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Role of ectonucleotide pyrophosphatase/phosphodiesterase 2 in the midline axis formation of zebrafish

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ABSTRACT

Lysophosphatidic acid (LPA) is a unique bioactive lysophospholipid that induces pleiotropic effects in various cell types and organisms by acting on its specific receptors. LPA is mainly synthetised extracellularly by the ectonucleotide pyrophosphatase/phosphodiesterase 2/autotaxin (enpp2). Altered LPA signalling is associated with embryonic abnormalities, suggesting critical roles for LPA during development. However, the role of LPA signalling during early embryogenesis is not well established. We demonstrate that enpp2/LPA signalling in the early zebrafish embryo results in altered axis and midline formation, defects in left right (L-R) patterning, ciliogenesis of the Kupffer’s vesicle (KV), through the modulation of cell migration during gastrulation in a lpar1–3 Rho/ROCK-dependant manner. Overall, this study demonstrates an essential role of enpp2/LPA signalling during early embryogenesis.

No MeSH data available.


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Pharmacological blocking of lpa1–3 or Rho/ROCK signalling rescue enpp2 overexpression induced midline phenotype.Representative WISH images of shha (A,B) and foxj1 expression (C-D) at 90% epiboly of enpp2 overexpressing embryos treated with the lpa1–3 antagonist Ki16425(A,C) and Rho/ROCK signalling inhibitor Y27632 (B,D). Enpp2 overexpressing embryos display expanded expression of shha (A,B) and foxj1 (C,D) illustrated by pointed in arrows. Quantifications of the of shha expression domain in enpp2 overexpressing embryo following vehicle or Ki16425 (E) or Y27632 treatment (F). The phenotype penetrance following enpp2 injection and Ki16425 treatment and Y27632 were measured at 90% epiboly (E,F). (E,F) The sample size (n) is stated as numerical value above each bar from at least two independent experiments. Data are mean ± SEM. Statistical analysis was established by t-test; *P < 0.05; **P < 0.01. n indicated above bars.
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f4: Pharmacological blocking of lpa1–3 or Rho/ROCK signalling rescue enpp2 overexpression induced midline phenotype.Representative WISH images of shha (A,B) and foxj1 expression (C-D) at 90% epiboly of enpp2 overexpressing embryos treated with the lpa1–3 antagonist Ki16425(A,C) and Rho/ROCK signalling inhibitor Y27632 (B,D). Enpp2 overexpressing embryos display expanded expression of shha (A,B) and foxj1 (C,D) illustrated by pointed in arrows. Quantifications of the of shha expression domain in enpp2 overexpressing embryo following vehicle or Ki16425 (E) or Y27632 treatment (F). The phenotype penetrance following enpp2 injection and Ki16425 treatment and Y27632 were measured at 90% epiboly (E,F). (E,F) The sample size (n) is stated as numerical value above each bar from at least two independent experiments. Data are mean ± SEM. Statistical analysis was established by t-test; *P < 0.05; **P < 0.01. n indicated above bars.

Mentions: We next addressed the role played by LPA receptors in the phenotypes observed after enpp2-overexpression. To this end we performed rescue experiments using the well-established LPA receptor (Lpa1–3) antagonist Ki1642547. Ki16425 is a specific lpa receptor antagonist that efficiently antagonize lpa1–3 but not lpa4, lpa6a, and lpa6b and it has been used to antagonise LPA signalling in zebrafish previously34. We incubated enpp2-overexpressing embryos immediately after RNA injection with increasing concentrations of Ki16425 and assessed the impact on phenotypes by using shha and foxj1 expression as a read-out at the end of gastrulation (Fig. 4). enpp2 injected embryos treated with Ki16425 showed a significant reduction of ennp2-induced CE and ciliogenesis phenotypes in a dose-dependent manner (Fig. 4A–C). Following enpp2 overexpression, the CE phenotype characterised by a short axis and a broadened shha expression was detected in 66.5 ± 7.8% of the injected embryos (Fig. 4E). However, following and Ki16425 treatment, the number of embryos displaying this phenotype decreased to 41.3 ± 4.9% (Fig. 4E). Similarly, declustered foxj1 expression was observed in 53 ± 3.8% of the vehicle control embryos but decreased to 28.3 ± 6.2% in Ki16425-treated embryos (Fig. 4E). The rescue effect was maintained until later stages of development as we observed significantly reduced morphological phenotypes and almost normal expression levels of ntl, shha and its downstream gene gli2 following Ki16425 treatments at 24 hours post fertilisation (hpf, Fig. 2, Suppl. Fig. 3).


Role of ectonucleotide pyrophosphatase/phosphodiesterase 2 in the midline axis formation of zebrafish
Pharmacological blocking of lpa1–3 or Rho/ROCK signalling rescue enpp2 overexpression induced midline phenotype.Representative WISH images of shha (A,B) and foxj1 expression (C-D) at 90% epiboly of enpp2 overexpressing embryos treated with the lpa1–3 antagonist Ki16425(A,C) and Rho/ROCK signalling inhibitor Y27632 (B,D). Enpp2 overexpressing embryos display expanded expression of shha (A,B) and foxj1 (C,D) illustrated by pointed in arrows. Quantifications of the of shha expression domain in enpp2 overexpressing embryo following vehicle or Ki16425 (E) or Y27632 treatment (F). The phenotype penetrance following enpp2 injection and Ki16425 treatment and Y27632 were measured at 90% epiboly (E,F). (E,F) The sample size (n) is stated as numerical value above each bar from at least two independent experiments. Data are mean ± SEM. Statistical analysis was established by t-test; *P < 0.05; **P < 0.01. n indicated above bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121889&req=5

f4: Pharmacological blocking of lpa1–3 or Rho/ROCK signalling rescue enpp2 overexpression induced midline phenotype.Representative WISH images of shha (A,B) and foxj1 expression (C-D) at 90% epiboly of enpp2 overexpressing embryos treated with the lpa1–3 antagonist Ki16425(A,C) and Rho/ROCK signalling inhibitor Y27632 (B,D). Enpp2 overexpressing embryos display expanded expression of shha (A,B) and foxj1 (C,D) illustrated by pointed in arrows. Quantifications of the of shha expression domain in enpp2 overexpressing embryo following vehicle or Ki16425 (E) or Y27632 treatment (F). The phenotype penetrance following enpp2 injection and Ki16425 treatment and Y27632 were measured at 90% epiboly (E,F). (E,F) The sample size (n) is stated as numerical value above each bar from at least two independent experiments. Data are mean ± SEM. Statistical analysis was established by t-test; *P < 0.05; **P < 0.01. n indicated above bars.
Mentions: We next addressed the role played by LPA receptors in the phenotypes observed after enpp2-overexpression. To this end we performed rescue experiments using the well-established LPA receptor (Lpa1–3) antagonist Ki1642547. Ki16425 is a specific lpa receptor antagonist that efficiently antagonize lpa1–3 but not lpa4, lpa6a, and lpa6b and it has been used to antagonise LPA signalling in zebrafish previously34. We incubated enpp2-overexpressing embryos immediately after RNA injection with increasing concentrations of Ki16425 and assessed the impact on phenotypes by using shha and foxj1 expression as a read-out at the end of gastrulation (Fig. 4). enpp2 injected embryos treated with Ki16425 showed a significant reduction of ennp2-induced CE and ciliogenesis phenotypes in a dose-dependent manner (Fig. 4A–C). Following enpp2 overexpression, the CE phenotype characterised by a short axis and a broadened shha expression was detected in 66.5 ± 7.8% of the injected embryos (Fig. 4E). However, following and Ki16425 treatment, the number of embryos displaying this phenotype decreased to 41.3 ± 4.9% (Fig. 4E). Similarly, declustered foxj1 expression was observed in 53 ± 3.8% of the vehicle control embryos but decreased to 28.3 ± 6.2% in Ki16425-treated embryos (Fig. 4E). The rescue effect was maintained until later stages of development as we observed significantly reduced morphological phenotypes and almost normal expression levels of ntl, shha and its downstream gene gli2 following Ki16425 treatments at 24 hours post fertilisation (hpf, Fig. 2, Suppl. Fig. 3).

View Article: PubMed Central - PubMed

ABSTRACT

Lysophosphatidic acid (LPA) is a unique bioactive lysophospholipid that induces pleiotropic effects in various cell types and organisms by acting on its specific receptors. LPA is mainly synthetised extracellularly by the ectonucleotide pyrophosphatase/phosphodiesterase 2/autotaxin (enpp2). Altered LPA signalling is associated with embryonic abnormalities, suggesting critical roles for LPA during development. However, the role of LPA signalling during early embryogenesis is not well established. We demonstrate that enpp2/LPA signalling in the early zebrafish embryo results in altered axis and midline formation, defects in left right (L-R) patterning, ciliogenesis of the Kupffer&rsquo;s vesicle (KV), through the modulation of cell migration during gastrulation in a lpar1&ndash;3 Rho/ROCK-dependant manner. Overall, this study demonstrates an essential role of enpp2/LPA signalling during early embryogenesis.

No MeSH data available.


Related in: MedlinePlus