Cross Sectional and Longitudinal Imaging Reveals Spatiotemporal Divergence in Morphogenesis and Cell Lineage Specification between in-vivo and in-vitro Mouse Embryo during Pre- and Peri-implantation

ABSTRACT In-vitro culture is an essential step in assisted reproductive technology. Previous studies using sectional approaches have reported the impacts of in-vitro technologies on mammalian embryogenesis and offspring development. However, systematic and longitudinal comparisons between in-vivo and in-vitro spatiotemporal morphogenesis and cell lineage specification remain incomplete. Moreover, the phototoxicity of laser confocal microscopes on embryos is not fully evaluated. This study aims to bridge this gap using 4D live imaging via a laser scanning confocal microscopy to capture dynamic embryo development from a CAG::H2B-GFP mouse line. The results showed that in-vitro culture under a confocal microscopy cumulatively slowed the increase in embryonic cell number and embryos size over time. Furthermore, in-vitro embryo compaction, cavitation, primitive cell migration and hatching deferred in both spatial pattern and temporal progression. Additionally, in-vitro and in-vivo cell lineages differed in spatial distribution and expansion rate. These findings demonstrate that in-vitro culture delays embryo development, alters morphological events and disrupts cell lineage specification at both spatial and temporal scales during pre- and early peri-implantation. This work provides insights on the detailed extent of similarities and differences between in-vivo and in-vitro embryogenesis, beneficial when translating in-vitro findings into clinical, industrial and ecological applications. Competing Interest Statement The authors have declared no competing interest. Footnotes Page 2, Abstract, a new sentence has been added to clarify the background of the present study. The new sentence reads: Moreover, the phototoxicity of laser confocal microscopes on embryos is not fully evaluated. Page 3, Introduction, Paragraphs 2 and 3 have been revised to further clarify the background of the study, aims, and main results. Page 26, in Resources and Materials section, the detailed institute where the study was conducted has been clarified. It reads now: Resources and materials used in this study at the University of Manchester (UoM) are summarised in Supplementary Tables S1-S4. Page 26, in Ethical Approval and Regulations for Animal Care section, the first sentence has been revised based on confirmed detailed and precise information obtained from the Operational Office at the University of Manchester. The revised sentence now reads: Ethical approval for the animals used for this work was granted by the UoM Animal Welfare and Ethical Review Committee. Page 26, Figure 11, the label IV in the first green box at the bottom has been clarified as in-vitro (control group) vs in-vitro (experimental group).