Cell polarity regulator PARD6B is essential for trophectoderm formation in the preimplantation mouse embryo

Abstract

In preimplantation mouse development, the first cell lineages to be established are the trophectoderm (TE) and inner cell mass. TE possesses epithelial features, including apical-basal cell polarity and intercellular junctions, which are crucial to generate a fluid-filled cavity in the blastocyst. Homologs of the partitioning defective (par) genes in Caenorhabditis elegans are critical regulators of cell polarity. However, their roles in regulating TE differentiation and blastocyst formation remain unclear. Here, the role of mouse Pard6b, a homolog of par-6 gene and a component of the PAR-atypical protein kinase C (aPKC) complex, was investigated. Pard6b expression was knocked down by microinjecting RNA interference construct into zygotes. Pard6b-knockdown embryos cleaved and compacted normally but failed to form the blastocyst cavity. The cavitation failure is likely the result of defective intercellular junctions, because Pard6b knockdown caused abnormal distribution of actin filaments and TJP1 (ZO-1) tight junction (TJ) protein and interfered with cavitation in chimeras containing cells from normal embryos. Defective TJ formation may be caused by abnormal cell polarization, because the apical localization of PRKCZ (aPKCzeta) was absent in Pard6b-knockdown embryos. Pard6b knockdown also diminished the expression of CDX2, a TE-lineage transcription factor, in the outer cells. TEAD4, a transcriptional activator that is required for Cdx2 expression and cavity formation, was not essential for the transcription of Pard6b. Taken together, Pard6b is necessary for blastocyst morphogenesis, particularly the development of TE-specific features-namely, the apical-basal cell polarity, formation of TJ, paracellular permeability sealing, and up-regulated expression of Cdx2.

FIG. 1.

Pard6b knockdown suppresses formation of the blastocyst cavity. A) Selection of effective Pard6b shRNA plasmids in P19 mouse embryonal carcinoma cells. Results of the qRT-PCR analysis of Pard6b expression in P19 cells that are transiently transfected with shRNA plasmids for 24 h (control encoding a nontarget sequence and five types of Pard6b shRNA) are shown. Pard6b mRNA levels were measured using primers for the protein-coding region (gray bars) and for the 3′ untranslated region (white bars) and normalized by Gapdh mRNA level. Bars indicate mean ± SD. B) Immunostaining for MYC-tagged PARD6B protein in P19 cells that are cotransfected with control or Pard6b shRNA 2 plasmid. The signal for MYC-tagged PARD6B protein is markedly reduced by Pard6b shRNA plasmid. Nuclei are stained with DAPI. C) Images taken by time-lapse video microscopy at three different time points in one set of experiments corresponding to the 8-cell embryos during compaction, morula, and blastocyst stage. Eight embryos on the left side of the images have been injected with control shRNA plasmid, whereas eight embryos on the right side have been injected with Pard6b shRNA plasmid. Time elapsed after the administration of hCG is indicated in hours (h). D) Comparison of the timing of blastocyst cavity formation between control shRNA plasmid-injected and Pard6b shRNA plasmid-injected embryos. Data are a compilation of three independent sets of experiments, and the total number of embryos examined is indicated (n). Bar = 100 μm (B and C).

FIG. 2.

Gene expression in Pard6b-knockdown and Tead4-knockdown embryos. A) Z-series projections of confocal images of embryos at the early blastocyst stage (100 h post-hCG) that were immunostained for histone protein. Arrows point to mitotic nuclei. Embryos had been injected with control shRNA plasmid and Pard6b shRNA plasmid. B) Bar graph summarizing the total number of nuclei per embryo (mean ± SD) as determined by histone staining. Mean number of mitotic nuclei per embryo are represented by the shaded portion of the bar. Total number of embryos examined per group is indicated (n). C) Quantitative RT-PCR analysis of shRNA plasmid-injected embryos at the early blastocyst stage (100 h post-hCG). Relative expression levels of Gapdh, Pard6b (3′ untranslated region [UTR]), Cdx2, Tead4, Pou5f1, and Nanog are shown as percentages of their expression levels in Pard6b shRNA plasmid-injected embryos relative to those in control shRNA plasmid-injected embryos. In each set of experiments, the expression level of each gene is normalized by that of Actb. Bars indicate mean ± SD. P values for Pard6b, Cdx2, and Nanog are based on Student t-test against Gapdh, indicating that the change in these genes by the Pard6b shRNA plasmid is statistically significant. D) Immunostaining for PARD6B protein in shRNA plasmid-injected embryos at the morula stage (90 h post-hCG). A single confocal optical section near the equator of a representative embryo is shown for each group. Graph shows a comparison of fluorescence intensity of PARD6B immunostaining between control shRNA plasmid-injected and Pard6b shRNA plasmid-injected embryos. Circles represent the intensity in individual embryos, and horizontal bars represent means (n = 12 for control, n = 13 for Pard6b shRNA plasmid). P value is based on Student t-test between the two groups, indicating that the reduction in PARD6B protein level by Pard6b shRNA plasmid is statistically significant. E) Knockdown of Tead4 reduces TE-lineage transcription factors and elevates pluripotency transcription factors. Results of the qRT-PCR analysis of shRNA plasmid-injected embryos at the early blastocyst stage (100 h post-hCG) are shown. Relative expression levels of Gapdh, Tead4, Cdx2, Pou5f1, Nanog, and Pard6b (3′ UTR) are shown as percentages of their expression levels in Tead4 shRNA plasmid-injected embryos relative to those in control shRNA plasmid-injected embryos. In each set of experiments, the expression level of each gene is normalized by that of Actb. Bars indicate mean ± SD. P values for Tead4, Cdx2, and Nanog are based on Student t-test against Gapdh, indicating that the change in these genes by Tead4 shRNA plasmid is statistically significant. Bar = 100 μm (A and D).

FIG. 3.

Knockdown of Pard6b impairs the localization of cytoplasmic actin filaments, TJP1, and PRKCZ, but not intercellular CDH1 and CLDN4, in the outer cells of the embryo. A and B) Immunostaining for CDH1 protein in shRNA-injected embryos (90 h post-hCG). Images are average intensity projections of three confocal optical sections near the equator. The distinct staining at cell-cell boundaries are observed in both control shRNA plasmid-injected and Pard6b shRNA plasmid-injected embryos, suggesting that the knockdown of Pard6b does not impair the expression and localization of CDH1 at cell-cell boundaries. C and D) Phalloidin-staining for actin filaments in shRNA-injected embryos at the early blastocyst stage (100 h post-hCG). Images are Z-series projections of confocal sections of embryos. In the control shRNA plasmid-injected embryo, actin filaments are enriched at the cell-cell boundaries, particularly near the apical edge, reflecting their association with the AJ. In the Pard6b shRNA plasmid-injected embryo, the localization of actin filaments along the apical edge of cell-cell boundaries is indistinct, and actin filaments appear to be more enriched throughout the apical cortex of outer cells. E and F) Immunostaining for CLDN4 protein in shRNA-injected embryos at the early blastocyst stage (100 h post-hCG). Embryos were imaged via fluorescence microscopy. Pard6b knockdown apparently does not disturb CLDN4 distribution, because it is localized similarly at the apical edge of cell-cell boundaries to form the TJ in control shRNA plasmid-injected and Pard6b shRNA plasmid-injected embryos. G and H) Immunostaining for TJP1 protein in shRNA-injected embryos at the early blastocyst stage (100 h post-hCG). Images are Z-series projections of confocal sections of embryos. In the control shRNA plasmid-injected embryo, TJP1 is localized at the apical edge of cell-cell boundaries. In the Pard6b shRNA plasmid-injected embryo, the localization of TJP1 is severely impaired such that the staining is weak or discontinuous along cell-cell boundaries. I and J) Immunostaining for PRKCZ protein in shRNA-injected embryos (90 h post-hCG). Single confocal optical sections near the equator are shown. In the control shRNA plasmid-injected embryo, the PRKCZ staining is enriched at the apical cortex of outer cells but weak at the cell-cell boundaries. In the Pard6b shRNA plasmid-injected embryo, the apical staining of PRKCZ is indistinct, whereas cytoplasmic staining is increased. Bar = 100 μm (A–D and G–J) and 50 μm (E and F).

FIG. 4.

Knockdown of Pard6b interferes with paracellular permeability sealing in the outer cells and impairs the blastocyst cavity formation. A) A schematic overview of the chimera experiment. Egfp-transgenic fertilized eggs are injected with control (nontarget) shRNA plasmid or Pard6b shRNA plasmid. At the 8-cell stage, the injected embryos are combined with nontransgenic uninjected embryos and allowed to develop as chimeras up to the blastocyst stage. As a comparison, nontransgenic, uninjected embryos are cultured by themselves as nonchimeras up to the same stage. B) Bright-field and fluorescence images of two representative chimeras for each shRNA plasmid injection group at 96 h post-hCG. Note that both control shRNA chimeras possess a large cavity (occupying >50% of the embryo in volume) and that EGFP-positive cells are found in both epithelial and nonepithelial portions. By contrast, Pard6b shRNA chimeras possess only small cavities (<50% of the embryo in volume; top) or have no cavity (bottom). Bar = 100 μm. C) Comparison of the efficiency of blastocyst cavity formation among two types of shRNA chimeras and nonchimeras. The definitions for large and small cavities are as described in B. Total numbers of embryos examined are indicated (n).

FIG. 5.

A) Double immunostaining for CDX2 (red) and POU5F1 (green) proteins in shRNA plasmid-injected embryos (100 h post-hCG). Z-series projections of confocal images of embryos are shown for each group. In the control shRNA plasmid-injected embryo, CDX2 is stained more intensely in the nuclei of outer cells than in the nuclei of inner cells, whereas POU5F1 is stained ubiquitously among all the nuclei. As a result, outer cell nuclei exhibit an orange color in the merge image (right). In the Pard6b shRNA plasmid-injected embryo, CDX2 is stained less intensely in all the nuclei, which as a result exhibit a green color in the merge image. Images were collected during the same confocal session using the same settings. B) Quantification of relative intensity of CDX2 to POU5F1 immunostaining in shRNA plasmid-injected embryos. Data are a compilation of two independent experiments; a total of 13 and 14 embryos were examined for the control and Pard6b shRNA groups, respectively. For each embryo, nuclei of five inner cells and five outer cells were arbitrarily chosen for fluorescence intensity measurement, and the mean ± SD from all the measurements for each group are presented in the graph. P value is based on Student t-test, indicating that the relative intensity of CDX2 to POU5F1 in outer cells is significantly reduced by the knockdown of Pard6b. Outer cell nucleus was identified as lacking adjacent nucleus at one side when images of the embryos were scanned in the z-axis via confocal microscopy. A nucleus was considered to be internal when it was surrounded by other nuclei, as observed when images of the embryos were scanned in the z-axis. C) Immunostaining for NANOG (green) and propidium iodide (PI) staining for nuclei (red) in shRNA plasmid-injected embryos (100 h post-hCG). Z-series projections of confocal images of embryos are shown for the control and Pard6b shRNA groups. Images were collected during the same confocal session using the same settings. D) Levels of NANOG protein in shRNA plasmid-injected embryos. NANOG fluorescence levels were normalized by PI fluorescence levels for each nucleus. Data are a compilation of two independent experiments; a total of six embryos were examined for each group. For each embryo, nuclei of five inner cells and five outer cells were arbitrarily chosen for measurement, and the mean ± SD from all the measurements for each group are presented in the graph. P value is based on Student t-test, indicating that the relative intensity of NANOG to PI in outer cells is significantly elevated by the knockdown of Pard6b. E) Increased incidence of apoptosis at the late blastocyst stage by the knockdown of Pard6b. The shRNA plasmid-injected embryos were analyzed at 124 h post-hCG for apoptosis by TUNEL (left) and stained with phalloidin for actin filaments (middle). Numbers at the right-bottom corner in the TUNEL images correspond to the TUNEL-positive area in the arbitrary unit that is depicted in the graph in F. The representative samples show that the TUNEL-positive area is larger in the Pard6b shRNA plasmid-injected embryos than in the control shRNA plasmid-injected embryos, both of which have been cultured in KSOM. However, the incidence of apoptosis resulting from the knockdown of Pard6b is reduced when the injected embryos have been cultured in ESCM from the 8-cell stage. F) Graph shows the mean ± SD of TUNEL-positive area in control shRNA plasmid-injected embryos cultured in KSOM, Pard6b shRNA plasmid-injected embryos cultured in KSOM, and Pard6b shRNA plasmid-injected embryos cultured in ESCM. Numbers of embryos examined are indicated (n). Bar = 50 μm (A and C) and 100 μm (E).