Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression

doi:10.1186/s12929-017-0351-y

. 2017 Jul 17;24(1):45.

doi: 10.1186/s12929-017-0351-y.

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression

Che-Yi Lin^{1

2}, Ming-Yuan Tsai^{3

2}, Yu-Hsiu Liu⁴, Yu-Fen Lu⁵, Yi-Chung Chen⁵, Yun-Ren Lai¹, Hsin-Chi Liao¹, Huang-Wei Lien⁶, Chung-Hsiang Yang⁶, Chang-Jen Huang⁶, Sheng-Ping L Hwang^{7

8

9}

Affiliations

¹ Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
² Present address: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
³ Graduate Institute of Life Sciences, National Defence Medical Center, National Defence University, Neihu, Taipei, Taiwan.
⁴ Department of Life Science, National Taiwan University, Taipei, Taiwan.
⁵ Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan.
⁶ Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
⁷ Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan. zoslh@gate.sinica.edu.tw.
⁸ Department of Life Science, National Taiwan University, Taipei, Taiwan. zoslh@gate.sinica.edu.tw.
⁹ Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan. zoslh@gate.sinica.edu.tw.

PMID: 28716076
PMCID: PMC5513281
DOI: 10.1186/s12929-017-0351-y

Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression

Che-Yi Lin et al. J Biomed Sci. 2017.

. 2017 Jul 17;24(1):45.

doi: 10.1186/s12929-017-0351-y.

Authors

Affiliations

¹ Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan.
² Present address: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
³ Graduate Institute of Life Sciences, National Defence Medical Center, National Defence University, Neihu, Taipei, Taiwan.
⁴ Department of Life Science, National Taiwan University, Taipei, Taiwan.
⁵ Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan.
⁶ Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
⁷ Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan. zoslh@gate.sinica.edu.tw.
⁸ Department of Life Science, National Taiwan University, Taipei, Taiwan. zoslh@gate.sinica.edu.tw.
⁹ Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, 11529, Taiwan. zoslh@gate.sinica.edu.tw.

PMID: 28716076
PMCID: PMC5513281
DOI: 10.1186/s12929-017-0351-y

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Klf8; Kupffer’s vesicle; L-R patterning; Spaw; Zebrafish.

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Conflict of interest statement

Ethics approval and consent to participate

All animal procedures were approved by the Institutional Animal Care and Use Committee of Academia Sinica (Protocol ID: 15-12-918).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

**Fig. 1**
Knockdown of zebrafish *klf8* caused defects in heart jogging and looping, and visceral organ positions. a *klf8*-MO1^atg or *klf8*-MO2^atg -injected embryos stained with *myl7* exhibited left (L)-jog, no-jog or right (R)-jog and were compared to stained wild-type or 4 mm-MO1-injected control embryos at 24 hpf. b *myl7* stained embryos injected with *klf8*-MO1^atg or *klf8*-MO2^atg displayed D-loop, no-loop or L-loop heart and were compared to wild type and control embryos at 48 hpf. c *myl7* stained embryos injected with *klf8*-MO1^atg or *klf8*-MO2^atg displayed D-loop, no-loop or L-loop heart and were compared to wild type and control embryos at 72 hpf. d At 54 hpf, *gata6* stained wild type or embryos injected with *klf8*-4 mm MO1, *klf8*-MO1^atg or *klf8*-MO2^atg exhibited organ positions that were classified as: (Normal) normal positions of liver-left, pancreas-right and intestine with left looping, (Intestine only) only intestine without left looping, (Reverse) reversed position of liver-right, pancreas-left and no looping intestine, or (Bilateral) bilateral extension of liver and pancreas. A, atrium; I, intestine; L, liver; P, pancreas; V, ventricle

**Fig. 2**
Genes encoding Nodal signalling components exhibited decreased or abolished expression in *klf8* morphants. The majority of *klf8*-MO1^atg or *klf8*-MO2^atg -injected embryos showed decreased or abolished *spaw* (*arrow*) expression in the left lateral plate mesoderm (LPM) at the 18 s stage (**a-f**). The majority of *klf8* morphants failed to express *lft1* in the left diencephalon (*arrowhead*) or heart (*asterisk*) (**g- i**), *lft2* in the left heart (*asterisk*) (**j-l**), and *pitx2* in the left LPM (*white arrow*) (**m-o**) at the 22 s stage. Dorsal views of embryos are shown. A, absent; B, bilateral; D, decreased; L, left; R, right

**Fig. 3**
Reduced *spaw* expression in *klf8* morphants was partially rescued by co-injection of *klf8* mRNA. Representative embryos showing *spaw* expression in the left LPM (spaw+) or absent *spaw* expression (spaw-) are shown (**a-d**). Percentages of embryos with asymmetric *spaw* expression or no *spaw* expression are shown with indicated treatments (e). Statistical significance was determined by Fisher’s Exact Test. *p < 0.05

**Fig. 4**
Overexpression of *klf8* mRNA caused bilateral expression of Nodal signalling component genes. Injection of *klf8* mRNA induced bilateral expression of *spaw* in the LPM (*arrow*) at the 18 s stage, in a dose-dependent manner (**a-e**). Embryos injected with *klf8*, but not *LacZ*, exhibited bilateral expression of *lft1* in the diencephalon (*arrowhead*) and heart (*asterisk*; **f-j**), *lft2* in the heart (*asterisk*; **k-o**), and *pitx2* in the LPM (*white arrow*) (**p-t**) at the 19–22 s stage. Expression of *ntl* in the notochord is similar in wild type and embryos injected with *LacZ* or *klf8* mRNA (**u-w**). A, absent; B, bilateral; L, left; R, right

**Fig. 5**
Knockdown of *klf8* affected dorsal forerunner cell number, KV cilia number and length. Cell number of dorsal forerunner cells (DFCs) was affected in *klf8-*MO1^atg- or *klf8*-MO2^atg -injected embryos as compared to *klf8*-4 mm MO1-injected *Tg(sox17:gfp)* control embryos (4 mm) at 75% epiboly (**a-d**). Images of KV cilia stained with acetylated tubulin antibody in *klf8* morphants (**g-i**) and control embryos (**e, f**) at 10s stage (**e-i**). Images of acetylated tubulin stained KV cilia and GFP stained DFCs in *Tg(sox17:gfp)* embryos injected with different *klf8* MOs (**l-n**), *klf8*-4 mm MO1 (k) or wild type (j) embryos at 10s stage (**j-n**). DFC number (o), KV cilia number (q), length (r) but not lumen area (p) were affected in *klf8* morphants as compared to control embryos. Statistical significance was determined by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. *Error bars* indicate standard deviation

**Fig. 6**
Generation of *klf8* mutants by CRISPR-Cas9 gene editing and the effect on *spaw* expression. a *klf8* genomic structure with *klf8* sgRNA (*blue lettering*) targeted to exon 2. Protospacer adjacent motif (PAM) sequence is shown in *red*. b Nucleotide and predicted amino acid sequences of *klf8* in wild type, *klf8* ^I17 and *klf8d25* mutants are shown. Deleted nucleotides are shown by a *red dashed line*, while inserted nucleotides are shown in *green lettering*. Representative images of embryos with different *spaw* expression patterns in the LPM at 18 s stage (*arrow*; **c-f**). g Percentage of embryos displayed left (L), right (R), decreased (D) or bilateral (B) expression of *spaw* in the LPM from intercross of respective *klf8* ^d25 or *klf8* ⁱ¹⁷ F2 heterozygous mutants. Deduced percentage of wild type (+/+), heterozygote (+/−) or homozygote (−/−) genotype of embryos from intercross of respective *klf8* ^d25 or *klf8* ⁱ¹⁷ F2 heterozygous mutants exhibited bilateral *spaw* expression pattern. Expression levels of *klf3* (**h, k**), *klf12a* (**i, l**) or *klf12b* (**j, m**) were compared between wild type and respective *klf8* ^d25 and *klf8* ⁱ¹⁷ F5 homozygous mutant embryos at 10–12 s stages (**h-m**). Knockdown of *klf12b* reduced the percentage of embryos with bilateral *spaw* expression in the LPM of *klf8* ^d25 F6 homozygous mutant embryos, but the reduction did not reach significance (p = 0.45 for the comparison between *klf8* ^d25 and *klf8* ^d25+ 5 ng *klf812b* MO, p = 0.05 for the comparison between *klf8* ^d25 and *klf8* ^d25+ 10 ng *klf812b* MO) (n). Statistical significance was determined by Student’s t-test. * p < 0.05. *Error bars* indicate standard deviation

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References

1. Bisgrove BW, Morelli SH, Yost HJ. Genetics of human laterality disorders: insights from vertebrate model systems. Annu Rev Genomics Hum Genet. 2003;4:1–32. doi: 10.1146/annurev.genom.4.070802.110428. - DOI - PubMed
1. Kosaki K, Casey B. Genetics of human left-right axis malformations. Semin Cell Dev Biol. 1998;9:89–99. doi: 10.1006/scdb.1997.0187. - DOI - PubMed
1. Hamada H, Meno C, Watanabe D, Saijoh Y. Establishment of vertebrate left-right asymmetry. Nat Rev Genet. 2002;3:103–113. doi: 10.1038/nrg732. - DOI - PubMed
1. Shiratori H, Hamada H. The left-right axis in the mouse: from origin to morphology. Development. 2006;133:2095–2104. doi: 10.1242/dev.02384. - DOI - PubMed
1. Hirokawa N, Tanaka Y, Okada Y. Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow. Cold Spring Harb Perspect Biol. 2009;1:a000802. doi: 10.1101/cshperspect.a000802. - DOI - PMC - PubMed

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[1] Bisgrove BW, Morelli SH, Yost HJ. Genetics of human laterality disorders: insights from vertebrate model systems. Annu Rev Genomics Hum Genet. 2003;4:1–32. doi: 10.1146/annurev.genom.4.070802.110428. - DOI - PubMed

[2] Bisgrove BW, Morelli SH, Yost HJ. Genetics of human laterality disorders: insights from vertebrate model systems. Annu Rev Genomics Hum Genet. 2003;4:1–32. doi: 10.1146/annurev.genom.4.070802.110428. - DOI - PubMed

[3] Kosaki K, Casey B. Genetics of human left-right axis malformations. Semin Cell Dev Biol. 1998;9:89–99. doi: 10.1006/scdb.1997.0187. - DOI - PubMed

[4] Kosaki K, Casey B. Genetics of human left-right axis malformations. Semin Cell Dev Biol. 1998;9:89–99. doi: 10.1006/scdb.1997.0187. - DOI - PubMed

[5] Hamada H, Meno C, Watanabe D, Saijoh Y. Establishment of vertebrate left-right asymmetry. Nat Rev Genet. 2002;3:103–113. doi: 10.1038/nrg732. - DOI - PubMed

[6] Hamada H, Meno C, Watanabe D, Saijoh Y. Establishment of vertebrate left-right asymmetry. Nat Rev Genet. 2002;3:103–113. doi: 10.1038/nrg732. - DOI - PubMed

[7] Shiratori H, Hamada H. The left-right axis in the mouse: from origin to morphology. Development. 2006;133:2095–2104. doi: 10.1242/dev.02384. - DOI - PubMed

[8] Shiratori H, Hamada H. The left-right axis in the mouse: from origin to morphology. Development. 2006;133:2095–2104. doi: 10.1242/dev.02384. - DOI - PubMed

[9] Hirokawa N, Tanaka Y, Okada Y. Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow. Cold Spring Harb Perspect Biol. 2009;1:a000802. doi: 10.1101/cshperspect.a000802. - DOI - PMC - PubMed

[10] Hirokawa N, Tanaka Y, Okada Y. Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow. Cold Spring Harb Perspect Biol. 2009;1:a000802. doi: 10.1101/cshperspect.a000802. - DOI - PMC - PubMed

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