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. 2013 May 14;8(5):e62756.
doi: 10.1371/journal.pone.0062756. Print 2013.

Phenotypical analysis of atypical PKCs in vivo function display a compensatory system at mouse embryonic day 7.5

Sebastian Seidl  1 , Ursula Braun, Norbert Roos, Shaohua Li, Timo H-W Lüdtke, Andreas Kispert, Michael Leitges
Affiliations

Phenotypical analysis of atypical PKCs in vivo function display a compensatory system at mouse embryonic day 7.5

Sebastian Seidl et al. PLoS One. .

Abstract

Background: The atypical protein kinases C (PKC) isoforms ι/λ and ζ play crucial roles in many cellular processes including development, cell proliferation, differentiation and cell survival. Possible redundancy between the two isoforms has always been an issue since most biochemical tools do not differentiate between the two proteins. Thus, much effort has been made during the last decades to characterize the functions of aPKCs using gene targeting approaches and depletion studies. However, little is known about the specific roles of each isoform in mouse development.

Methodology/principal findings: To evaluate the importance of PKCι in mouse development we designed PKCι deletion mutants using the gene targeting approach. We show that the deletion of PKCι, results in a reduced size of the amniotic cavity at E7.5 and impaired growth of the embryo at E8.5 with subsequent absorption of the embryo. Our data also indicate an impaired localization of ZO-1 and disorganized structure of the epithelial tissue in the embryo. Importantly, using electron microscopy, embryoid body formation and immunofluorescence analysis, we found, that in the absence of PKCι, tight junctions and apico-basal polarity were still established. Finally, our study points to a non-redundant PKCι function at E9.5, since expression of PKCζ is able to rescue the E7.5 phenotype, but could not prevent embryonic lethality at a later time-point (E9.5).

Conclusion: Our data show that PKCι is crucial for mouse embryogenesis but is dispensable for the establishment of polarity and tight junction formation. We present a compensatory function of PKCζ at E7.5, rescuing the phenotype. Furthermore, this study indicates at least one specific, yet unknown, PKCι function that cannot be compensated by the overexpression of PKCζ at E9.5.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Histological overview of E7.5 and E8.5 embryos.
Embryos were isolated at indicated time points and embedded in paraffin. (A) Sagittal sections of wt and PKCιΔ/Δ embryos at E7.5 (right). Phase contrast image of the corresponding embryo (left) (B) Sagittal sections of wt and PKCιΔ/Δ embryos at day E8.5 (right). Phase contrast image of the corresponding embryo (left). Abbreviations: ac, amniotic cavity; all, allantois; am, amnion; ec, ectoplacental cavity; fnf, forebrain neural-fold; hb, hindbrain; mb, midbrain; h, heart; ps, primitive streak; S = somites; tl; tiny lumina.
Figure 2
Figure 2. Whole-mount in situ hybridization analysis reveals an anterior-posterior body axis but a lack of a cardiovascular system in E8.5 embryos.
Either wt (wt) or PKCι- deficient embryos were analyzed using a set of different markers: Otx2, orthodenticle homeobox 2; Fgf8, fibrobalst growth factor 8; Tcf15, transcription factor 15; Tbx20, T- box 20; Pax2, paired box gene 2; T, Brachyury; Bmp4, bone morphogenetic protein 4; Gja5, gap junction alpha-5 protein.
Figure 3
Figure 3. Immunofluorescence analysis of embryoid bodies (EBs) from either wt or PKCιΔ/Δ.
(A) EBs from WT and PKCιΔ/Δ ES cells were stained with Rhodamin-Phalloidin (red, F-actin) and an antibody against Laminin-α1 (green, Lm α1). (B) Phase-contrast picture of 5 days old WT and PKCιΔ/Δ EBs. (C) Statistical analysis of lumen formation of 5 days and 7 days old EBs from WT and PKCιΔ/Δ ES cells. (D) EBs from WT and PKCιΔ/Δ ES cells were stained with antibodies against cleaved caspase-3 (green, cleaved cas-3) and perlecan (red). (E) WB analysis of activated caspase-3 at different time-points in WT and PKCιΔ/Δ EBs. (F) Immunofluorescence analysis of EBs from either WT or PKCιΔ/Δ ES cells including localization of E-cadherin, MUPP1, Par-3 and GM130 (green), perlecan and laminin α1 (red) (G) EBs from WT and PKCιΔ/Δ ES cells were stained with antibodies against perlecan (green) and aPKCzeta (red, PKCζ). (H) WB analysis of PKCζ in 4 days and 5 days old WT and PKCιΔ/Δ EBs. All membranes were scanned and the intensities of the single bands were calculated. Values represent rations of PKCζ to actin. Nucleus staining was performed with DAPI. Scale is as indicated.
Figure 4
Figure 4. Immunofluorescence analysis of marker proteins in PKCιΔ/Δ embryos.
Analysis were performed using the indicated antibodies. (A) Paraffin sections showing the localization of E-cadherin (E-cad) in the wt and PKCιΔ/Δ embryo. Scale bars: 100 µm (B) Immunofluorescence analysis of aPKC localization at the apical pole of wt and PKCι deficient embryos in high magnification. Scale bars: 20 µm (C) Immunofluorescence analysis of proteins involved in cellular polarity (E-cad, E-cadherin; Occl., occludin; ZO-1, Zonula Occludens-1). Localization of occludin is indicated by open arrows and the localization of ZO-1 by solid arrows. Scale bars: 20 µm.
Figure 5
Figure 5. Electron microscopic analysis of PKCι deficient embryos.
Embryos were isolated at E7.5. (A) Apical junctional complex (AJC) formation in wt and PKCι deficient (PKCιΔ/Δ) embryos was compared. Functional complexes are indicated: tight junction (TJ), adherens junctions (AJs) and desmosomes (DSs). The amniotic cavity is indicated by an asterix. (B) Arrows indicate intracellular space in the apicobasal membrane in PKCι wt and PKCιΔ/Δ embryos. The orientation of the images is comparable to the images in A. (C) Arrows indicate the F-actin bundles parallel to the cell-membrane in wt and PKCιΔ/Δ embryos. EM analysis was performed as described in Materials and methods. Representative micrographs are presented. Bar is equivalent to 200 micrometer (200 µm).
Figure 6
Figure 6. Overexpression of PKCζ rescues PKCι KO phenotype.
(A) Comparison of the appearance of embryos, that are either wt, heterozygous for the rescue allele (PKCιζRes/+) or homozygous for the rescue (PKCιζRes/ζRes) allele embryos at E9.5. (B) Table containing the total numbers of analyzed embryos at day E7.5 and E9.5 and the corresponding percentage of the genotypes. (C) Western blot analysis of E7.5 embryos using an antibody against pan-aPKC. Each lane contains the total protein amount of a whole embryo. (D) Western blot analysis of day E9.5 embryos using indicated antibodies. Each lane contains adjusted (β-actin) protein amounts from single embryos.
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