Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band ∼22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as “claudin-1” and “claudin-2”, respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

Mouse pre-implantation development gives rise to the blastocyst, which is made up of at least three distinct cell types: the trophectoderm (TE) that surrounds a cavity, and an inner cell mass (ICM) comprising the primitive endoderm (PE) and epiblast (EPI). However, the underlying mechanisms involved in patterning the cleavage-stage embryo are still unresolved. By analyzing the distribution of the transcription factors Oct4 (Pou5f1), Cdx2 and Nanog at precisely defined stages in pre-implantation development, we were able to identify critical events leading to the divergence of TE, EPI and PE lineages. We found that Oct4 is present in all cells until late blastocyst, gradually disappearing from the TE thereafter. The expression patterns of both Cdx2 and Nanog exhibit two specific phases, culminating in their restriction to TE and EPI, respectively. In the first phase, starting after compaction, blastomeres show highly variable Cdx2 and Nanog protein levels. Importantly, the variability in Nanog levels is independent of position within the morula, whereas Cdx2 variability may originate from asymmetric cell divisions at the 8-cell stage in a non-stereotypic way. Furthermore, there is initially no reciprocal relationship between Cdx2 and Oct4 or between Cdx2 and Nanog protein levels. In the second phase, a definite pattern is established, possibly by a sorting process that accommodates intrinsic and extrinsic cues. Based on these results, we propose a model in which early embryonic mouse patterning includes stochastic processes, consistent with the highly regulative capacity of the embryo. This may represent a feature unique to early mammalian development.

It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterised the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.

During pre-implantation development, the mammalian embryo self-organizes into the blastocyst consisting of an epithelial layer encapsulating the inner-cell mass (ICM), which gives rise to all embryonic tissues1. In mice, oriented cell division, apico-basal polarity and acto-myosin contractility are thought to contribute to the formation of the ICM2–5. However, how these processes work in concert remains unclear. Here, we show that asymmetric segregation of the apical domain generates blastomeres with different contractility, which triggers their sorting into inner and outer positions. 3D physical modeling of embryo morphogenesis reveals that cells internalize only when differences in surface contractility exceed a predictable threshold. We validate this prediction using biophysical measurements and successfully re-direct cell sorting within the developing blastocyst using maternal myosin (Myh9) knockout chimeric embryos. Finally, we find that loss of contractility causes blastomeres to show ICM-like markers regardless of their position. In particular, contractility controls Yap sub-cellular localization6, raising the possibility that mechanosensing occurs during blastocyst lineage specification. We conclude that contractility couples the positioning and fate specification of blastomeres. We propose that this ensures the robust self-organization of blastomeres into the blastocyst, which confers remarkable regulative capacities to mammalian embryos.

Mammalian embryos initiate morphogenesis with compaction, which is essential for specifying the first lineages of the blastocyst. The 8-cell-stage mouse embryo compacts by enlarging its cell-cell contacts in a Cdh1-dependent manner. It was therefore proposed that Cdh1 adhesion molecules generate the forces driving compaction. Using micropipette aspiration to map all tensions in a developing embryo, we show that compaction is primarily driven by a twofold increase in tension at the cell-medium interface. We show that the principal force generator of compaction is the actomyosin cortex, which gives rise to pulsed contractions starting at the 8-cell stage. Remarkably, contractions emerge as periodic cortical waves when cells are disengaged from adhesive contacts. In line with this, tension mapping of mzCdh1(-/-) embryos suggests that Cdh1 acts by redirecting contractility away from cell-cell contacts. Our study provides a framework to understand early mammalian embryogenesis and original perspectives on evolutionary conserved pulsed contractions.