Transcriptional control of apical protein clustering drives de novo cell polarity establishment in the early mouse embryo

Background:

All higher eukaryotic organisms develop from a single-celled zygote, which undergoes active binary divisions and cell fate determination leading to differentiation into different tissues and organs. This is achieved through tightly regulated lineage specification events, determination of embryonic axes and gene expression at specific developmental time-points.

The first fate specification in the mammalian embryo gives rise to the inner cell mass (ICM), which develops into the fetus and the yolk sac, and the outer extra-embryonic trophectoderm (TE), which forms the placenta. This determination of ICM and TE fate requires cell polarization. In mouse, this event is observed around the 8-cell-stage when the cells acquire an apical domain, surrounded by an actomyosin ring.

There is a lack of knowledge pertaining to the mechanistic details and genetic regulators that define cell polarization and apical domain formation. To address this gap, the authors have employed various embryological, gene-editing and live-imaging tools to uncover the mechanism of cell polarization and the key players involved in the first cell fate decision made by the embryo.

Key findings:

A newly formed zygote harbours maternal transcripts. However, as the embryo develops, zygotic transcription takes over developmental control. Since the authors were interested in understanding the initial fate determination, they hypothesized that zygotic transcription may have a significant role in apical domain formation. Using chemical inhibition and cytoplasmic resection, they increased and decreased the concentration of zygotic transcripts before cell polarization, respectively. While transcription inhibition led to a failure in the positioning of polarity proteins at the apical region, resection of cytoplasm (resulting in higher cytoplasmic mRNA concentration) led to the advancement of cell polarization. Further, transcription inhibition in the cytoplasm-resected cells led to simultaneous polarization of these cells and the controls. This suggests the involvement of zygotic transcription in cell polarization.

To identify the specific factors involved in apical domain formation, they analyzed publically available single-cell RNA sequencing and ATAC-seq data and shortlisted 124 genes (118 polarity regulators and 6 transcription factors). Downregulation of each of these candidate genes revealed that only depletion of Tfap2c and Tead4 transcription factors prevents cell polarization at the 8-cell stage.

To confirm the cell polarization inducing ability of Tfap2c and Tead4, the mRNA of these genes was overexpressed (both together and individually) at the 2-cell stage and the authors observed premature cell protrusions enriched in apical polarity proteins. However, these protrusions were small and lacked an actomyosin enclosed apical domain. Therefore, the authors overexpressed RhoA (which is required for the activation of actomyosin and hence apical domain formation) at the 4-cell stage and found a fully formed apical domain. This domain then followed the zippering process to seal boundaries, which is an essential step for blastocyst formation.

Though Tfap2c/Tead4/RhoA has proven to be sufficient for the formation of a functional apical domain, the precocious formation of embryonic structures which do not align with actual developmental time may cause hazardous effects to the embryo. However, in this study the advancement of cell polarization and apical domain formation did not seem to affect embryonic development. In fact, the authors found that the embryos enter into the next stage, i.e. express differentiation transcription factors for TE like cdx2 and gata3. Taken together, these experiments suggest that Tfap2c/Tead4/RhoA is sufficient to induce the formation of a functional apical domain and advance the timing of the cell differentiation program.

Now that we are aware of the factors crucial for the formation of the apical domain, the authors wished to find the mechanism involved in apical domain formation. Through imaging, they found that the polarity proteins first cluster in the centre of the cell contact-free surface concomitant with actin exclusion (centralization step). Then, they expand to form a concentrated patch which is surrounded by ring-like actomyosin (expansion step). They observed that apical protein clustering occurred during actin remodelling. Then, to determine the role of actin cytoskeleton remodelling in apical protein clustering and centralization, they chemically inhibited actin and found that apical protein clustering was affected. They also found that in embryos lacking Tfap2c and Tead4, the centralization step failed, suggesting the key role of Tfap2c and Tead4 in the initiation of the apical domain.

To understand the mechanistic details of Tfap2c-Tead4 regulated apical domain formation, they performed RNA sequencing of 8-cell stage embryos lacking Tfap2c and Tead4 together and individually. Differential gene expression analysis revealed that a significant number of genes downregulated were associated with actin polymerization. When the authors downregulated two of the candidate genes they found that it hampered apical domain formation. Hence, Tfap2c and Tead4 regulate the actin network which in turn aids apical protein centralization and expansion. These events along with the expression of RhoA, which activates actomyosin, contribute to the successful formation of a functional apical domain to execute the first cell fate decision.

What I like about the preprint:

The authors have provided a clear picture of apical domain formation and its polarization, which marks the initiation of the first fate determination event of an embryo. The authors have analyzed the intricate details of the formation of a functional apical domain, from finding the crucial molecular players involved to the demonstration of the biophysical features and mechanism. Also, their use of multiple controls for every experiment strengthens their data and provides elaborate details of the ICM and TE fate-determining event. It is also interesting to note that the authors have for the first time demonstrated controlled premature cell polarization and functional apical domain formation in the mouse embryo.

Questions for the authors:

1. Your findings show that Tfap2c and Tead4 perform similar functions from the onset of the cell polarization to the formation of a functional apical domain. However, unlike Tfap2c, Tead4 alone was unable to enhance the timing of cell polarization. Can you please give us your thoughts on that?
2. Do the authors think that cross-supplementation experiments would provide more insights into the redundancy of Tfap2c and Tead4?
3. Advancing cell polarization advances the cell determination program as evidenced by the premature expression of TE differentiation transcription factors, Cdx2 and Gata3. Did the authors check for the differentiation markers of ICM expressed in the apolar cells as well?
4. It is known that early embryonic development involves widespread epigenetic modifications. Did the authors find any epigenetic modifiers highly regulated at the 2-8 cell stage which facilitates the transcriptional activation of tfap2c or tead4?

Tags: apical domain, cell-polarity, fate determination

doi: https://doi.org/10.1242/prelights.19838

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