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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

Localization of the nuclear β-catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium iodide (PI) (red) and β-catenin antibody (green). (A2–E2) images of β-catenin staining only. (A3–E3) Drawing of (A1–E1) images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso; mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear β-catenin was preferentially present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No β-catenin was found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear β-catenin was detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase, and nuclear β-catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; Vg, vegetal side.
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fig004: Localization of the nuclear β-catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium iodide (PI) (red) and β-catenin antibody (green). (A2–E2) images of β-catenin staining only. (A3–E3) Drawing of (A1–E1) images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso; mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear β-catenin was preferentially present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No β-catenin was found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear β-catenin was detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase, and nuclear β-catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; Vg, vegetal side.

Mentions: Figure 4 shows the Hpβ-catenin distribution in embryos from the 16- to 56-cell stages. At the 16-cell stage, nuclear β-catenin was usually detected only in the macromeres (Mac) (Fig. 4A), although in some batches of embryos it was detectable also in the micromeres, but its fluorescence intensity was always much weaker than in the macromeres (data not shown). In the late 16-cell stage (Fig. 4B), the macromeres and mesomeres entered the next cleavage cycle (anaphase chromosomes were visible in Mac) and the four micromeres (mic) were still in the interphase. At the early 28-cell stage, the micromeres were in metaphase with more condensed stain with PI, while Mac had already divided and attained the telophase. It is noticeable that β-catenin accumulated around the newly formed metaphase chromosomal plates of mic (Fig. 4C1, C2). When the Mac and meso had completed the 5th division and formed interphase nuclei, the micromeres belatedly entered anaphase of the 5th division, displaying nuclear-β-catenin-like distribution (Fig. 4D). Nuclear β-catenin was still detected only in the daughter cells of the macromeres. At the 56-cell stage (Fig. 4E), four small (s-mic) and four large micromeres (l-mic) were formed, and the macromere descendants were located in the veg1 and veg2 cell layers. Nuclear β-catenin was now detected in the interphase nuclei of both s-mic and l-mic, and as well as in the veg2 cell layer. A small weak signal seemed to be present in the nuclei of veg1 cells. However, nuclei of animal cells (an) always exhibited no β-catenin signal.Figure 3


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)

Localization of the nuclear β-catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium iodide (PI) (red) and β-catenin antibody (green). (A2–E2) images of β-catenin staining only. (A3–E3) Drawing of (A1–E1) images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso; mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear β-catenin was preferentially present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No β-catenin was found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear β-catenin was detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase, and nuclear β-catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; Vg, vegetal side.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig004: Localization of the nuclear β-catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium iodide (PI) (red) and β-catenin antibody (green). (A2–E2) images of β-catenin staining only. (A3–E3) Drawing of (A1–E1) images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso; mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear β-catenin was preferentially present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No β-catenin was found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear β-catenin was detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase, and nuclear β-catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; Vg, vegetal side.
Mentions: Figure 4 shows the Hpβ-catenin distribution in embryos from the 16- to 56-cell stages. At the 16-cell stage, nuclear β-catenin was usually detected only in the macromeres (Mac) (Fig. 4A), although in some batches of embryos it was detectable also in the micromeres, but its fluorescence intensity was always much weaker than in the macromeres (data not shown). In the late 16-cell stage (Fig. 4B), the macromeres and mesomeres entered the next cleavage cycle (anaphase chromosomes were visible in Mac) and the four micromeres (mic) were still in the interphase. At the early 28-cell stage, the micromeres were in metaphase with more condensed stain with PI, while Mac had already divided and attained the telophase. It is noticeable that β-catenin accumulated around the newly formed metaphase chromosomal plates of mic (Fig. 4C1, C2). When the Mac and meso had completed the 5th division and formed interphase nuclei, the micromeres belatedly entered anaphase of the 5th division, displaying nuclear-β-catenin-like distribution (Fig. 4D). Nuclear β-catenin was still detected only in the daughter cells of the macromeres. At the 56-cell stage (Fig. 4E), four small (s-mic) and four large micromeres (l-mic) were formed, and the macromere descendants were located in the veg1 and veg2 cell layers. Nuclear β-catenin was now detected in the interphase nuclei of both s-mic and l-mic, and as well as in the veg2 cell layer. A small weak signal seemed to be present in the nuclei of veg1 cells. However, nuclei of animal cells (an) always exhibited no β-catenin signal.Figure 3

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