorana, Cousin Xavier, Allamand Valérie
Sorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, Paris F-75013, France, Institut de Myologie, Paris F-75013, France.
Fish Ecophysiology Group, Ifremer, L'Houmeau F-17137, France.
Hum Mol Genet. 2015 Dec 1;24(23):6624-39. doi: 10.1093/hmg/ddv368. Epub 2015 Sep 11.
Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains.
胶原蛋白VI（COLVI）是一种在结缔组织中普遍表达的蛋白质，对结构完整性、细胞黏附、迁移和存活至关重要。在哺乳动物中已识别出六个不同的基因，它们编码六条COLVI链，这些链组装成两条“短”链（α1、α2）和一条“长”链（理论上为α3 - 6中的任何一条）。在人类中，由于COL6A1 - 3基因的突变，最广泛表达的异源三聚体（α123）出现缺陷，导致一组异质性的神经肌肉疾病，统称为COLVI相关肌肉疾病。对于最近描述的α4 - 6链的功能知之甚少，目前尚未检测到相关突变。在本研究中，我们鉴定了斑马鱼中两条与哺乳动物α4链同源性最高的新型COLVI长链；因此，我们将相应的基因命名为col6a4a和col6a4b。这些直系同源物代表了哺乳动物Col6a4 - 6基因 的祖先。通过原位杂交和RT - qPCR，我们发现col6a4b与其他col6a基因相比具有独特的表达动力学。使用针对col6a4a、col6a4b和col6a2的吗啉代反义寡核苷酸，我们分别模拟了部分和完全COLVI缺陷。所有吗啡啉处理的胚胎均呈现肌肉结构改变和运动能力受损。虽然细胞凋亡没有大幅增加，但所有吗啡啉处理的胚胎中自噬诱导均存在缺陷。此外，这些吗啡啉处理的胚胎中运动神经元轴突生长异常。重要的是，col6a4a和col6a4b吗啡啉处理的胚胎之间出现了一些表型差异，表明只有部分功能冗余。总体而言，我们的结果进一步证实了COLVI在斑马鱼肌肉发育中的重要性，并可能为与最近描述的COLVI链缺乏相关的潜在人类表型提供重要线索。
