Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, Syracuse, NY 13210, USA.
Development. 2011 Jan;138(1):45-54. doi: 10.1242/dev.052985. Epub 2010 Nov 23.
The vertebrate body plan features a consistent left-right (LR) asymmetry of internal organs. In several vertebrate embryos, motile cilia generate an asymmetric fluid flow that is necessary for normal LR development. However, the mechanisms involved in orienting LR asymmetric flow with previously established anteroposterior (AP) and dorsoventral (DV) axes remain poorly understood. In zebrafish, asymmetric flow is generated in Kupffer's vesicle (KV). The cellular architecture of KV is asymmetric along the AP axis, with more ciliated cells densely packed into the anterior region. Here, we identify a Rho kinase gene, rock2b, which is required for normal AP patterning of KV and subsequent LR development in the embryo. Antisense depletion of rock2b in the whole embryo or specifically in the KV cell lineage perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ LR asymmetries. Analyses of KV architecture demonstrated that rock2b knockdown altered the AP placement of ciliated cells without affecting cilia number or length. In control embryos, leftward flow across the anterior pole of KV was stronger than rightward flow at the posterior end, correlating with the normal AP asymmetric distribution of ciliated cells. By contrast, rock2b knockdown embryos with AP patterning defects in KV exhibited randomized flow direction and equal flow velocities in the anterior and posterior regions. Live imaging of Tg(dusp6:memGFP)(pt19) transgenic embryos that express GFP in KV cells revealed that rock2b regulates KV cell morphology. Our results suggest a link between AP patterning of the ciliated Kupffer's vesicle and LR patterning of the zebrafish embryo.
脊椎动物的身体形态具有内部器官一致的左右（LR）不对称性。在几种脊椎动物胚胎中，运动纤毛产生不对称的流动，这对于正常的 LR 发育是必要的。然而，用于定向具有先前建立的前后（AP）和背腹（DV）轴的 LR 不对称流动的机制仍知之甚少。在斑马鱼中，不对称流是在 Kupffer 囊（KV）中产生的。KV 的细胞结构沿 AP 轴是不对称的，更多的纤毛细胞密集地排列在前部区域。在这里，我们鉴定了一个 Rho 激酶基因 rock2b，它是 KV 的正常 AP 模式形成和随后胚胎中 LR 发育所必需的。在整个胚胎或特异性地在 KV 细胞谱系中反义耗尽 rock2b 扰乱了侧中胚层的不对称基因表达，并破坏了器官的 LR 不对称性。对 KV 结构的分析表明，rock2b 敲低改变了纤毛细胞的 AP 位置，而不影响纤毛数量或长度。在对照胚胎中，左向流动穿过 KV 的前极强于右向流动在后端，与纤毛细胞的正常 AP 不对称分布相关。相比之下，KV 中具有 AP 模式缺陷的 rock2b 敲低胚胎表现出随机的流动方向和前区和后区的相等流动速度。Tg（dusp6：memGFP）（pt19）转基因胚胎的活体成像，该胚胎在 KV 细胞中表达 GFP，揭示了 rock2b 调节 KV 细胞形态。我们的结果表明，纤毛 Kupffer 囊的 AP 模式形成与斑马鱼胚胎的 LR 模式形成之间存在联系。
