Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
Present address: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
J Biomed Sci. 2017 Jul 17;24(1):45. doi: 10.1186/s12929-017-0351-y.
Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos.
Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody.
Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed. Interestingly, klf8 mutant embryos, generated by a CRISPR-Cas9 system, showed bilateral spaw expression in the LPM at late somite stages. This observation may be partly attributed to compensatory upregulation of klf12b, because klf12b knockdown reduced the percentage of klf8 mutants exhibiting bilateral spaw expression.
Our results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer's vesicle morphogenesis and spaw expression in the left LPM.
尽管脊椎动物是左右对称的生物体，但它们的内部器官沿左右轴呈不对称分布。左右轴不对称模式的破坏经常发生在人类遗传疾病中。在斑马鱼胚胎中，类似于小鼠节点的 Kupffer 泡通过在左侧侧板中诱导 Nodal 相关基因 spaw 的不对称表达来打破对称性中胚层（LPM）。然后，Spaw 刺激自身和下游基因的转录，包括 lft1、lft2 和 pitx2，专门在大脑半球、心脏和 LPM 的左侧。这一发育步骤对于建立随后的不对称器官定位至关重要。在这项研究中，我们评估了 Krüppel 样因子 8（klf8）在调节斑马鱼胚胎左右不对称模式中的作用。
通过 morpholino 反义寡核苷酸介导的敲低和 CRISPR-Cas9 系统破坏斑马鱼 klf8 的表达。进行全胚胎原位杂交以评估 Nodal 信号成分的基因表达模式以及心脏和内脏器官的位置。在 Tg(sox17:gfp)胚胎中评估背侧前体细胞数量，并通过使用乙酰化微管蛋白抗体的免疫细胞化学分析分析 Kupffer 泡中纤毛的长度和数量。
斑马鱼 klf8 形态发生缺陷的心脏跳动、环和内脏器官定位均存在缺陷。在 18-22 个阶段，klf8 形态发生缺陷的胚胎中，左侧 LPM、大脑半球和心脏中编码 Nodal 信号成分（spaw、lft1、lft2 和 pitx2）的基因表达减少。klf8 mRNA 与 klf8 morpholino 的共注射部分挽救了 spaw 的表达。此外，klf8 但不是 klf8△zf 过表达胚胎在晚期体节阶段表现出 Nodal 信号成分的双侧表达失调。在 10 个阶段，klf8 形态发生缺陷的胚胎中，Kupffer 泡中的纤毛长度和数量减少，而在 75%的胚环期，观察到更少的背侧前体细胞。有趣的是，通过 CRISPR-Cas9 系统产生的 klf8 突变体胚胎在晚期体节阶段在 LPM 中表现出双侧 spaw 表达。这一观察结果可能部分归因于 klf12b 的代偿性上调，因为 klf12b 的敲低降低了表现出双侧 spaw 表达的 klf8 突变体的百分比。
我们的结果表明，斑马鱼 Klf8 通过调节 Kupffer 泡形态发生和左侧 LPM 中 spaw 的表达来调节左右不对称模式。
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