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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.


Related in: MedlinePlus

Enpp2 overexpression alters midline axis formation in the early embryogenesis of zebrafish.Developmental series of WISH at designated stages were performed using enpp2 antisense riboprobe. (A) Lateral view and animal/dorsal view of WISH early zebrafish embryos at designated stages. (B) Representative pictures of control, mild (slight delay and smooth somite borders) and severe (developmental delay and midline axial defect) phenotypes in enpp2 injected embryos. (C,D) Quantification of phenotype variant and penetrance (detectable phenotype) following injection with different doses of enpp2 RNA and normalized to control. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM from at least four independent experiments. Statistical analysis was established by one-way ANOVA; *P < 0.05; ***P < 0.001. Dead embryos were normalised to uninjected embryos (E) Representative WISH pictures of control and enpp2 injected embryos with midline axis gene probes ntl or shha riboprobes (F). >50 embryos used in each experiment. Scale bars: 200 μm.
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f1: Enpp2 overexpression alters midline axis formation in the early embryogenesis of zebrafish.Developmental series of WISH at designated stages were performed using enpp2 antisense riboprobe. (A) Lateral view and animal/dorsal view of WISH early zebrafish embryos at designated stages. (B) Representative pictures of control, mild (slight delay and smooth somite borders) and severe (developmental delay and midline axial defect) phenotypes in enpp2 injected embryos. (C,D) Quantification of phenotype variant and penetrance (detectable phenotype) following injection with different doses of enpp2 RNA and normalized to control. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM from at least four independent experiments. Statistical analysis was established by one-way ANOVA; *P < 0.05; ***P < 0.001. Dead embryos were normalised to uninjected embryos (E) Representative WISH pictures of control and enpp2 injected embryos with midline axis gene probes ntl or shha riboprobes (F). >50 embryos used in each experiment. Scale bars: 200 μm.

Mentions: We cloned the full-length enpp2 (NM_200603.1)34 to generate probes for whole mount in situ hybridization (WISH). WISH analysis of the developing zebrafish embryos showed that enpp2 mRNA expression is dynamically regulated during development, which was also confirmed by quantitative RT-PCR analysis (Fig. 1A, Suppl. Fig. 1). Enpp2 was maternally deposited (sphere) and expressed at a very low level in the yolk syncytial layer (YSL) during the early gastrulation stage (50% epiboly and shield) (Fig. 1A, Suppl. Fig. 1A,B). At the end of gastrulation (tail bud), enpp2 was notably expressed in the midline axis and its levels continued to increase during the segmentation period (10 somite stage, 15 somite stage) (Fig. 1A, Suppl. Fig. 1), suggesting a potential role in midline and axis formation. In order to assess the role of enpp2 during embryogenesis, we injected increasing amounts of capped enpp2 mRNA into the zebrafish embryo (1–4 cell stage), determined the morphology (Fig. 1B) and measured the phenotype penetrance (Fig. 1C). The overexpression of enpp2 resulted in significant axis defects and in a kinked notochord in a dose-dependent manner (Fig. 1B–F). This was accompanied by aberrant somite shapes, highlighted by the lack of chevron-shaped somites and shortened body length (Fig. 1B). Embryos injected with enpp2 mRNA at increasing concentrations exhibited dose-dependent penetrance of phenotypes (25 pg: 19.3 ± 6.8%; 50 pg: 25.6 ± 9.6%; 100 pg: 60.7 ± 2.7%; 200 pg: 60.5 ± 4.3%, Fig. 1D). These phenotypes suggest midline and axis defects during embryogenesis. To examine this further, we performed WISH to assess the expression patterns of midline markers shha and ntl to mark the notochord of the zebrafish embryo at 10 somite stage and 24 hours post fertilization, respectively (Fig. 1E,F). The majority of enpp2 -overexpressing embryos displayed abnormal pattern of shha and ntl expression, characterized by kinked, patchy or expanded patterns, or expression in multiple buds (duplicated), which indicated a notochord defect. This finding suggests a role of enpp2 in regulating the midline formation and its impact on the expression of the midline axis genes shha and ntl.


Role of ectonucleotide pyrophosphatase/phosphodiesterase 2 in the midline axis formation of zebrafish
Enpp2 overexpression alters midline axis formation in the early embryogenesis of zebrafish.Developmental series of WISH at designated stages were performed using enpp2 antisense riboprobe. (A) Lateral view and animal/dorsal view of WISH early zebrafish embryos at designated stages. (B) Representative pictures of control, mild (slight delay and smooth somite borders) and severe (developmental delay and midline axial defect) phenotypes in enpp2 injected embryos. (C,D) Quantification of phenotype variant and penetrance (detectable phenotype) following injection with different doses of enpp2 RNA and normalized to control. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM from at least four independent experiments. Statistical analysis was established by one-way ANOVA; *P < 0.05; ***P < 0.001. Dead embryos were normalised to uninjected embryos (E) Representative WISH pictures of control and enpp2 injected embryos with midline axis gene probes ntl or shha riboprobes (F). >50 embryos used in each experiment. Scale bars: 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121889&req=5

f1: Enpp2 overexpression alters midline axis formation in the early embryogenesis of zebrafish.Developmental series of WISH at designated stages were performed using enpp2 antisense riboprobe. (A) Lateral view and animal/dorsal view of WISH early zebrafish embryos at designated stages. (B) Representative pictures of control, mild (slight delay and smooth somite borders) and severe (developmental delay and midline axial defect) phenotypes in enpp2 injected embryos. (C,D) Quantification of phenotype variant and penetrance (detectable phenotype) following injection with different doses of enpp2 RNA and normalized to control. The sample size (n) is stated as numerical value above each bar. Data are mean ± SEM from at least four independent experiments. Statistical analysis was established by one-way ANOVA; *P < 0.05; ***P < 0.001. Dead embryos were normalised to uninjected embryos (E) Representative WISH pictures of control and enpp2 injected embryos with midline axis gene probes ntl or shha riboprobes (F). >50 embryos used in each experiment. Scale bars: 200 μm.
Mentions: We cloned the full-length enpp2 (NM_200603.1)34 to generate probes for whole mount in situ hybridization (WISH). WISH analysis of the developing zebrafish embryos showed that enpp2 mRNA expression is dynamically regulated during development, which was also confirmed by quantitative RT-PCR analysis (Fig. 1A, Suppl. Fig. 1). Enpp2 was maternally deposited (sphere) and expressed at a very low level in the yolk syncytial layer (YSL) during the early gastrulation stage (50% epiboly and shield) (Fig. 1A, Suppl. Fig. 1A,B). At the end of gastrulation (tail bud), enpp2 was notably expressed in the midline axis and its levels continued to increase during the segmentation period (10 somite stage, 15 somite stage) (Fig. 1A, Suppl. Fig. 1), suggesting a potential role in midline and axis formation. In order to assess the role of enpp2 during embryogenesis, we injected increasing amounts of capped enpp2 mRNA into the zebrafish embryo (1–4 cell stage), determined the morphology (Fig. 1B) and measured the phenotype penetrance (Fig. 1C). The overexpression of enpp2 resulted in significant axis defects and in a kinked notochord in a dose-dependent manner (Fig. 1B–F). This was accompanied by aberrant somite shapes, highlighted by the lack of chevron-shaped somites and shortened body length (Fig. 1B). Embryos injected with enpp2 mRNA at increasing concentrations exhibited dose-dependent penetrance of phenotypes (25 pg: 19.3 ± 6.8%; 50 pg: 25.6 ± 9.6%; 100 pg: 60.7 ± 2.7%; 200 pg: 60.5 ± 4.3%, Fig. 1D). These phenotypes suggest midline and axis defects during embryogenesis. To examine this further, we performed WISH to assess the expression patterns of midline markers shha and ntl to mark the notochord of the zebrafish embryo at 10 somite stage and 24 hours post fertilization, respectively (Fig. 1E,F). The majority of enpp2 -overexpressing embryos displayed abnormal pattern of shha and ntl expression, characterized by kinked, patchy or expanded patterns, or expression in multiple buds (duplicated), which indicated a notochord defect. This finding suggests a role of enpp2 in regulating the midline formation and its impact on the expression of the midline axis genes shha and ntl.

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