Bailly, Y. | Search bioRxiv

bioRxiv2025

Single-cell omics reveal distinct gene regulatory dynamics underpinning embryonic and extraembryonic lineage functions during pig blastocyst development

The late-stage development of the blastocyst before implantation is a unique feature of ungulates. During this period, the epiblast proliferates and remains pluripotent for several days before gastrulation begins. Simultaneously, extra-embryonic tissues undergo significant growth, elongating to several tens of centimeters. However, the mechanisms that regulate and synchronize these processes remain poorly understood. In this study, we analyzed transcriptomic and epigenomic data at the single-cell level from early and late porcine blastocyst cells, spanning from the hatched blastocyst stage (E7) to early (E9) and late ovoid blastocyst (E11). We characterized 15,370 blastocyst cells, clustering them into distinct embryonic and extra-embryonic populations with specific chromatin accessibility landscapes. For each population, we inferred gene regulatory networks based on enhancer-driven gene regulation modules (eRegulons) and validated them through motif occupancy visualization. Our findings reveal strong dynamics in gene regulatory module activity within extra-embryonic tissues at the onset of blastocyst elongation, while gene regulatory activity in the epiblast remains relatively stable.

developmental biology↗

bioRxiv2023

Cell specification and functional interactions in the pig blastocyst inferred from single cell transcriptomics

The early embryonic development of the pig comprises a long in utero pre- and peri-implantation development, which dramatically differs from mouse and human. During this pro-tracted peri-implantation period, an intimate dialogue between the embryo and the uterus is established through a complex series of paracrine signals. It leads to concomitant drastic changes in the embryonic morphology and uterine receptivity to implantation. From day 7 after fertilization, the spherical blastocyst elongates as ovoid, tubular and filamentous wich transforms from a 0.5-1 mm diameter sphere to a 1000 mm long filamentous blastocyst at day 16. At the same time, the inner cell mass moves up to the trophoblast as the Raubers layer disappears and evolves as an embryonic disc that is directly exposed to molecules present in the uterine fluids. These drastic changes occurring before implantation are driven and coordinated by interactions between embryonic cells and maternal tissues. To better understand the biology of pig embryos before implantation, we generated a large dataset of single-cell RNAseq at different embryonic stages (early and hatched blastocyst, spheroid and ovoid conceptus) and proteomic datasets from corresponding uterine fluids. These data were cleaned, filtered and represent a total of 34,888 cells. We first characterised the embryonic and extra-embryonic cell populations and their evolution, and identified population-specific markers of the three main populations (epiblast, trophectoderm and hypoblast). Our analysis also confirmed known functions and predicted new biological functions associated with these cell populations.We then inferred gene regulatory networks working on modules of gene regulation (regulon) and selected those that were specifically active in each embryonic population. We confirmed the relevance of the identified regulons by a meta-analysis of two other scRNAseq datasets (porcine and human preimplantation embryos). We then linked these regulons to signalling pathways and biological processes. Our results confirm the molecular specificity and functionality of the three main cell populations and identify novel stage-specific subpopulations. In particular, we discovered two previously unknown subpopulations of the trophectoderm, one characterised by the expression of LRP2, which could represent a subpopulation of progenitor cells, and the other, expressing many pro-apoptotic markers, which could correspond to the cells of the Raubers layer. We also provide new insights into the biology of these populations, their reciprocal functional interactions and the molecular dialogue established with the maternal uterine environment.

developmental biology↗

bioRxiv2018

Molecular and functional heterogeneity of cerebellar granule cell terminals expands temporal coding in molecular layer interneurons.

In the cerebellum, molecular layer interneurons (MLIs) play an essential role in motor behavior by exerting precise temporal control of Purkinje cells, the sole output of the cerebellar cortex. The recruitment of MLIs is tightly controlled by the release of glutamate from granule cells (GCs) during high-frequency activities. Here we study how single MLIs are recruited by their distinct unitary GC inputs during burst of GC stimulations. Stimulation of individual GC-MLI synapses revealed four classes of connections segregated by their profile of short-term plasticity. Each class of connection differentially drives MLI recruitment. Molecular and ultrastructural analyses revealed that GC-MLI synaptic diversity is underlain by heterogeneous expression of synapsin II at individual GC terminals. In synapsin II knock-out mice, the number of classes is reduced to profiles associated with slow MLI recruitment. Our study reveals that molecular diversity across GC terminals enables diversity in temporal coding by MLIs and thereby influences the processing of sensory information by cerebellar networks.

neuroscience↗