A Single-Cell Transcriptomic Atlas of Sheep Symmetry Breaking, Gastrulation and Conceptus Elongation

P. Ramos-Ibeas¹; L. González-Brusi¹; N. Martínez de los Reyes¹; M. Carvajal-Serna^{1, 2}; P. Marigorta¹; L. Simpson³; R. Alberio³; P. Bermejo-Álvarez¹

¹Animal Reproduction Department, INIA, CSIC, Madrid, Spain

²Biochemistry and Molecular and Cellular Biology Department, IUCA, Zaragoza University, Zaragoza, Spain

³ School of Biosciences, University of Nottingham, United Kingdom

Abstract Text:

In ungulates, symmetry breaking and gastrulation, involving the differentiation of three germ layers from the epiblast, occur in a flat embryonic disc (ED) during conceptus elongation, concomitant with the massive proliferation of the extraembryonic membranes. Our aim was to analyse lineage development during symmetry breaking, gastrulation and conceptus elongation by single-cell RNA sequencing in sheep. Using the Evercode^TM WT Mini kit (Parse Biosciences), we obtained scRNAseq profiles from 80 embryos collected in vivo from superovulated ewes at embryonic days (E) 11 (spherical; n = 15), E11.5 (spherical and ovoid; n = 25), E12.5 (tubular and filamentous; n = 25 isolated EDs + extraembryonic membranes [EEMs]) and E13.5 (filamentous; n = 15 isolated EDs + EEMs). Transcriptomes of 13,847 cells passed quality controls, with a median of 4,729 genes detected per cell. Unbiased cluster of all in vivo embryos (UMAP) and known cell-type markers allowed us to identify 16 cell populations, as well as novel sheep cell-type marker genes. Two distinct trophectoderm (TE) populations were detected. Immature TE cells expressed higher CDX2, while mature TE cells, characterized by high interferon-tau (TP-1P8), PAG11, FURIN, and PTGS2, emerged and proliferated from E11.5, showing enriched GO terms related to lipid metabolism and transport, and the PPAR signalling pathway. The hypoblast was divided into parietal and visceral populations. Within the parietal hypoblast, a distal subpopulation expressing high PDGFRA, TDGF1, and BMP7 was detected, while proximal parietal hypoblast cells upregulated PRDM1, BMP2 and BMP6. NODAL, EOMES and OTX2 were expressed throughout the visceral hypoblast, while the anterior visceral hypoblast (AVH), marking the onset of symmetry breaking, emerged at E11, and disappeared at E12.5. At E11.5, the AVH specifically expressed CER1, LEFTY1 and DKK1. At these stages, active BMP, NODAL, WNT, and FGF signalling was detected between the epiblast, hypoblast, and the TBXT-positive primitive streak (PS), which emerged from the epiblast at E11.5. Mesoderm cells, expressing BMP4, HAND1, COL3A1, and SNAI2, were detected from E11.5 and proliferated rapidly from E12.5 to E13.5. A population of ETV2/KDR+ hematoendothelial progenitors appeared at E13.5. CDH1/FOXA2+ anterior primitive streak (APS) cells could be distinguished from the PS at E12.5, as well as notochord cells expressing high NOTO, GSC, FOXA2, and TBXT at E13.5. Definitive endoderm (DE) cells, expressing FOXA2, PRDM1, SOX17, BMP7 and POU5F1, emerged from the APS at E12.5 and proliferated at a slower pace than mesoderm cells. Primordial germ cells (PGCs), expressing POU5F1, NANOG, KIT, SOX17, and TFAP2C, emerged at E12.5, while GABRP- and TFAP2C-positive amnion cells, as well as GRLH2 and GRLH3-positive anterior surface ectoderm cells, were detected at E13.5. This single-cell molecular map of lineage differentiation during sheep gastrulation and conceptus elongation uncovers the timing of formation, molecular markers, and cellular origins of critical structures within the sheep conceptus. It also provides valuable insights to facilitate symmetry breaking, gastrulation and conceptus elongation in vitro.

Work supported by PID2021-122153NA-I00 and StG 757886-ELONGAN.