Temporally resolved early BMP-driven transcriptional cascade during human amnion specification

Reviewer #1 (Public Review):

Sekulovski et al present an interesting and timely manuscript describing the temporal transition from epiblast to amnion. The manuscript builds on their previous work describing this process using stem cell models.

They suggest a multi-step process initiated by BMP induction of GATA3, followed by expression of TFAP2A, followed by ISL1/HAND1 in parallel with loss of pluripotency markers. This transition was reproduced through IF analysis of CS6/7 NHP embryo.

There are significant similarities in the expression of trophectoderm and the amnion. There are also ample manuscripts showing trophoblast induction following BMP stimulation of primed pluripotent stem cells. The authors should ensure that the amnion indeed is only amnion and not trophectoderm (or the amount of contribution to trophectoderm). As an extension, does the amnion character remain after the 48h BMP4 treatment, and is a trophectoderm-like state adopted as suggested by Ohgushi et al 2022?

The functional data does not support a direct function of GATA3 prior to TFAP2A and the authors suggest compensatory mechanisms from other GATAs. If so, which GATAs are expressed in this system, with and without GATA3 targeting? Would it not be equally likely that the other early genes could be the key drivers of amnion initiation, such as ID2?

The targeting of TFAP2A displays a very interesting phenotype which suggests that amnion and streak share an initial trajectory but where TFAP2A is necessary to adopt amnion fate. It would again be important to ensure that this alternative fate is indeed in streak and not misannotated alternative lineages, including trophoblast.

Is TBXT induced in this setting as well as in the wt situation during amnion induction? This should be displayed as in Figure 3D and would be nice to be complimented by NHP IF analysis.

The authors should address why they get different results from Castillo-Venzor et al 2023 DOI 10.26508/lsa.202201706

Reviewer #2 (Public Review):

In this study, Sekulovski and colleagues report refinements to an in vitro model of human amnion formation. Working with 3D cultures and BMP4 to induce differentiation, the authors chart the time course of amnion induction in human pluripotent stem cells in their system using immunofluorescence and RNA-seq. They carry out validation through comparison of their data to existing embryo datasets, and through immunostaining of post-implantation marmoset embryos. Functional experiments show that the transcription factor TFAP2C drives the amnion differentiation program once it has been initiated.

There is currently great interest in the development of in vitro models of human embryonic development. While it is known that the amnion plays an important structural supporting role for the embryo, its other functions, such as morphogen production and differentiation potential, are not fully understood. Since a number of aspects of amnion development are specific to primates, models of amniogenesis will be valuable for the study of human development. Advantages of this model include its efficiency and the purity of the cell populations produced, a significant degree of synchrony in the differentiation process, benchmarking with single-cell data and immunocytochemistry from primate embryos, and identification of key markers of specific phases of differentiation. Weaknesses are the absence of other embryonic tissues in the model, and overinterpretation of certain findings, in particular relating bulk RNA-seq results to scRNA-seq data from published analyses of primate embryos and results from limited (though high quality) embryo immunostainings.

Reviewer #3 (Public Review):

In this work, the authors tried to profile time-dependent changes in gene and protein expression during BMP-induced amnion differentiation from hPSCs. The authors depicted a GATA3 - TFAP2A - ISL1/HAND1 order of amniotic gene activation, which provides a more detailed temporary trajectory of amnion differentiation compared to previous works. As a primary goal of this study, the above temporal gene/protein activation order is amply supported by experimental data. However, the mechanistic insights on amniotic fate decision, as well as the transcriptomic analysis comparing amnion-like cells from this work and other works remain limited. While this work allows us to see more details of amnion differentiation and understand how different transcription factors were turned on in a sequence and might be useful for benchmarking the identity of amnion in ex utero cultured human embryos/embryoids, it provides limited insights on how amnion cells might diverge from primitive streak / mesoderm-like cells, despite some transcriptional similarity they shared, during early development.