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

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’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.


Related in: MedlinePlus

Enpp2 modulates nodal-related asymmetry gene expressions, KV formation and ciliogenesis.Representative images of WISH of the control and enpp2-injected zebrafish embryos at 15–21ss with lefty2 (A) and spaw (C) antisense riboprobes respectively. The midline/notochord is highlighted by ntl expression. The determination of left, bilateral, absence, and right groups was based on the location of lefty2 or spaw expression compared to ntl (midline). The quantification of lefty2 and spaw expression distribution embryos is shown in (B) and (D) respectively. (B) The proportion of lefty2 expression on the left, bilateral, absence and right side are 67.3%, 4.5%, 28.2%, and 0% respectively in the control embryos and 54%, 12.6%, 28.7%, and 4.6% respectively in the enpp2 overexpressed embryos. (D) The proportion of spaw expression on the left, bilateral, absence and right side are 86%, 4%, 8%, and 2% respectively in the control embryos and 61.3%, 21.8%, 12.7%, 4.2% respectively in the enpp2-overexpressed embryos. (E) Representative images of the KV morphology in control- and in enpp2 overexpressing embryos (5–8ss). (F) Quantification KV size in enpp2 overexpressed embryos compared to control. (G) Whole-mount antibody staining of zona occludens 1 (ZO-1) that labels cell junctions and outlines the KV in control enpp2 injected embryos. (H) Representative confocal Z-stack images of cilia in the KV, marked with arl13b GFP, highlighting cilia morphology and distribution in control- and enpp2 overexpressing embryos at 5–8ss. (I) Quantification of the cilia length between control- and enpp2 overexpressing embryos. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM Statistical analysis was established by t-test; ***P < 0.001. n indicated above bars. White circles highlight the KV lumen. (J) Enpp2 overexpression altered the expression pattern of foxj1, a master regulator of ciliogenesis, observed at 90% epiboly embryo.
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f2: Enpp2 modulates nodal-related asymmetry gene expressions, KV formation and ciliogenesis.Representative images of WISH of the control and enpp2-injected zebrafish embryos at 15–21ss with lefty2 (A) and spaw (C) antisense riboprobes respectively. The midline/notochord is highlighted by ntl expression. The determination of left, bilateral, absence, and right groups was based on the location of lefty2 or spaw expression compared to ntl (midline). The quantification of lefty2 and spaw expression distribution embryos is shown in (B) and (D) respectively. (B) The proportion of lefty2 expression on the left, bilateral, absence and right side are 67.3%, 4.5%, 28.2%, and 0% respectively in the control embryos and 54%, 12.6%, 28.7%, and 4.6% respectively in the enpp2 overexpressed embryos. (D) The proportion of spaw expression on the left, bilateral, absence and right side are 86%, 4%, 8%, and 2% respectively in the control embryos and 61.3%, 21.8%, 12.7%, 4.2% respectively in the enpp2-overexpressed embryos. (E) Representative images of the KV morphology in control- and in enpp2 overexpressing embryos (5–8ss). (F) Quantification KV size in enpp2 overexpressed embryos compared to control. (G) Whole-mount antibody staining of zona occludens 1 (ZO-1) that labels cell junctions and outlines the KV in control enpp2 injected embryos. (H) Representative confocal Z-stack images of cilia in the KV, marked with arl13b GFP, highlighting cilia morphology and distribution in control- and enpp2 overexpressing embryos at 5–8ss. (I) Quantification of the cilia length between control- and enpp2 overexpressing embryos. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM Statistical analysis was established by t-test; ***P < 0.001. n indicated above bars. White circles highlight the KV lumen. (J) Enpp2 overexpression altered the expression pattern of foxj1, a master regulator of ciliogenesis, observed at 90% epiboly embryo.

Mentions: Establishment of the midline is important for L-R asymmetry and loss of enpp2/LPA signaling has been linked to asymmetry phenotypes31353637. To establish if increased enpp2/LPA signaling alters the L-R patterning of the zebrafish embryo, we examined the expression of the nodal-related asymmetric genes, lefty1/2 and southpaw (spaw) in the lateral plate mesoderm (LPM) during somite stages in control wild type and enpp2-overexpressing zebrafish. As shown in Fig. 2A–D, lefty1/2 and spaw were mainly expressed on the left side of the LPM during mid-late somitogenesis (15–21ss) in the control wild type zebrafish, which is consistent with previous reports3839. However, following enpp2 overexpression, the zebrafish displayed a more randomized expression pattern of these genes, either right-sided, absent, or bilaterally expressed in the LPM compared to control (Fig. 2A–D). In addition, the spaw expression domain was shifted posteriorly in some of the enpp2 overexpression embryos suggesting that migration of the lateral plate mesoderm is delayed or perturbed (Fig. 2A–D). Taken together, this demonstrated that enpp2 overexpression modulates L-R patterning and maintenance of the expression of the nodal-related asymmetric genes lefty1/2 and spaw.


Role of ectonucleotide pyrophosphatase/phosphodiesterase 2 in the midline axis formation of zebrafish
Enpp2 modulates nodal-related asymmetry gene expressions, KV formation and ciliogenesis.Representative images of WISH of the control and enpp2-injected zebrafish embryos at 15–21ss with lefty2 (A) and spaw (C) antisense riboprobes respectively. The midline/notochord is highlighted by ntl expression. The determination of left, bilateral, absence, and right groups was based on the location of lefty2 or spaw expression compared to ntl (midline). The quantification of lefty2 and spaw expression distribution embryos is shown in (B) and (D) respectively. (B) The proportion of lefty2 expression on the left, bilateral, absence and right side are 67.3%, 4.5%, 28.2%, and 0% respectively in the control embryos and 54%, 12.6%, 28.7%, and 4.6% respectively in the enpp2 overexpressed embryos. (D) The proportion of spaw expression on the left, bilateral, absence and right side are 86%, 4%, 8%, and 2% respectively in the control embryos and 61.3%, 21.8%, 12.7%, 4.2% respectively in the enpp2-overexpressed embryos. (E) Representative images of the KV morphology in control- and in enpp2 overexpressing embryos (5–8ss). (F) Quantification KV size in enpp2 overexpressed embryos compared to control. (G) Whole-mount antibody staining of zona occludens 1 (ZO-1) that labels cell junctions and outlines the KV in control enpp2 injected embryos. (H) Representative confocal Z-stack images of cilia in the KV, marked with arl13b GFP, highlighting cilia morphology and distribution in control- and enpp2 overexpressing embryos at 5–8ss. (I) Quantification of the cilia length between control- and enpp2 overexpressing embryos. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM Statistical analysis was established by t-test; ***P < 0.001. n indicated above bars. White circles highlight the KV lumen. (J) Enpp2 overexpression altered the expression pattern of foxj1, a master regulator of ciliogenesis, observed at 90% epiboly embryo.
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f2: Enpp2 modulates nodal-related asymmetry gene expressions, KV formation and ciliogenesis.Representative images of WISH of the control and enpp2-injected zebrafish embryos at 15–21ss with lefty2 (A) and spaw (C) antisense riboprobes respectively. The midline/notochord is highlighted by ntl expression. The determination of left, bilateral, absence, and right groups was based on the location of lefty2 or spaw expression compared to ntl (midline). The quantification of lefty2 and spaw expression distribution embryos is shown in (B) and (D) respectively. (B) The proportion of lefty2 expression on the left, bilateral, absence and right side are 67.3%, 4.5%, 28.2%, and 0% respectively in the control embryos and 54%, 12.6%, 28.7%, and 4.6% respectively in the enpp2 overexpressed embryos. (D) The proportion of spaw expression on the left, bilateral, absence and right side are 86%, 4%, 8%, and 2% respectively in the control embryos and 61.3%, 21.8%, 12.7%, 4.2% respectively in the enpp2-overexpressed embryos. (E) Representative images of the KV morphology in control- and in enpp2 overexpressing embryos (5–8ss). (F) Quantification KV size in enpp2 overexpressed embryos compared to control. (G) Whole-mount antibody staining of zona occludens 1 (ZO-1) that labels cell junctions and outlines the KV in control enpp2 injected embryos. (H) Representative confocal Z-stack images of cilia in the KV, marked with arl13b GFP, highlighting cilia morphology and distribution in control- and enpp2 overexpressing embryos at 5–8ss. (I) Quantification of the cilia length between control- and enpp2 overexpressing embryos. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM Statistical analysis was established by t-test; ***P < 0.001. n indicated above bars. White circles highlight the KV lumen. (J) Enpp2 overexpression altered the expression pattern of foxj1, a master regulator of ciliogenesis, observed at 90% epiboly embryo.
Mentions: Establishment of the midline is important for L-R asymmetry and loss of enpp2/LPA signaling has been linked to asymmetry phenotypes31353637. To establish if increased enpp2/LPA signaling alters the L-R patterning of the zebrafish embryo, we examined the expression of the nodal-related asymmetric genes, lefty1/2 and southpaw (spaw) in the lateral plate mesoderm (LPM) during somite stages in control wild type and enpp2-overexpressing zebrafish. As shown in Fig. 2A–D, lefty1/2 and spaw were mainly expressed on the left side of the LPM during mid-late somitogenesis (15–21ss) in the control wild type zebrafish, which is consistent with previous reports3839. However, following enpp2 overexpression, the zebrafish displayed a more randomized expression pattern of these genes, either right-sided, absent, or bilaterally expressed in the LPM compared to control (Fig. 2A–D). In addition, the spaw expression domain was shifted posteriorly in some of the enpp2 overexpression embryos suggesting that migration of the lateral plate mesoderm is delayed or perturbed (Fig. 2A–D). Taken together, this demonstrated that enpp2 overexpression modulates L-R patterning and maintenance of the expression of the nodal-related asymmetric genes lefty1/2 and spaw.

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