International Journal of Molecular Sciences Article Identification of Intercellular Crosstalk between Decidual Cells and Niche Cells in Mice Jia-Peng He , Qing Tian, Qiu-Yang Zhu and Ji-Long Liu * Guangdong Laboratory for Lingnan Modern Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; [email protected] (J.-P.H.); [email protected] (Q.T.); [email protected] (Q.-Y.Z.) * Correspondence: [email protected] Abstract: Decidualization is a crucial step for human reproduction, which is a prerequisite for embryo implantation, placentation and pregnancy maintenance. Despite rapid advances over recent years, the molecular mechanism underlying decidualization remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of a mouse uterus during decidualization. By analyzing the undecidualized inter-implantation site of the uterus as a control, we were able to identify global gene expression changes associated with decidualization in each cell type. Additionally, we identified intercellular crosstalk between decidual cells and niche cells, including immune cells, endothelial cells and trophoblast cells. Our data provide a valuable resource for deciphering the molecular mechanism underlying decidualization. Keywords: decidualization; mouse; single-cell RNA-seq; transcriptional changes Citation: He, J.-P.; Tian, Q.; Zhu, Q.-Y.; Liu, J.-L. Identification 1. Introduction of Intercellular Crosstalk between Decidualization is a crucial step for human reproduction. It is a prerequisite for em- Decidual Cells and Niche Cells in bryo implantation, placentation and pregnancy maintenance [1]. Decidualization initiates Mice. Int. J. Mol. Sci. 2021, 22, 7696. spontaneously in the secretory phase of the menstrual cycle, which is tightly controlled by https://doi.org/10.3390/ ovarian estrogen and progesterone [2]. Upon decidualization, uterine stromal cells change ijms22147696 into large epithelioid cells and secrete two protein markers, decidual prolactin (dPRL) and insulin-like growth factor-binding protein 1 (IGFBP1) [3]. Impaired decidualization may Academic Editor: Satoshi Kishigami lead to intrauterine growth restriction (IUGR), repeated pregnancy loss (RPL) and severe pre-eclampsia (sPE) [4]. Therefore, it is imperative to understand the molecular mechanism Received: 23 June 2021 of decidualization. Accepted: 16 July 2021 Due to ethical restrictions, studies on human decidualization are limited to the in vitro Published: 19 July 2021 level. The in vivo investigation of decidualization heavily relies on mice. Slightly different from humans, the decidual reaction in mice is an embryo-dependent process. Prolactin Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in family 8 subfamily a member 2 (Prl8a2), a paralog of human dPRL, is a well-established published maps and institutional affil- marker for mouse decidualization [5]. By using uterus-specific gene knockout mice, a iations. number of genes have been proved to be required for decidualization [6]. Alternatively, several studies have analyzed global gene expression changes associated with decidualiza- tion by using high-throughput transcriptomic approaches [5,7–10]. The limitation of these studies is that the bulk tissue was used. The decidua is a complex tissue consisting of many cell types, including decidual cells, stromal cells, endothelial cells and various immune Copyright: © 2021 by the authors. cells. Thus, bulk-tissue methods were unable to accurately capture cell-type-specific gene Licensee MDPI, Basel, Switzerland. expression changes. Moreover, the contribution of crosstalk between different cell types This article is an open access article distributed under the terms and was ignored. conditions of the Creative Commons With advances in the single-cell RNA-seq techniques, it is now possible to analyze the Attribution (CC BY) license (https:// global gene expression profile within highly heterogeneous tissues at a single-cell level [11]. creativecommons.org/licenses/by/ In the present study, by using the state-of-the-art single-cell RNA-seq approach, we re- 4.0/). solved all the cell types at the implantation site (decidualized) and the inter-implantation Int. J. Mol. Sci. 2021, 22, 7696. https://doi.org/10.3390/ijms22147696 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 17 Int. J. Mol. Sci. 2021, 22, 7696 2 of 17 we resolved all the cell types at the implantation site (decidualized) and the inter-implan- sitetation (undecidualized, site (undecidualized, served served as control) as cont ofrol) the of mouse the mouse uterus uterus on gestational on gestational day eight. day Consequently,eight. Consequently, we were we ablewere to able identify to identify differential differential expression expression changes changes associated associated with decidualizationwith decidualization in all in the all the cell cell types. types. Additionally, Additionally, we we predicted predicted intercellular intercellular crosstalkcrosstalk betweenbetween thethe decidualdecidual cellscells andand thethe nicheniche cells.cells. Our study provides a valuable resourceresource forfor understandingunderstanding thethe molecularmolecular mechanismsmechanisms underlyingunderlying decidualization.decidualization. 2.2. Results 2.1.2.1. A Single-Cell Atlas of MouseMouse Uterus on GestationalGestational DayDay EightEight To createcreate aa cell-typecell-type resolvedresolved mapmap ofof aa mousemouse uterusuterus uponupon decidualization,decidualization, we per- formedformed single-cellsingle-cell RNA-seqRNA-seq analysisanalysis (Figure(Figure1 1A).A). The The implantation implantation site site (IS, (IS, decidualized decidualized uterus)uterus) andand the the inter-implantation inter-implantation site site (IIS, (IIS undecidualized, undecidualized uterus, uterus served, served as a control)as a control) were collectedwere collected from gestationalfrom gestational day eight day (GD8) eight (Figure(GD8) 1(FigureB). The 1B). whole The uterus, whole which uterus, consists which ofconsists the endometrium, of the endometrium, the myometrium the myometrium and the and perimetrium, the perimetrium, was subjected was subjected to single-cell to sin- dissociationgle-cell dissociation (Figure 1(FigureC). The 1C). embryo The embryo at the IS at was the alsoIS was kept. also Single-cell kept. Single-cell RNA-seq RNA-seq data weredata were generated generated by using by using the 10 ×theGenomics 10× Genomics platform. platform. After qualityAfter quality control, control, a total ofa total 15,087 of cells15087 (6793 cells for (6793 the for IIS andthe IIS 8294 and for 8294 the IS)for werethe IS) obtained were obtained (Figure1 (FigureD,E). In 1D,E). order toInvalidate order to thisvalidate single-cell this single-cell RNA-seq RNA-seq dataset, we dataset, also generated we also generated a bulk RNA-seq a bulk RNA-seq dataset using dataset the using same samplesthe same (Figure samples1F). (Figure The cell-averaged 1F). The cell-averaged single-cell RNA-seqsingle-cell data RNA-seq were highlydata were accordant highly withaccordant the conventional with the conventional bulk RNA-seq bulk data RNA-se (r =q 0.7465 data for(r = the0.7465 IIS andfor the r = 0.7653IIS and for r = the 0.7653 IS), indicativefor the IS), of indicative the high of quality the high of our quality single-cell of our RNA-seq single-cell data. RNA-seq data. Figure Figure 1.1.Single-cell Single-cell transcriptome transcriptome analysis analysis of of the the decidualized decidualized mouse mouse uterus uterus on on gestational gestational day day 8. (A 8.) ( AA flowchart) A flowchart overview over- ofview this of study. this study. IIS, inter-implantation IIS, inter-implantati siteon (undecidualized site (undecidualized uterus, uterus, served served as control); as control); IS, implantation IS, implantation site (decidualizedsite (decidual- uterus).ized uterus). (B) A ( photographB) A photograph of mouse of mouse uterus uterus on gestational on gestational day 8. Theday positions8. The positions of embryo of embryo implantation implantation sites were sites marked were marked with an arrow. (C) Cell viability analysis of single-cell suspension. Cells were stained with the AO/PI solution. with an arrow. (C) Cell viability analysis of single-cell suspension. Cells were stained with the AO/PI solution. Cells in Cells in green were live and cells in yellow were dead. A representative photo was provided, and a bar plot was shown green were live and cells in yellow were dead. A representative photo was provided, and a bar plot was shown on the on the right. n = 3; * p < 0.05. (D,E) Single-cell RNA-seq data pre-processing and quality control. Cells with detected genes right.of fewern = than 3; * p 200< 0.05. or more (D,E )than Single-cell 6000 were RNA-seq removed. data pre-processingOnly cells with and total quality mitochondrial control. Cells gene with expression detected below genes 25% of fewer were thankept. 200 (F) orScatter more plots than 6000showing were the removed. correlation Only between cells with single-cell total mitochondrial RNA-seq and gene bulk expression RNA-seq. below For single-cell 25% were RNA-seq kept. (F) Scatterdata, gene plots expression showing thelevels correlation were averaged between and single-cell normalized RNA-seq as transcript and bulk per RNA-seq. million (TPM). For single-cell For bulk RNA-seqRNA-seq data,data, genegene expressionexpression