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. 2009 Aug 25;106(34):14426-31.
doi: 10.1073/pnas.0901332106. Epub 2009 Aug 11.

Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical-basal polarity

Hirotaka Tao  1 , Makoto Suzuki, Hiroshi Kiyonari, Takaya Abe, Toshikuni Sasaoka, Naoto Ueno
Affiliations

Mouse prickle1, the homolog of a PCP gene, is essential for epiblast apical-basal polarity

Hirotaka Tao et al. Proc Natl Acad Sci U S A. .

Abstract

Planar cell polarity (PCP) genes are essential for establishing planar cell polarity in both invertebrate and vertebrate tissues and are known to regulate cellular morphogenesis and cell movements during development. We focused on Prickle, one of the core components of the PCP pathway, and deleted one of two mouse prickle homologous genes, mpk1. We found that the deletion of mpk1 gene resulted in early embryonic lethality, between embryonic day (E)5.5 and E6.5, associated with failure of distal visceral endoderm migration and primitive streak formation. The mpk1(-/-) epiblast tissue was disorganized, and analyses at the cellular level revealed abnormal cell shapes, mislocalized extracellular matrix (ECM) proteins, and disrupted orientation of mitotic spindles, from which loss of apico-basal (AB) polarity of epiblast cells are suspected. Furthermore, we show mpk1 genetically interacts with another core PCP gene Vangl2/stbm in the epiblast formation, suggesting that PCP components are commonly required for the establishment and/or the maintenance of epiblast AB polarity. This was further supported by our finding that overexpression of DeltaPET/LIM (DeltaP/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKCzeta, and expanded the apical domain of ectoderm cells. Our results demonstrate a role for mpk1 in AB polarity formation rather than expected role as a PCP gene.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression pattern of mpk1 in postimplantation embryos. (A–D′) Expression of mpk1 transcripts in postimplantation embryos. (E and E′) Immunostaining for Pk1-specifc protein (red) within the cytoplasm of the E5.5+ epiblast. Nuclei were stained with Draq5 (green). ex, extraembryonic region; em, embryonic region; ect, ectoderm; ps, primitive streak; mes, mesoderm; ve, visceral endoderm; ac, proamniotic cavity. Anterior is to the left in B–D′. (Scale bars, 50 μm in B–D.)
Fig. 2.
Fig. 2.
Morphogenetic defects in the mpk1−/− mutant embryo. (A–D) Whole-mount E6.5 control embryo and mpk1−/− mutant; (C and D) sagittal sections of E6.5 control and mpk1−/− mutant. (E and F[prime]) Fluorescent images of nuclei (stained by TOTO-3, green) in transverse sections in control and mpk1−/− mutant embryos. (E′ and F′) Insets are higher-magnification images of the epiblast indicated by F-actin (red) and nuclei (green). Arrowheads indicate the boundary between the extraembryonic and embryonic regions. eec, ectoplacental cone; ac, proaminiotic cavity; epi, epiblast. (Scale bar, 50 μm in A–D.) (G) Quantification of epiblast nuclei in the control and mpk1−/− mutant. Values are from 3 independent experiments, n = 20–30 nuclei per experiments. Error bars represent the s.e.m.; asterisk denotes a significant difference (P < 0.05), compared with the control (n = 3, each genotypes).
Fig. 3.
Fig. 3.
Marker analysis in the mpk1−/− mutant. Whole-mount in situ hybridization analysis of control (A–H) and mpk−/− mutant (A′–H′) embryos at E5.5–6.75. The number of analyzed mpk1−/− mutant embryos were following; E5.5, Hex, n = 5/5; Dkk1, n = 4/5; E6.5, Wnt3, n = 4/5; Bmp4, n = 5/6; E5.5, Wnt3, n = 4/5, Bmp4, n = 3/3; E5.5, Nodal, n = 3/3; E6.75, Nodal, n = 5/7. Arrowheads indicate the boundary between the embryonic and extraembryonic regions. Anterior is to the left in all panels.
Fig. 4.
Fig. 4.
Loss of mpk1 leads to disrupted ECM deposition and abnormal orientation of cell division. (A and B) The epiblast nuclei were labeled using BrdU (black), and mitotic cells were labeled with anti-phospho-histone H3 antibody (green) on transverse sections of control and mpk1−/− mutant (n = 3, each genotypes). (C–F) Double immunofluorescence for laminin (green) and pHH3 (red) on confocal sections of the control and mpk1−/− mutant embryos at E5.5 and E6.5. (n = 3, each genotypes). Nuclei were stained with Draq5 (blue). (G and H) Quantification of the different orientations of the mitotic spindle at the apical surface in the control and mpk1−/− mutant (n = 3, each genotypes). Error bars represent the s.e.m. (I and J′) Immunofluorescence for γ-tubulin (red dots) and nuclei (green) on confocal sections of the control and mpk1−/− epiblast (n = 4, each genotypes). Anterior is to the left in A and B. Apical side is down in C–J′. epi, epiblast; ve, visceral endoderm; ac, proaminiotic cavity.
Fig. 5.
Fig. 5.
mpk1−/− mutant epiblast have defective cytoskeletal actin polarization and abnormal localization of E-cadherin and PKCζ. (A and B) Transverse sections, and (C and D) sagittal sections of E5.5 control and mpk1−/− mutant embryos. Accumulation of F-actin at the apical surface of epiblast was observed in control (A, arrowheads), but was ectopically dispersed in the mpk1−/− mutant (B and C, arrowheads). (E–I′) Immunofluorescence for E-cadherin (E, F, I, and J) and PKCζ (G and H) on confocal sections of the control and mpk1−/− epiblast at E5.5–6.5. (E–H) Polarized localization of E-cadherin was PKCζ were observed in control, but not in mpk1−/− mutant at E6.25–E6.5. (J and J′) In E5.5, mpk1−/− mutant, localization of E-cadherin was ectopically dispersed in the cytoplasm (J, white arrowheads). Bodipy-ceramide was used for visualize outlines cell contours. Each experiment was carried out at least with two embryos. epi, epiblast; ve, visceral endoderm; ac, proaminiotic cavity; N, nuclei.
Fig. 6.
Fig. 6.
Overexpression of ΔP/L disrupts the apical and basolateral domains in the presumptive ectoderm of the Xenopus embryo. (A–D) ΔP/L caused partial pigment defects and/or expanded cells (B, asterisks) compared with controls (A). (C and D) FLAG-tagged β-globin or FLAG-tagged ΔP/L (1 ng each) was injected. (E) The effect of ΔP/L overexpression, which was scored blind, is shown. (F–K) Immunostaining with AB markers is shown in each panel. (F, H, and J) The β-globin-injected embryos were entirely normal. The number of analyzed ΔP/L overexpressing embryos were following; (G) n = 18; (I) n = 20; (K) n = 15. (G) The injection of ΔP/L caused the abnormal localization of PKCζ in the apical region. Inset is another image. These images are of single planes of optical sections (F and G). (I) The injection of ΔP/L caused the ectopic localization of ZO-1 to the basolateral side (red arrowheads). (K) The localization of β1-integrin to the basolateral region was not altered by the injection of ΔP/L. (L) The quantification of namely uneven localization of PKCζ at the apical membrane in the ΔP/L-injected embryo compared with the control (n = 8, each injected cells) (see SI Materials and Methods). (M) The quantification of ectopic ZO-1 in the ΔP/L-injected embryo compared with β-globin-expressing cells (embryos); n = 291 (13), 196 (7), 197 (10), ΔP/L-expressing cells (embryos); n = 110 (9), 169 (10), 204 (12). This experiment was carried out 3 times. Error bars, the s.e.m.; asterisk denotes a significant difference (P < 0.05), compared with the control. Apical is at the top (A–D, F–K).
Fig. 7.
Fig. 7.
Genetic interaction between mpk1 and Vangl2/stbm in epiblast organization. (A and B′) Immunofluorescence for PKCζ on transverse sections of the control and mpk1+/−;Vangl2Lp/+ epiblast. (C) Relative fluorescence of immunostaining with an anti-PKCζ antibody measured by ImageJ (see SI Materials and Methods). (D) The quantification of epiblast nuclei shape in the wild-type (+/+;+/+) (n = 3), mpk1+/−;Vangl2+/+ (+/−;+/+) (n = 3), mpk1+/+;Vangl2Lp/+ (+/+;Lp/+) (n = 5) and mpk1+/−;Vangl2Lp/+ (+/−;Lp/+) (n = 5). Values are from 3 independent experiments, n = 20–30 nuclei per experiments. Error bars represent the s.e.m.; asterisk denotes a significant difference (P < 0.05), compared with wild type. (E) The quantification of the AB polarity defects in the mpk1+/−;Vangl2Lp/+ mutant. The number of embryos for the control and mpk1+/−;Vangl2Lp/+ mutant genotypes are indicated at the top.
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