Abstract
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 Tg(sox17: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.
Highlights
Vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis
Klf8 deficiency affects morphogenesis of Kupffer’s vesicle and asymmetric charon expression Since asymmetric flow, generated by rotation of cilia within KV, is essential to initiate left-right asymmetric patterning, and KV is derived from dorsal forerunner cells (DFCs), we investigated whether klf8 knockdown affected cilia or DFC number during KV morphogenesis
The majority (61% for MO1atg, 57% for MO2atg) of embryos injected with different klf8-Morpholino oligomer (MO) revealed symmetric charon expression with reduced expression area around KV as compared to wild type and control embryos at the 10s stage (Additional file 4: Figure S4). These results indicate that KV morphogenesis, cilia length, cilia number and asymmetric charon expression were affected in klf8 knockdown embryos
Summary
Vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Spaw stimulates transcription of itself and downstream genes, including lft, lft, and pitx, in the left side of the diencephalon, heart and LPM This developmental step is essential to establish subsequent asymmetric organ positioning. In order to study the various processes that establish left-right asymmetry in a laboratory setting, several vertebrates, including mice and zebrafish, have been utilized Based on these animal studies, the major developmental processes which establish asymmetry are known to include: symmetry-breaking in the node, the transfer of asymmetric Nodal expression from the node to the left lateral plate mesoderm (LPM), asymmetric expression of Nodal and downstream genes in the left LPM, and the completion of left-right asymmetric organ morphogenesis [3, 4]. Selfenhancement and lateral-inhibition systems involving NODAL, LEFTY1 and LEFTY2 reinforce the asymmetric distribution and restrict Nodal gene expression to the left side of the organism [9]
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