Klf8 regulates left-right asymmetric patterning through modulation of Kupffer’s vesicle morphogenesis and spaw expressionReport as inadecuate

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer’s vesicle morphogenesis and spaw expression - Download this document for free, or read online. Document in PDF available to download.

Journal of Biomedical Science

, 24:45

First Online: 17 July 2017Received: 05 April 2017Accepted: 07 July 2017


BackgroundAlthough 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.

MethodsZebrafish 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 Tgsox17:gfp embryos and the length and number of cilia in Kupffer’s vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody.

ResultsHeart 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.

ConclusionsOur results demonstrate that zebrafish Klf8 regulates left-right asymmetric patterning by modulating both Kupffer’s vesicle morphogenesis and spaw expression in the left LPM.

KeywordsZebrafish Klf8 Spaw L-R patterning Kupffer’s vesicle AbbreviationsASELeft-side specific enhancers

CRISPR-CasClustered regularly interspaced short palindromic repeats- CRISPR-associated system

CtBPC-terminal binding protein

DFCsDorsal forerunner cells

HpfHours post fertilization

klf8krüppel-like factor 8

KVKupffer’s vesicle



LPMLateral plate mesoderm

MOMorpholino oligomer

NdrNodal-related genes

RTRoom temperature

RT-PCRReverse transcription PCR

RT-qPCRReverse transcription quantitative real-time PCR


Electronic supplementary materialThe online version of this article doi:10.1186-s12929-017-0351-y contains supplementary material, which is available to authorized users.

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Author: Che-Yi Lin - Ming-Yuan Tsai - Yu-Hsiu Liu - Yu-Fen Lu - Yi-Chung Chen - Yun-Ren Lai - Hsin-Chi Liao - Huang-Wei Lien - Chu

Source: https://link.springer.com/

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