A Novel Zebrafish ret Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for mapk10 as a Modifier of Enteric Nervous System Phenotype Severity.
A Novel Zebrafish ret Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for mapk10 as a Modifier of Enteric Nervous System Phenotype Severity.
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DOI：
10.1371/journal.pgen.1006439
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发表时间：
2016-11
期刊：
影响因子：
4.5
通讯作者：
Pachnis V
中科院分区：
文献类型：
--
作者：
Heanue TA;Boesmans W;Bell DM;Kawakami K;Vanden Berghe P;Pachnis V
先天性巨结肠(HSCR)是以远端结肠无肠神经元和严重的肠道运动障碍为特征的疾病。为了了解HSCR的病理生理学和遗传学，我们开发了一种独特的斑马鱼模型，允许结合遗传、发育和体内生理研究。我们发现，RET突变体斑马鱼表现出HSCR的细胞、生理和遗传特征，包括缺乏肠道神经元，蠕动减少，以及杂合状态下不同的表型表达。我们使用UAS-GAL4二元遗传系统进行实时成像实验，以驱动ENS前体细胞中荧光蛋白的表达。我们证明ENS祖细胞在RET杂合子胚胎中迁移的速度减慢，而在增殖或存活方面没有变化，这确立了这是远端无神经节细胞增多症的主要致病机制。我们使用实际肠道运动的实时成像显示，在ret突变体中，肠道运动严重受损，在ret杂合子幼虫中部分受损，并在神经元位置和有组织的肠道运动之间建立了明确的相关性。我们利用部分穿透性ret杂合子表型作为敏感背景来测试候选修饰基因的影响。我们产生了Mapk10功能缺失突变体，显示出肠道神经元数量减少。值得注意的是，我们发现在ret杂合子中引入MAPK10突变增强了ENS缺陷，支持MAPK10作为HSCR易感基因。我们的研究表明，Rit杂合斑马鱼是一种致敏模型，与现有的小鼠模型相比具有许多显著的优势，可以探索HSCR的病理生理学和复杂的遗传学。先天性巨结肠(HSCR)是一种常见的先天性肠道动力障碍，出生时就被诊断为远端肠道缺乏肠神经元，导致肠梗阻，需要救命手术。HSCR表现出复杂的遗传模式，其遗传学基础尚不完全清楚。尽管人类遗传学家对此进行了很好的研究，并用小鼠进行了建模，但关于这种疾病的细胞和遗传原因，以及神经元丢失和肠道运动障碍之间的关系，仍然存在重大问题。在这里，我们使用可接近的、透明的斑马鱼来解决这些悬而未决的问题。我们发现，RET突变体斑马鱼表现出HSCR的关键特征，包括缺乏肠道神经元，肠道动力降低和表型表达不同。利用活体成像，我们证明了肠道神经前体细胞定植于肠道的迁移速度减慢是导致神经元缺陷的主要缺陷。斑马鱼可能有这种成像，但老鼠没有。通过对斑马鱼幼体肠道运动的直接检测，我们在神经元和运动模式之间建立了明确的相关性。最后，我们发现MAPK10突变恶化了ret突变体的肠神经元缺失，这表明MAPK10突变可能增加了对HSCR的易感性。我们展示了在斑马鱼中模拟人类遗传病的许多好处，并增进了我们对HSCR的理解。
Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR. Hirschsprung Disease (HSCR) is a common congenital intestinal motility disorder diagnosed at birth by absence of enteric neurons in the distal gut, leading to intestinal obstruction that requires life-saving surgery. HSCR exhibits complex inheritance patterns and its genetic basis is not fully understood. Although well studied by human geneticists, and modelled using mouse, significant questions remain about the cellular and genetic causes of the disease and the relationship between neuron loss and defective intestinal motility. Here we use accessible, transparent zebrafish to address these outstanding questions. We establish that ret mutant zebrafish display key features of HSCR, including absence of intestinal neurons, reduced gut motility and varying phenotype expressivity. Using live imaging, possible in zebrafish but not in mouse, we demonstrate that decreased migration speed of enteric neuron progenitors colonising the gut is the principal defect leading to neuron deficits. By direct examination of gut motility in zebrafish larvae, we establish a clear correlation between neurons and motility patterns. Finally, we show that mapk10 mutations worsen the enteric neuron deficit of ret mutants, indicating that mutations in MAPK10 may increase susceptibility to HSCR. We show many benefits of modelling human genetic diseases in zebrafish and advance our understanding of HSCR.
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