Evolutionary transcriptomics implicates HAND2 in the origins of implantation and regulation of gestation length

  1. Mirna Marinić
  2. Katelyn Mika
  3. Sravanthi Chigurupati
  4. Vincent J Lynch

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Abstract

The developmental origins and evolutionary histories of cell types, tissues, and organs contribute to the ways in which their dysfunction produces disease. In mammals, the nature, development and evolution of maternal-fetal interactions likely influence diseases of pregnancy. Here we show genes that evolved expression at the maternal-fetal interface in Eutherian mammals play essential roles in the evolution of pregnancy and are associated with immunological disorders and preterm birth. Among these genes is HAND2 , a transcription factor that suppresses estrogen signaling, a Eutherian innovation allowing blastocyst implantation. We found dynamic HAND2 expression in the decidua throughout the menstrual cycle and pregnancy, gradually decreasing to a low at term. HAND2 regulates a distinct set of genes in endometrial stromal fibroblasts including IL15 , a cytokine also exhibiting dynamic expression throughout the menstrual cycle and gestation, promoting migration of natural killer cells and extravillous cytotrophoblasts. We demonstrate that HAND2 promoter loops to an enhancer containing SNPs implicated in birth weight and gestation length regulation. Collectively, these data connect HAND2 expression at the maternal-fetal interface with evolution of implantation and gestational regulation, and preterm birth.

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  1. Version published to 10.7554/elife.61257 on eLife
  2. eLife

    ###Reviewer #3

    PREreview of "Evolutionary transcriptomics implicates HAND2 in the origins of implantation and regulation of gestation length"

    Authored by Mirna Marinić et al. and posted on bioRxiv DOI: 10.1101/2020.06.15.152868

    Review authors in alphabetical order: Monica Granados, Katrina Murphy, Maria Sol Ruiz, Daniela Saderi

    This review is the result of a virtual, live-streamed journal club organized and hosted by PREreview and eLife. The discussion was joined by 17 people in total, including researchers from several regions of the world, the last preprint author, and the event organizing team.

    Overview and take-home message:

    In this preprint, Marinić et al. begin the beautiful exploration of gene involvement at the maternal-fetal interface of pregnancy evolution with a look at the importance of a known early-pregnancy gene, HAND2. The research team's findings shown through uterine models and a combination of cell, gene, and data analysis demonstrate HAND2's roles in supporting progesterone in placental mammals by down-regulating estrogen in time for implantation, and through IL15 signaling, where both the promotion of immune and placental cell migration as well as up-regulation of estrogen at the end of term for a healthy gestation length is noted. This important work also sheds some light on progesterone's role in non-placental mammal pregnancy where estrogen continues to be produced throughout the pregnancy. Although this work is an important addition to the field of pregnancy evolution, there are some points that need clarification and a few minor concerns that could be addressed in the next version. These are outlined below.

    Positive feedback:

    1. The selection of HAND2 as a hypothetical regulator of gestation was based on previous knowledge, but the authors supported this selection after an extensive phylogenetic analysis of genes expressed in the endometria of pregnant/gravid organisms from several Eutherian and non-Eutherian species.

    2. Several participants evaluated the results as encouraging for looking into other models such as organoids (as stated by the manuscript), and as a great start for a deeper understanding of pregnancy evolution via the study of gene expression.

    3. The potential implications of these results in the field of abnormalities in pregnancy/infertility were also mentioned as relevant.

    4. Definitely recommended for peer review because this is a great start for a deeper understanding of genes involved in the evolution of pregnancy!

    5. I think the fact that there could be a mechanism involved in HAND2 that ends gestation is really interesting.

    6. Cool to learn that HAND2 expression was specific to fibroblasts and the fibroblasts influence signaling in other cell types.

    7. A proposal of a new hypothesis based on "evolutionary" observations.

    8. Enjoyed learning from the author that a uterus is a counter-intuitive place with immune cells making up half the cells to allow for tolerance towards the pregnancy process.

    9. The methods section was quite detailed; including a GitHub repository and on page 17, a data availability statement for images, genes, and related data. I found the manuscript really interesting. Enjoyed it very much!

    10. In general, the manuscript was easy to follow and figures were logically arranged.

    Concerns:

    Areas that could use more clarification:

    1. It was helpful to hear from the author that the known HAND2 gene wasn't knocked out in mice, so it was an easy early pregnancy gene to start with.

    2. To reproduce the study, there were a couple of questions around the production of the conditioned media including, how long were the cells incubated in the media and what was the volume of the media used. Can more details be shared in the next version?

    3. Can you further explain why the opossum was used to measure the estrogen levels?

    4. Please explain why the researchers decided on the TPM=2 expression cut-off.

    -We heard from the author that genes with TPM less than 2 are functioning in the cell; this might be nice to add in the next version.

    1. Can you include your thoughts on why mammals have evolved this way? This might be a good addition to the discussion.

    2. I think that given the technical model limitations present in the study of the uterus, and in the study of different species, it would deserve some comments about limitations in order to highlight these great findings.

    3. The relationship between ESR1 and HAND2 is a little unclear. Is ESR1 expression correlated with HAND2 expression in all species studied?

    Acknowledgments: We thank all participants for attending the live-streamed preprint journal club. We are especially grateful for both the last author's contributions to the discussion and for those that engaged in providing constructive feedback.

    Below are the names of participants who wanted to be recognized publicly for their contribution to the discussion:

    Monica Granados | PREreview | Leadership Team | Ottawa, ON

    María Sol Ruiz | CONICET-University of Buenos Aires | Postdoctoral Researcher | Buenos Aires Argentina

    Katrina Murphy | PREreview | Project Manager | Portland, OR

  3. eLife

    ###Reviewer #2

    The manuscript "Evolutionary transcriptomics implicates HAND2 in the origins of implantation and regulation of gestation length" by Marinić et al. uses an innovative expression dataset in an evolutionary framework to identify a set of transcripts whose endometrial expression emerged at the eutherian stem lineage. One of these is the transcription factor HAND2. Using both existing datasets and experimental data they build a model of the activity of HAND2 and its associated protein IL15 at the maternal-fetal interface and implicate the proteins in both the evolution and disorders of pregnancy. I highly recommend this manuscript. This work illustrates the utility of evolutionary analysis for elucidating functional mechanisms of complex disorders. The authors support their evolutionary analysis with a thorough characterization, including additional experimental data, of their hypothesized gene association. This work substantially contributes to our knowledge of the evolution and diseases of pregnancy.

    I have only two point of inquiry that I believe the authors should address in the manuscript:

    1. Of the 149 genes that unambiguously evolved endometrial expression why was only HAND2 analyzed? I am not suggesting that each gene be followed up with this level of rigor but would you hypothesize that each of the genes you identified play a role in eutherian reproduction? Or are there other major innovations that some of these genes may be associated with? How frequently would this pattern occur by chance?

    2. Figures 2F and 4F - there appears to be a gap in the data points during the third trimester (which looks like it says "thirdr"). Is there still a negative trend if each section is analyzed independently as if they were independent datasets? Aka could this linear trend be composed of two separate trends instead?

  4. eLife

    ###Reviewer #1

    Parsing mechanisms of disease from the perspective of evolutionary biology is an interesting approach. This perspective may be particularly advantageous when focussing the 'bigger picture' as it is perhaps less constrained by details that tend to preoccupy more conventional disease-focussed studies, such as clinical phenotyping, timing of biopsies, sample size, validation studies etc. In this study, Marinić and colleagues made use of a wealth of publicly available data sets to argue for a role of HAND2-IL15 axis in endometrial cells in implantation and, more importantly, the onset of parturition. The observation that enhancer regions in both HAND2 and IL15 harbour SNPs associated with gestational length/preterm birth renders the study timely and compelling. However, to my knowledge, the impact of these SNPs on the expression of either gene is not known. Further, the lack of validation studies on clinical samples renders the proposed mechanism plausible but speculative, as acknowledged by the authors. There are several other issues that require clarification:

    1. Fig. 1C appears interesting but there is no comparator or controls. Without comparison, for example the histotrophic phase, it appears difficult to conclude that estrogen signaling genuinely persists during pregnancy in the opossum. pESR1 staining in the tissue section is ubiquitous with no evidence of nuclear localisation, raising concerns about antibody specificity. KI67 staining may be more informative?

    2. The authors used a large single-cell RNA-seq data set to map HAND2 expression at the human maternal-fetal interface in the first-trimester of pregnancy (Vento-Tormo et al. 2018). They demonstrate that HAND2 expression is confined to 3 maternal subsets, termed endometrial stromal fibroblast (ESF) 1 and 2 and decidual stromal cells (DSC). If I am not mistaken, in the Vento-Tormo paper, these populations of cells were labelled decidual stromal cells 1-3 (DS1-3), emphasizing that all these cells were decidualized, as expected in pregnancy. Vento-Tormo et al. further demonstrated that the differences in gene expression between DS subsets relate to their topography in the maternal tissue. Hence, it is confusing that the authors changed the terminology of these subsets, giving the erroneous impression of two undifferentiated ESF populations and a single DS/DSC population in pregnancy. By doing so, the inference seems to be that T-HESC, a telomerase-transformed endometrial stromal cell line used in functional studies, is a good model of ESF populations in vivo, which is doubtful.

    3. Fig. 2G. The authors state that 'We also used previously published gene expression datasets (see Methods) to explore if HAND2 was associated with disorders of pregnancy and found significant HAND2 dysregulation in the endometria of women with infertility (IF) and recurrent spontaneous abortion (RSA) compared to fertile controls' - This bold statement is based on microanalysis of merely 5 biopsies in each group. Considering the intrinsic temporo-spatial heterogeneity of the cycling endometrium, this sample size is grossly inadequate. The microarray study was published in 2011. In fact there are several more recent and more robust datasets available (e.g. 115 IF biopsies in GSE58144 and 20 RM biopsies in GPL11154). These comments also apply to Figure 4G.

    4. The authors also state 'HAND2 was not differentially expressed in ESFs or DSCs from women with preeclampsia (PE) compared to controls (Figure 2G).' It is unclear which dataset this was based on. The authors' claim seems to indicate that this was single-cell data? In any case, the sample size is again grossly inadequate to draw robust conclusions without further validation in a much larger cohort of samples.

    5. Figure 3. The authors decided to knockdown HAND2 in T-HESC, a telomerase-transformed endometrial stromal cell line, and performed RNA-seq 48 h later. The cells were not decidualized or even treated with progesterone. Hence, the rationale for this experiment, and its relevance to the in vivo situation, is genuinely lost on me. See also comment regarding the renaming of DS subsets into ESF. In an undifferentiated state, these cells are not representative of gestational cells (with the possible exception that decidual senescence is characterised by progesterone resistance, i.e. re-activation of genes that are suppressed by progesterone). More importantly, as HAND2 is critical for the identity of these cells, perhaps knockdown triggers a stress response? For example, from the data presented in Supplementary Table 6 (it would be helpful to add gene names), on of the strongest up-regulated gene upon HAND2 knockdown is BLCAP2 [Log2(FC): 10.2], which encodes a protein that reduces cell growth by stimulating apoptosis.

    6. The authors illustrated the importance of examining the right cellular state: knockdown HAND2 in T-HESC increases IL15 expression whereas it is well established that HAND2 knockdown in decidual cells decreases IL15 expression. Further, IL15 is strongly induced upon decidualization and previous studies on primary endometrial stromal cells demonstrated that IL15 secretion is undetectable in undifferentiated cells whereas it is abundantly secreted upon decidualization (PMID: 31965050). Thus, to be informative, the authors should repeat HAND2 KD in decidualizing T-HESC and measure IL15 secretion in both states, with and without HAND2 knockdown.

    7. Fig. 3B - it is unclear what is compared here: genes deregulated upon HAND2 knockdown in T-HESC versus knockdown NR2F2, FOXO1 and GAT2 in decidualized primary cultures? If this is the case, the comparison is not informative as it involves two different cell states. It is surprising that FOSL2 was not included in this analysis.

    8. I do not understand the relevance of the experiments described in Figure 5 to the context of gestation length or preterm birth. Trophoblast invasion will have been completed in the 2nd trimester of pregnancy - what is the purpose/message of these experiments? What is the level of IL15 secreted by these cells? Again the T-HESC appears not decidualized - so, what is the relevance to either the midluteal implantation window or gestation?

    9. What is the evolution of IL15 expression at the maternal-fetal interface? Does it parallel HAND2?

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    Parsing mechanisms of disease from the perspective of evolutionary biology is a powerful approach. The manuscript by Marinić et al. uses an innovative expression dataset in an evolutionary framework to identify a set of transcripts whose endometrial expression emerged at the eutherian stem lineage. One of these is the transcription factor HAND2. Using both existing datasets and experimental data the authors build a model of the activity of HAND2 and its associated protein IL15 at the maternal-fetal interface and implicate the proteins in both the evolution and disorders of pregnancy. The work illustrates the utility of evolutionary analysis for elucidating functional mechanisms of complex disorders and substantially contributes to our knowledge of the evolution and diseases of pregnancy.

  6. Version published to 10.1101/2020.06.15.152868v2 on bioRxiv
  7. Version published to 10.1101/2020.06.15.152868v1 on bioRxiv