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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

In vitro differentiation of PKD2-kinase-dead inactivation.(A) Scheme illustrating schedule of EB culture in both PKD2 wild type (WT) and PKD2 kinase-dead (KD) iPSCs. (B–E) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Endoderm -FoxA2; Ectoderm -Pax6, -Tubb3. (F–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: c-kit, PDGFR. (H–I) qPCR of early cardiac markers: Tbx5 and Isl1. (J–L) qPCR analysis of vascular markers CD34, CD31 and vWF. (M–N) qPCR of myocardial markers Myl2a and Myh6. (O) Immunostaining of differentiating ESC cultures for CD31 at day 14. Genotypes are indicated in the figure. All experiments were performed n = 3 in replicates. 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 3.
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f6: In vitro differentiation of PKD2-kinase-dead inactivation.(A) Scheme illustrating schedule of EB culture in both PKD2 wild type (WT) and PKD2 kinase-dead (KD) iPSCs. (B–E) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Endoderm -FoxA2; Ectoderm -Pax6, -Tubb3. (F–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: c-kit, PDGFR. (H–I) qPCR of early cardiac markers: Tbx5 and Isl1. (J–L) qPCR analysis of vascular markers CD34, CD31 and vWF. (M–N) qPCR of myocardial markers Myl2a and Myh6. (O) Immunostaining of differentiating ESC cultures for CD31 at day 14. Genotypes are indicated in the figure. All experiments were performed n = 3 in replicates. 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 3.

Mentions: To substantiate the above findings, we made use of a previously reported PKD2 loss-of-function mouse model. In this mouse strain two critical serine residues (i.e. Ser 706, 710) in the kinase domain of PKD2 have been replaced by alanine leading to the expression of a kinase-dead mutant. Therefore, catalytic activity of PKD2 cannot be detected and the mouse recapitulates the phenotype of a PKD2 gene trap line4041. Mouse embryonic fibroblasts isolated form this mouse strain served as a somatic template to generate mutant induced pluripotent stem cell lines. Albeit the experiment did not aim to investigate the impact of PKD2 on pluripotency, we noted fewer Alkaline-Phosphatase (AP)-postive cultures in the reprogrammed kinase-dead fibroblasts (Fig. 5A). However, further studies need to focus on this aspect in more detail. We manually selected several induced pluripotent stem cell clones according to their dome-shaped compact colony morphology (Fig. 5B). These iPSC lines displayed all hallmarks of pluripotency such as marker expression of SSEA1 and Oct3/4 (Fig. 5C) and were capable to differentiate into all three germ layers: endoderm (Sox17), mesoderm (T) and ectoderm (Tubb3) (Fig. 5D). The presence of the mutated PKD2 locus was confirmed in two independent clones via PCR (Fig. 5E). Next, we applied two clones to subsequent spontaneous differentiation as outlined in Fig. 6A. qPCR analyses revealed increased mesendodermal marker expression in the early differentiation phase until day 4 (Brachyury, FoxA2,Fig. 6B,C), while ectodermal markers Pax6, Tubb3) showed decreased expression (Fig. 6D,E). Thus, PKD2-KD iPSCs display the expected, entirely opposite germ layer segregation pattern compared to an early activation of PKD2 (see Fig. 3B–J). Markers of the hemangioblast and the tripotent cardiovascular progenitor showed a similar pattern (Fig. 6F–I). These findings were confirmed with a second PKD2-KD iPSCs clone (Supplemental Fig. 2A-F). Later on, a trend towards decreased terminal differentiation of the cardiovascular lineage was observed as demonstrated by various markers gene expression (Fig. 6J–N). On protein level, CD31 positive cells were fewer and more scattered distributed in PKD2-KD EBs compared to WT, but appeared more organized in structure (data not shown, Fig. 6O).


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)

In vitro differentiation of PKD2-kinase-dead inactivation.(A) Scheme illustrating schedule of EB culture in both PKD2 wild type (WT) and PKD2 kinase-dead (KD) iPSCs. (B–E) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Endoderm -FoxA2; Ectoderm -Pax6, -Tubb3. (F–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: c-kit, PDGFR. (H–I) qPCR of early cardiac markers: Tbx5 and Isl1. (J–L) qPCR analysis of vascular markers CD34, CD31 and vWF. (M–N) qPCR of myocardial markers Myl2a and Myh6. (O) Immunostaining of differentiating ESC cultures for CD31 at day 14. Genotypes are indicated in the figure. All experiments were performed n = 3 in replicates. 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 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: In vitro differentiation of PKD2-kinase-dead inactivation.(A) Scheme illustrating schedule of EB culture in both PKD2 wild type (WT) and PKD2 kinase-dead (KD) iPSCs. (B–E) qPCR analysis depicting expression levels of different germ layers markers: Mesoderm - Brachyury (Bry); Endoderm -FoxA2; Ectoderm -Pax6, -Tubb3. (F–G) qPCR analysis illustrating expression of hemangioblast and cardiovascular progenitor markers: c-kit, PDGFR. (H–I) qPCR of early cardiac markers: Tbx5 and Isl1. (J–L) qPCR analysis of vascular markers CD34, CD31 and vWF. (M–N) qPCR of myocardial markers Myl2a and Myh6. (O) Immunostaining of differentiating ESC cultures for CD31 at day 14. Genotypes are indicated in the figure. All experiments were performed n = 3 in replicates. 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 3.
Mentions: To substantiate the above findings, we made use of a previously reported PKD2 loss-of-function mouse model. In this mouse strain two critical serine residues (i.e. Ser 706, 710) in the kinase domain of PKD2 have been replaced by alanine leading to the expression of a kinase-dead mutant. Therefore, catalytic activity of PKD2 cannot be detected and the mouse recapitulates the phenotype of a PKD2 gene trap line4041. Mouse embryonic fibroblasts isolated form this mouse strain served as a somatic template to generate mutant induced pluripotent stem cell lines. Albeit the experiment did not aim to investigate the impact of PKD2 on pluripotency, we noted fewer Alkaline-Phosphatase (AP)-postive cultures in the reprogrammed kinase-dead fibroblasts (Fig. 5A). However, further studies need to focus on this aspect in more detail. We manually selected several induced pluripotent stem cell clones according to their dome-shaped compact colony morphology (Fig. 5B). These iPSC lines displayed all hallmarks of pluripotency such as marker expression of SSEA1 and Oct3/4 (Fig. 5C) and were capable to differentiate into all three germ layers: endoderm (Sox17), mesoderm (T) and ectoderm (Tubb3) (Fig. 5D). The presence of the mutated PKD2 locus was confirmed in two independent clones via PCR (Fig. 5E). Next, we applied two clones to subsequent spontaneous differentiation as outlined in Fig. 6A. qPCR analyses revealed increased mesendodermal marker expression in the early differentiation phase until day 4 (Brachyury, FoxA2,Fig. 6B,C), while ectodermal markers Pax6, Tubb3) showed decreased expression (Fig. 6D,E). Thus, PKD2-KD iPSCs display the expected, entirely opposite germ layer segregation pattern compared to an early activation of PKD2 (see Fig. 3B–J). Markers of the hemangioblast and the tripotent cardiovascular progenitor showed a similar pattern (Fig. 6F–I). These findings were confirmed with a second PKD2-KD iPSCs clone (Supplemental Fig. 2A-F). Later on, a trend towards decreased terminal differentiation of the cardiovascular lineage was observed as demonstrated by various markers gene expression (Fig. 6J–N). On protein level, CD31 positive cells were fewer and more scattered distributed in PKD2-KD EBs compared to WT, but appeared more organized in structure (data not shown, Fig. 6O).

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