Trophectoderm formation: regulation of morphogenesis and gene expressions by RHO, ROCK, cell polarity, and HIPPO signaling

Abstract

In brief: Trophectoderm is the first tissue to differentiate in the early mammalian embryo and is essential for hatching, implantation, and placentation. This review article discusses the roles of Ras homolog family members (RHO) and RHO-associated coiled-coil containing protein kinases (ROCK) in the molecular and cellular regulation of trophectoderm formation.

Abstract: The trophectoderm (TE) is the first tissue to differentiate during the preimplantation development of placental mammals. It constitutes the outer epithelial layer of the blastocyst and is responsible for hatching, uterine attachment, and placentation. Thus, its formation is the key initial step that enables the viviparity of mammals. Here, we first describe the general features of TE formation at the morphological and molecular levels. Prospective TE cells form an epithelial layer enclosing an expanding fluid-filled cavity by establishing the apical-basal cell polarity, intercellular junctions, microlumen, and osmotic gradient. A unique set of genes is expressed in TE that encode the transcription factors essential for the development of trophoblasts of the placenta upon implantation. TE-specific gene expressions are driven by the inhibition of HIPPO signaling, which is dependent on the prior establishment of the apical-basal polarity. We then discuss the specific roles of RHO and ROCK as essential regulators of TE formation. RHO and ROCK modulate the actomyosin cytoskeleton, apical-basal polarity, intercellular junctions, and HIPPO signaling, thereby orchestrating the epithelialization and gene expressions in TE. Knowledge of the molecular mechanisms underlying TE formation is crucial for assisted reproductive technologies in human and farm animals, as it provides foundation to help improve procedures for embryo handling and selection to achieve better reproductive outcomes.

Figure 1.

The trophectoderm of the blastocyst. (A) Left: A photograph of a mouse blastocyst at around 3 days after fertilization. Right: A schematic drawing of the blastocyst, highlighting the key morphological features. (B) Morphological transformation of the mouse embryo from the 2-cell stage to the hatching blastocyst. (C) A photograph of a hatching blastocyst around 4 days after fertilization. Note that the expansion of the blastocyst cavity has opened the zona pellucida. Scale bars, 50 μm.

Figure 2.

Morphogenesis of the trophectoderm. (A) Establishment of the apical-basal polarity in outside cells through differential localizations of the apical and basal domain proteins. (B) Top: Formation of the tight junction at the boundaries between the apical and basal domains. Bottom: A projected confocal microscopy image of a mouse blastocyst that is immunohistochemically stained for the TJP1 protein. Scale bar, 50 μm. (C) Emergence and coalescence of microlumens into a single blastocyst cavity. (D) Expansion of the blastocyst cavity by active intake of sodium ions, followed by the influx of water along the osmotic gradient.

Figure 3.

Differential regulation of HIPPO signaling between outside and inside cells. In polar outside cells, AMOT is sequestered to the apical domain, which inactivates HIPPO signaling. As a result, YAP is unphosphorylated and translocates into the nucleus to interact with TEAD4 to activate transcription of the target genes. In non-polar inside cells, AMOT is located at the site of cell-cell contact, where it associates with NF2 and LATS to phosphorylate YAP. Phosphorylated YAP is either retained in the cytoplasm or degraded, and thus is unable to interact with TEAD4 to activate transcription.

Figure 4.

Tight junction in Tead4-knockdown embryos. (A) Mouse embryos that have been injected with control (nontarget) shRNA plasmid or Tead4 shRNA plasmid. Bright field images show that Tead4-knockdown prevents the blastocyst cavity formation. Fluorescence images show immunohistochemical (IHC) staining of the TJP1 protein as continuous lines along the apical edge of the cell-cell borders in both control and Tead4-knockdown embryos. Transmission electron microscopy (TEM) images show electron dense structures (white arrows) at the apical border of cell-cell contact, characteristic of the tight junction, in both control and Tead4-knockdown embryos. Scale bars, 50 μm (left), 20 μm (middle), and 0.2 μm (right). (B) Top: A schematic overview of the chimera experiment. GFP-transgenic fertilized eggs are injected with control, Pard6b, or Tead4 shRNA plasmid. At the 8-cell stage, the injected embryos are combined with non-transgenic uninjected embryos and allowed to develop as chimeras up to the blastocyst stage. Bottom: Bright field and fluorescence images of a representative chimera for each shRNA injection group. Note that control and Tead4 shRNA chimeras possess a large cavity, implicating the presence of intact paracellular sealing by the tight junction. By contrast, Pard6b shRNA chimera fails to form a blastocyst cavity. Scale bar, 50 μm. The data shown are from unpublished studies of the authors (V.B.A. and Y.M.).

Figure 5.

Regulation of the trophectoderm morphogenesis and gene expressions by RHO and ROCK. (A) General molecular characteristics of RHO and ROCK. See the text for details. (B) Impact of RHO or ROCK inhibition on the distributions of the apical and basal domain proteins and YAP protein in outside cells. (C) Models depicting the molecular mechanisms by which RHO and ROCK regulate two aspects of trophectoderm formation: morphogenesis and gene expression. See the text for details.