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Ca²⁺ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.

Yazaki I, Tsurugaya T, Santella L, Chun JT, Amore G, Kusunoki S, Asada A, Togo T, Akasaka K - Zygote (2014)

Bottom Line: To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I).Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage.Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences,Tokyo Metropolitan University,Minamiohsawa 1-1,Hachiohji-shi,Tokyo 192-0397,Japan.

ABSTRACT
Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

No MeSH data available.


Related in: MedlinePlus

Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were treated with 25 μM GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 μM (B) or 5 μM (C) during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos. Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C).
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fig006: Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were treated with 25 μM GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 μM (B) or 5 μM (C) during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos. Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C).

Mentions: The earliest function of the micromeres appears to be endoderm induction, as the signal is emitted from the micromeres during the 16–60-cell stages (Ransick & Davidson, 1995). We have examined whether the Ca2+ influx and PKC activity are essential for this micromere signal. As the Ca2+ influx at the micromere formation is suppressed with GdCl3, an inhibitor of stretch-activated ion channels (Yazaki et al., 2004), we exposed P. lividus embryos to 25 μM GdCl3 and examined the frequency of the embryos that clearly exhibited the expected phenotypes: ingression of the primary mesenchyme cells (PMCs) and gastrulation (Fig. 6A). We found that gastrulation was delayed by GdCl3 when it was applied at the 4th and 5th cleavages, but not before or after the period. When GdCl3 was added from the 5th cleavage, which included micromere cleavage to form small and large micromeres during treatment, gastrulation was delayed to a lesser extent than when treatment was carried out at the 4th cleavage (Fig. 6A). By contrast, the ingression of PMCs into the blastocoel was not altered in all treatment schemes.Figure 6


Ca²⁺ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.

Yazaki I, Tsurugaya T, Santella L, Chun JT, Amore G, Kusunoki S, Asada A, Togo T, Akasaka K - Zygote (2014)

Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were treated with 25 μM GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 μM (B) or 5 μM (C) during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos. Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4416383&req=5

fig006: Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were treated with 25 μM GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 μM (B) or 5 μM (C) during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos. Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C).
Mentions: The earliest function of the micromeres appears to be endoderm induction, as the signal is emitted from the micromeres during the 16–60-cell stages (Ransick & Davidson, 1995). We have examined whether the Ca2+ influx and PKC activity are essential for this micromere signal. As the Ca2+ influx at the micromere formation is suppressed with GdCl3, an inhibitor of stretch-activated ion channels (Yazaki et al., 2004), we exposed P. lividus embryos to 25 μM GdCl3 and examined the frequency of the embryos that clearly exhibited the expected phenotypes: ingression of the primary mesenchyme cells (PMCs) and gastrulation (Fig. 6A). We found that gastrulation was delayed by GdCl3 when it was applied at the 4th and 5th cleavages, but not before or after the period. When GdCl3 was added from the 5th cleavage, which included micromere cleavage to form small and large micromeres during treatment, gastrulation was delayed to a lesser extent than when treatment was carried out at the 4th cleavage (Fig. 6A). By contrast, the ingression of PMCs into the blastocoel was not altered in all treatment schemes.Figure 6

Bottom Line: To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I).Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage.Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences,Tokyo Metropolitan University,Minamiohsawa 1-1,Hachiohji-shi,Tokyo 192-0397,Japan.

ABSTRACT
Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

No MeSH data available.


Related in: MedlinePlus