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A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A - Sci Rep (2015)

Bottom Line: In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer.Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs.Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Ulm University, Ulm, Germany.

ABSTRACT
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

No MeSH data available.


Related in: MedlinePlus

Effects of early PKD2 overexpression from day 0–4.(A) Scheme illustrating treatment regimen of iPKD2 ES cells from the beginning of EB culture until cell harvest for qPCR analysis. (B–D) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Ectoderm - Pax6; Endoderm -FoxA2. (E–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: hemangioblast/angioblast - Flk1; hemangioblast - c-kit; cardiac -PDGFR. (H,I,K,L) qPCR analysis illustrating expression of early cardiac markers, (H,I) Tbx5 and Isl1 and late cardiac markers (K,L) Myh6, Myl2a. (J) Tubb3 marks neuronal differentiation at later time points. (M) α-actinin staining of Conditions Dox – and Dox 0–4 at day 14 of differentiation. Cultures were stimulated as illustrated in (A). (N–O) qPCR analysis illustrating expression of late vascular markers, CD34, CD31 in Dox – and Dox 0–14 conditions. (P) CD31 staining at day 14 of differentiation. Cultures were stimulated as illustrated in (A). Time points as indicated in the figure and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 1.
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f3: Effects of early PKD2 overexpression from day 0–4.(A) Scheme illustrating treatment regimen of iPKD2 ES cells from the beginning of EB culture until cell harvest for qPCR analysis. (B–D) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Ectoderm - Pax6; Endoderm -FoxA2. (E–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: hemangioblast/angioblast - Flk1; hemangioblast - c-kit; cardiac -PDGFR. (H,I,K,L) qPCR analysis illustrating expression of early cardiac markers, (H,I) Tbx5 and Isl1 and late cardiac markers (K,L) Myh6, Myl2a. (J) Tubb3 marks neuronal differentiation at later time points. (M) α-actinin staining of Conditions Dox – and Dox 0–4 at day 14 of differentiation. Cultures were stimulated as illustrated in (A). (N–O) qPCR analysis illustrating expression of late vascular markers, CD34, CD31 in Dox – and Dox 0–14 conditions. (P) CD31 staining at day 14 of differentiation. Cultures were stimulated as illustrated in (A). Time points as indicated in the figure and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 1.

Mentions: In pluripotent stem cells, germ layer segregation occurs around days 2/31953, while the onset of vasculogenesis is ignited around day 3 by the emergence of the hemangioblast, a common progenitor of the endothelial and hematopoietic lineage54. The hemangioblast is defined by the co-expression of the vascular endothelial growth factor receptor (Flk1/VEGFR-2) and the mesodermal transcription factor brachyury (T). A second progenitor population arises shortly after the development of the hemangioblast55. These cardiovascular progenitors are tripotent giving rise to cardiomyocytes, endothelial cells and smooth muscle cells and co-express Mesp1, brachyury, PDGFRa and c-kit56. Using embryoid body (EB) -based differentiation, mimicking early cell fate decision in the embryo, we assessed the effects of overexpression of PKD2 on marker genes of all three germ layers, but also on above mentioned progenitor population markers. In a first approach, PKD2 was activated during the first 4 days of differentiation (Fig. 3A) to investigate changes in germ layer formation and vasculogenesis54. In PKD2 overexpressing cells, we observed a downregulation of early markers for mesoderm and endoderm [Brachyury, FoxA2 (Fig. 3B,C)] and upregulation of the early ectoderm marker Pax6 (Fig. 3D). In line, markers labelling both the hemangioblast and the tripotent cardiovascular progenitor were repressed [Flk1, cKit, Pdgfr (Figure 3E-G)]. In addition, early cardiac and endothelial markers were downregulated at day 4 [Tbx5, Isl1 (Fig. 3H,I)]. These findings were confirmed with a second, independently targeted iPKD2 ESC line (Supplemental Fig. 1A-F).


A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A - Sci Rep (2015)

Effects of early PKD2 overexpression from day 0–4.(A) Scheme illustrating treatment regimen of iPKD2 ES cells from the beginning of EB culture until cell harvest for qPCR analysis. (B–D) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Ectoderm - Pax6; Endoderm -FoxA2. (E–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: hemangioblast/angioblast - Flk1; hemangioblast - c-kit; cardiac -PDGFR. (H,I,K,L) qPCR analysis illustrating expression of early cardiac markers, (H,I) Tbx5 and Isl1 and late cardiac markers (K,L) Myh6, Myl2a. (J) Tubb3 marks neuronal differentiation at later time points. (M) α-actinin staining of Conditions Dox – and Dox 0–4 at day 14 of differentiation. Cultures were stimulated as illustrated in (A). (N–O) qPCR analysis illustrating expression of late vascular markers, CD34, CD31 in Dox – and Dox 0–14 conditions. (P) CD31 staining at day 14 of differentiation. Cultures were stimulated as illustrated in (A). Time points as indicated in the figure and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493579&req=5

f3: Effects of early PKD2 overexpression from day 0–4.(A) Scheme illustrating treatment regimen of iPKD2 ES cells from the beginning of EB culture until cell harvest for qPCR analysis. (B–D) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Ectoderm - Pax6; Endoderm -FoxA2. (E–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: hemangioblast/angioblast - Flk1; hemangioblast - c-kit; cardiac -PDGFR. (H,I,K,L) qPCR analysis illustrating expression of early cardiac markers, (H,I) Tbx5 and Isl1 and late cardiac markers (K,L) Myh6, Myl2a. (J) Tubb3 marks neuronal differentiation at later time points. (M) α-actinin staining of Conditions Dox – and Dox 0–4 at day 14 of differentiation. Cultures were stimulated as illustrated in (A). (N–O) qPCR analysis illustrating expression of late vascular markers, CD34, CD31 in Dox – and Dox 0–14 conditions. (P) CD31 staining at day 14 of differentiation. Cultures were stimulated as illustrated in (A). Time points as indicated in the figure and treatment regimen as indicated in the figure. All experiments were performed n = 3 in replicates. Scale bars 20 μm. Significances were calculated using R. Raw p values were adjusted using Bonferroni correction (§p < 0.05; §§p < 0.01; §§§p < 0.001). Adjusted p-values are listed in Suppl. Table 1.
Mentions: In pluripotent stem cells, germ layer segregation occurs around days 2/31953, while the onset of vasculogenesis is ignited around day 3 by the emergence of the hemangioblast, a common progenitor of the endothelial and hematopoietic lineage54. The hemangioblast is defined by the co-expression of the vascular endothelial growth factor receptor (Flk1/VEGFR-2) and the mesodermal transcription factor brachyury (T). A second progenitor population arises shortly after the development of the hemangioblast55. These cardiovascular progenitors are tripotent giving rise to cardiomyocytes, endothelial cells and smooth muscle cells and co-express Mesp1, brachyury, PDGFRa and c-kit56. Using embryoid body (EB) -based differentiation, mimicking early cell fate decision in the embryo, we assessed the effects of overexpression of PKD2 on marker genes of all three germ layers, but also on above mentioned progenitor population markers. In a first approach, PKD2 was activated during the first 4 days of differentiation (Fig. 3A) to investigate changes in germ layer formation and vasculogenesis54. In PKD2 overexpressing cells, we observed a downregulation of early markers for mesoderm and endoderm [Brachyury, FoxA2 (Fig. 3B,C)] and upregulation of the early ectoderm marker Pax6 (Fig. 3D). In line, markers labelling both the hemangioblast and the tripotent cardiovascular progenitor were repressed [Flk1, cKit, Pdgfr (Figure 3E-G)]. In addition, early cardiac and endothelial markers were downregulated at day 4 [Tbx5, Isl1 (Fig. 3H,I)]. These findings were confirmed with a second, independently targeted iPKD2 ESC line (Supplemental Fig. 1A-F).

Bottom Line: In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer.Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs.Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Ulm University, Ulm, Germany.

ABSTRACT
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

No MeSH data available.


Related in: MedlinePlus