Temporally resolved single cell transcriptomics in a human model of amniogenesis

  1. Nikola Sekulovski
  2. Amber E Carleton
  3. Anusha A Rengarajan
  4. Chien-Wei Lin
  5. Lauren L Juga
  6. Allison E Whorton
  7. Jenna Kropp Schmidt
  8. Thaddeus Golos
  9. Kenichiro Taniguchi

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Abstract

Amniogenesis is triggered in a collection of pluripotent epiblast cells as the human embryo implants. To gain insights into the critical but poorly understood transcriptional machinery governing amnion fate determination, we examined the evolving transcriptome of a developing human pluripotent stem cell-derived amnion model at the single cell level. This analysis revealed several continuous amniotic fate progressing states with state-specific markers, which include a previously unrecognized CLDN10+ amnion progenitor state. Strikingly, we found that expression of CLDN10 is restricted to the amnion-epiblast boundary region in the human post-implantation amniotic sac model as well as in a peri-gastrula cynomolgus macaque embryo, bolstering the growing notion that, at this stage, the amnion-epiblast boundary is a site of active amniogenesis. Bioinformatic analysis of published primate peri-gastrula single cell sequencing data further confirmed that CLDN10 is expressed in cells progressing to amnion. Additionally, our loss of function analysis shows that CLDN10 promotes amniotic but suppresses primordial germ cell-like fate. Overall, this study presents a comprehensive amniogenic single cell transcriptomic resource and identifies a previously unrecognized CLDN10+ amnion progenitor population at the amnion-epiblast boundary of the primate peri-gastrula.

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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    Using human pluripotent stem cell-based Gel-3D model of amniogenesis this study investigates the transcriptional dynamics of amnion differentiation at single cell level. Seven cell clusters are identified that emerge over four days of differentiation, including progressive amniotic fates precursors, among them CLDN10 progenitor located on the boundary of amnion and epiblast, and primordial germ cell-like fate. Mutational studies support the role of CLDN10 in promoting amniotic but limiting primordial germ cell-like.

    Major Comments:

    This generally clearly presented study significantly advances our understanding of human/NHP amniogenesis and should be of broad interest with relevance to human reproduction. However, the there are several questions about how experiments were performed, analyzed, presented and interpreted that need to be answered.

    1. The presented antibody stainings while beautiful are presented without sufficient quantification. A single representative (?) cyst is shown. Please provide information about how many cysts on average have been analyzed in how many experiments, expression levels of should be quantified to support conclusions such as: Line 116-118 "a subset of cells within the cysts displays reduced expression of NANOG, while TFAP2A expression becomes weakly activated"; or Line 129 "the transition from pluripotent to amnion cell types occurs progressively over the cyst, starting from focal initiation sites".
    2. As the the scRNA-seq experiment is one of the main advances of this study and it explores the temporal dynamics and transitional cell populations during amniogenesis this experiment should be performed with two independent biological replicates to investigate the variability of the amniogenesis in this model in terms of the proportion of the 7 distinct cell populations the authors identified in this analysis.
    3. Another interesting parallel between the amnion model and the CS7 human gastrula is most Tyser "Epiblast" cells are seen in the "pluripotency-exiting" population of the amnion model. However, pluripotency exit is a hallmark of epiblast as it initiates gastrulation and primitive streak formation/mesendoderm differentiation. This should be analyzed and discussed further, especially that the authors see in the amnion model some cells expressing TBXT at low level.
    4. How do the authors explain/interpret the difference in CLDN10 expression at RNA and protein level?
    5. Two hESC CLDN10 mutant lines are presented in Figure S4, which are transheterozygous for framesfhit mutations. However, it is not clear how (guideRNAs), in which position of the gene these mutations were generated and what is predicted mutant protein product of each allele. Please provide, gene structure, gRNA position and predicted protein product cartoons. As we do not know the antigen recognized by CLDN10 antibody, these are critical considerations.
    6. What are the consequences of these mutations on CLDN10 transcript? qPCR and also scRNA-seq data the authors have.
    7. Please indicate in the experiments using CLDN10 mutant lines, which KO line has been used for specific experiment and whether same/different results have been obtained with the two lines.
    8. The excess of PGCL cells in CLDN10 KO Gel-3D amnion model is an important observation, but not fully supported by the data. We are presented with single images of mutant cysts at different stages of amniogenesis. Additional data and the number of SOX17+ cells in WT and mutant cysts at should be provided.
    9. The authors propose an interesting concept of CLDN10 at the boundary between the amnion and the epiblast promoting amniogenesis and limiting hHPGLC formation. They speculate about the role of tight junction in this process in agreement with increased hHPGLC formation upon ZO1 reduction in another hPSC model. However, surprisingly little discussion is provided about signaling implications of the reported amniogenic transcriptional cascade, and signals emanating from the different amnion progression cell types. Given the important role of BMP in the formation of amnion and hPGCs, notable is increasing expression of BAMBI in progenitor cell types and high expression in specified and maturing clusters. The expression of signaling pathway components should be analyzed and discussed in more depth.

    Additional comments:

    1. It is not easy to discern the numbers of the seven populations that are detected at D1-D4 from Figure 1C. A panel in Figure 1 illustrating this would be informative.
    2. The similarity of the "Ectoderm" cluster from the CS7 human gastrula Tyser et al., 2021 to extraembryonic cell type with amnion/trophectoderm characteristics in hESC 2D-gastruloid model has been reported by Minn et al., Stem Cell Reports, 2021 and this should be acknowledged.

    Referees cross-commenting

    There is consensus among the reviewers that this is a novel and important work, but additional experiments and their rigorous quantification is needed. Attending to the reviewers comments will significantly elevate this exciting work.

    Significance

    Occurring upon implantation of human blastocyst, amniogenesis, or formation of the amniotic sac from the pluripotent epiblast, is still poorly understood but essential process of human embryogenesis. The key morphogenetic aspects of amniogenesis, i.e. epithelial polarization of epiblast into a cyst and subsequently differentiation of the portion of the cyst abutting the trophectoderm proximal to the uterus into squamous epithelium is in part modeled by the hESC-based amnion models in which BMP stimulation plays a crucial role. In the Gel-3D amnion model model deployed here, no exogenous BMP is added, however, BMP signaling is activated in the cells by a mechanosensitive cue provided by the soft substrate; hESCs initially form a cyst of epithelial cells expressing pluripotent markers that initiate transcriptional cascade and within 4 days of culture differentiate into a cyst of squamous-amnion-like epithelium.

    This work expands on the previous studies by investigating the transcriptional dynamics of amnion differentiation at single cell level combined with additional antibody stainings and compare their findings to distinct cell types in a Carnegie stage 7 human embryo (Tyser et al., 2021) and relevant non-human primate datasets. Based on the resulting data the authors posit contiguous amniogenic cell states: pluripotency-exiting, early progenitor, late progenitor, specified and maturing. Moreover, they also uncover that this model of amniogenesis also produces primordial germ cell-like (hPGC-L) and mesoderm-like cells. A notable finding is that high levels of CLDN10 mark a later transient progenitor state, but CLDN10 expression is downregulated more differentiated cells. Moroever, the authors posit that CLDN10 is a marker of the progenitor population, expression of which is restricted to the boundary between the amnion and the epiblast of the cynomolgus macaque peri-gastrula. Functional interrogation of CLDN10 using hESC mutant lines in the Gel-3D amnion model shows reduced amniogenesis and excess of hPGC-L cells. The authors propose that the CLDN10 the amnion-epiblast boundary is a site of active amniogenesis but limits hPGC-L. This work advances our understanding of amniogenesis, strengthens the concept that amnion and PGC progressing cells initially share acommon intermediate lineage, provides a valuable transcriptomic dataset and should be of broad interest with relevance to human development and reproduction.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Sekulovski et al characterize the transcriptome of human pluripotent stem cells differentiating to an amnion fate in 3D, using single-cell RNA sequencing. This leads to the identification of CLDN10+ cells as amnion progenitors. When CLDN10 is eliminated, amniogenesis is compromised. Moreover, analysis of CLDN10 localization in cynomolgus macaque embryos reveals that this progenitor population is located at the boundary between the epiblast and the amnion.

    Major comments:

    The key results are convincing and supported by clear experiments. However, additional controls, quantifications, and clarifications are needed as follows:

    • The authors identify five amnion-progressing states in vitro and mention that each of these states also shows transcriptional similarities to cell types in a CS7 embryo (Tyser et al, 2021). How do the authors interpret this result? Would this mean that there are amnion cells at all different maturation stages present at a specific time point in development? Given that the available in vivo reference is derived from a single human embryo, it is more likely that the true in vivo counterpart of these states is not captured in the embryo data.
    • The authors stain the 3D amnion model at different stages and conclude that "amniogenesis initiates focally and spreads laterally". This cannot be concluded from the data provided. The images in Figure 1 simply show heterogeneity in the levels of TFAP2A. To support their claims, the authors would need to perform time-lapse experiments using a TFAP2A reporter line.
    • The authors conclude that CLDN10+ cells give rise to amnion during gastrulation of cynomolgus macaque embryos. The data provided does not prove that CLDN10+ cells are the amnion progenitors in vivo.
    • CLDN10 KO cells form amnion cysts like control cells by day 3. However, by day 4 the cysts lose expression of the amnion marker ISL1 and become disorganized. To characterize the epithelial (or lack of) phenotype, the authors should include membrane/polarity/adhesion immunostainings. Is the disorganization observed at day 4 associated with the progressive changes in cell identity, or is it a time-dependent phenotype? The authors should include human PSC cysts as a control. This would allow them to determine whether the role of CLDN10 is specific to amnion cells.
    • Figure 2: is there a correlation between the levels of CLDN10 and TFAP2A based on the scRNAseq data and the immunofluorescence stainings? The IF data would benefit from quantifications.
    • Figure 4: the experiment has not been quantified. What is the % of PGCLCs in WT and KO cells? What are the levels of ISL1 in WT and KO cells? What is the localization of epithelial determinants in WT and KO cells? Is there an anti-correlation between CLDN10 and ISL1?

    Referees cross-commenting

    I think there is a general consensus that additional quantifications and careful analyses are needed before this paper is accepted for publication. I agree with the comments raised by the other reviewers.

    Significance

    This manuscript is a follow-up work of Sekulovski et al, 2024. In this recent manuscript, the authors already provided a temporally resolved transcriptomic characterization of in vitro amniogenesis. The key difference between the two articles is that while Sekulovski et al, 2024 performed a bulk RNAseq experiment, in the current manuscript a single-cell RNAseq experiment has been done. It is fundamental to clearly define what new findings have been obtained thanks to the single-cell experiment, which could not have been obtained using the bulk transcriptomics data. This is a particularly important point given the robustness and synchrony of the model. For example, the authors had already identified five amnion states in vitro in their previous publication. Is CLDN10 differentially expressed in the progenitor population based on the bulk RNAseq data? Are the same dynamics of expression recapitulated? The title of the manuscript does not mention CLDN10 but rather focuses on transcriptional profiling at the single-cell level. In my opinion, the key novelty of this manuscript is the identification of CLDN10 and the role it plays during amniogenesis. Focusing the manuscript on the dynamic transcriptional profile diminishes the novelty, as this had already been done by the authors at the bulk level. Globally, this manuscript provides additional information of the poorly understood process of amniogenesis that will be interesting for those working on early human embryogenesis.

    My area of expertise is early mammalian embryo development and stem cells. I do not have the computational background to evaluate the bioinformatic analyses of the manuscript in-depth.

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    Referee #1

    Evidence, reproducibility and clarity

    This is a novel and important and interesting study that uses one of the best amniogeneisis form PSCs int he field. The authors do scRNA-seq during 4 day course to understand different populations emerging during amniogeneisis, and they identify CLDN10 as a marker for newly emerging new amion cells, and then use their model and monkey real embryos to prove the CLDN10+ population at the amnion-epiblast border. In the final part, the authors knockout CLDN10 and claim it compromises amniogenesis and favours formation.

    Significance

    This is a well conducted study, and conclusions are novel and super exciting and IMPORTANT!!!. I have one-2 major comments to strengthen conclusions in the last part, and will help make this excellent study become superb and a landmark study.

    1. it is not really clear what is the phenotype of CLDN10 KO cells. is amniogenesis totally inhibited? can the authors do scRNA-seq on the KO cells and compare them to WT cells? There is no quantitation to amnion or PGC formation efficiency ? how many structures where analyzed?
    2. in continuation with the above The claim that PGC formation is enhanced in KO is not strong. PGCs should be stained for NANOS3 and blimp1 specific marker and not only SOX17 which can also be a Pre marker. Then quantification should be properly done.
  5. Version published to 10.1101/2023.09.07.556553v3 on bioRxiv
  6. Version published to 10.1101/2023.09.07.556553v2 on bioRxiv
  7. Version published to 10.1101/2023.09.07.556553v1 on bioRxiv