Adams Dany S, Robinson Kenneth R, Fukumoto Takahiro, Yuan Shipeng, Albertson R Craig, Yelick Pamela, Kuo Lindsay, McSweeney Megan, Levin Michael
The Forsyth Center for Regenerative and Developmental Biology, and Department of Developmental Biology, Harvard School of Dental Medicine, 140 The Fenway, Boston, MA 02115, USA.
Development. 2006 May;133(9):1657-71. doi: 10.1242/dev.02341. Epub 2006 Mar 22.
Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H+ flux. A pharmacological screen implicated the H+-pump H+-V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H+-V-ATPase subunits during the first three cleavages. H+-flux across plasma membranes is also asymmetric at the four- and eight-cell stages, and this asymmetry requires H+-V-ATPase activity. Abolishing the asymmetry in H+ flux, using a dominant-negative subunit of the H+-V-ATPase or an ectopic H+ pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H+-V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (Vmem) regulation. Varying either pH or Vmem, independently of direct manipulation of H+-V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H+-V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H+-V-ATPase induces heterotaxia in both species; in chick, H+-V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H+-V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH- and Vmem-dependent model of the early physiology of LR patterning.
左右不对称偏差是一种引人入胜且在医学上具有重要意义的现象。我们对一种新的、保守的、早期的生物物理事件进行了分子遗传学和生理学特征描述，该事件对于正确的不对称性至关重要：氢离子通量。一项药理学筛选表明，氢离子泵H⁺-V-ATP酶与非洲爪蟾的不对称性有关，它在早期不对称标记物的上游发挥作用。免疫组织化学显示，在最初的三次卵裂过程中，H⁺-V-ATP酶亚基存在肌动蛋白依赖性不对称性。在四细胞和八细胞阶段，跨质膜的氢离子通量也是不对称的，这种不对称性需要H⁺-V-ATP酶的活性。使用H⁺-V-ATP酶的显性负性亚基或异位氢离子泵消除氢离子通量的不对称性，会使胚胎内脏位置随机化，且不会导致任何其他缺陷。为了理解H⁺-V-ATP酶的作用机制，我们分离了它的两种生理功能，即细胞质pH值调节和膜电压（Vmem）调节。独立于对H⁺-V-ATP酶的直接操作来改变pH值或Vmem，都会导致正常不对称性的破坏，这表明这两种功能都发挥了作用。V-ATP酶抑制也消除了血清素正常的早期定位，从功能上连接了这两条早期不对称途径。H⁺-V-ATP酶在不对称性中的作用在鸡和斑马鱼中是保守的。抑制H⁺-V-ATP酶会在这两个物种中诱导内脏反位；在鸡中，H⁺-V-ATP酶活性在音猬因子（Shh）的上游；在鱼中，它在库普弗囊泡和无翅型MMTV整合位点家族成员a（Spaw）表达的上游。我们的数据表明H⁺-V-ATP酶活性在构建脊椎动物左右轴中发挥作用，并揭示了其活性的上游和下游机制。我们提出了一种依赖于pH值和Vmem的左右模式形成早期生理学模型。
