The EMBO Workshop ‘Imaging mouse development’ assembled an emerging community of researchers with the common goal of the four dimensional reconstruction of mammalian development. The conference sought to explore longstanding questions such as ‘How is the body plan established in the early embryo?’ or ‘How is morphogenesis coordinated with lineage specification?’ During their four days here at EMBL, participants pondered these exact questions along with the speakers, learned all about the recent developments in the field, and had fun during social activities – especially during the pub quiz, where we’ve seldom seen a more enthusiastic crowd!

During the poster sessions, participants had the opportunity to present their research to their peers, and from these, two winners were chosen by the community. Additionally, two speakers were chosen for best talk. We’d like to congratulate Madeleine, Konstantinos, Yash, and Audrey for their exceptional work!

YAP regulation in the preimplantation embryo and its role in linking the first and second cell fate decisions

Presenter: Madeleine Chalifoux

Authors: Madeleine Chalifoux, Maria Avdeeva, Stanislav Shvartsman, Eszter Posfai

Abstract:

A key determinant of the trophectoderm (TE) versus inner cell mass (ICM) fate decision is the localization of a transcriptional coactivator, Yes-associated protein (YAP). When localized to the nucleus, YAP can activate or repress TE and ICM specific transcription factor expression, respectively. One input known to regulate YAP subcellular localization is cell polarity; however, it is unknown whether YAP responds to additional inputs during preimplantation development, such as mechanics and cell cycle. Additionally, how YAP dynamics are interpreted by cells to result in the timing of Sox2 expression in ICM cells remains to be investigated.

Here, we use genetically engineered reporter mouse lines for YAP and SOX2, along with light sheet microscopy, to investigate the dynamic mechanisms by which cells acquire their distinct fate during preimplantation development. We find that YAP behavior can be explained through degradation of maternal protein, relative exposed surface area, and cell cycle-associated nuclear volume dynamics. Contrary to longstanding expectation within the field, we find that YAP does not respond to mechanical perturbations in the early embryo. We also show that nuclear YAP exclusion acts permissively in allowing for Sox2 expression and sets the timing for Sox2 expression. Furthermore, early Sox2 expression has important consequences for epiblast (EPI) versus primitive endoderm (PE) fate specification, suggesting that the origins of the second cell fate decision can be traced back to YAP-dependent cell dynamics at play during the first cell fate decision.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Investigating the role of calcium oscillations in the peri-gastrulation mammalian embryo

Presenter: Konstantinos Miti

Authors: Shifaan Thowfeequ, Konstantinos Miti, Matthew Stower, Richard Tyser, Shankar Srinivas

Abstract:

Growing evidence supports that in addition to the classic morphogen-mediated signalling pathways, cells can use electrical signalling to communicate and regulate tissue patterning. Calcium ions (Ca2+) have been implicated in a variety of developmental stages, ranging from fertilization to organogenesis. However, our knowledge of their role during early
post-implantation mammalian development is limited due to the inaccessibility of the embryos for imaging Ca2+ dynamics. In this study, we aimed to characterise and determine the role of Ca2+ oscillations at these stages to understand whether they have the potential to mediate morphogenetic processes. To obtain high resolution volumetric information about Ca2+ oscillations, we used the GCaMP6f mouse line and Lattice Light-sheet microscopy. We find that implantation marks the onset of widespread Ca2+ oscillations, with a significant rise in the frequency of intracellular Ca2+ oscillations in extra-embryonic tissues, while embryonic epiblast (Epi) cells remained mostly quiescent. At the onset of gastrulation, embryos exhibit decreased Ca2+ oscillations in the visceral endoderm (VE) but not the extraembryonic ectoderm. Robust oscillations are present in the primitive streak itself. By 7.25 dpc, mesodermal progenitor cells establish a stable and frequent periodicity of Ca2+ oscillations as they undergo epithelial-to-mesenchymal transition and migrate as the mesodermal wings towards the anterior midline. We were also able to identify large multicellular Ca2+ waves within the Epi, that appear to be triggered by VE cells. These oscillations propagated with an apical-to-basal directionality within individual Epi cells. Ca2+ oscillations are dependent on Ca2+ release from internal endoplasmic reticulum stores, as shown by the inhibition of ER specific SERCA re-uptake channels. These results indicate that tissue-specific patterned Ca2+ oscillations exist in the early embryo opening up the possibility that they influence patterning.

Due to the confidentiality of the unpublished data, we cannot share the poster.

Spatial patterning of mitochondrial energy production in mouse oocytes

Presenter: Yash Rana

Authors: Yash Rana, Daniel Needleman

Abstract:

Mitochondrial metabolism fuels mammalian oocyte maturation and early embryo development. The spatiotemporal variations in mitochondrial localization and metabolic activities pattern energy production within cells, precise control of which is crucial to mammalian embryo development and fertility. Despite its importance, we currently lack a
mechanistic understanding of the biophysical processes that give rise to these emergent patterns in mammalian embryos.

In this talk, I will share my ongoing work on deciphering the mechanism behind the formation of subcellular spatial patterns of mitochondrial metabolism in metaphase II arrested mouse oocytes, the first stage of development. At this non-equilibrium steady state, there exists a spatial gradient of mitochondrial metabolism with distance from the meiotic spindle, but the causes and consequences of this gradient are unknown. To decipher the biophysical mechanism underlying the origin of this gradient, I am integrating quantitative microscopy with biochemical and mechanical perturbations, with the aim of interpreting the data using the lens of quantitative biophysical theory.

My work indicates that individual cells contain functionally distinct mitochondria, whose metabolic state is associated with their actin-dependent mobility. The spatial location of the meiotic spindle dictates the direction of mitochondrial motion, which, coupled with the observed metabolism-dependent mitochondrial motility, may explain how the self-organization of mitochondria gives rise to large-scale spatial patterning of energy production. These results have implications for understanding the role of sub-cellular energy fluxes in early mammalian development. More broadly, I hope this work helps lead to a quantitative understanding of the spatiotemporal patterning of thermodynamics fluxes in cells and a predictive theory of mitochondrial self-organization.

Due to the confidentiality of the unpublished data, we cannot share the poster.

3DMOUSEneST: a method using higher harmonic generation microscopy for three-dimensional, label-free evaluation of decidualization efficacy

Presenter: Audrey Savolainen

Authors: Audrey Savolainen, Emmi Kapiainen, Veli-Pekka Ronkainen, Valerio Izzi, Martin M. Matzuk, Diana Monsivais, Renata Prunskaite-Hyyryläinen

Abstract:

Early embryonic lethality is highly prevalent in transgenic mouse models, however, convenient methods to screen embryo implantation success and maternal uterine response in mice are currently lacking. We present 3D Microscopy for Optically cleared Unlabeled implantation Site Evaluation using Second and Third harmonic generation (3DMOUSEneST), a novel application of label-free higher harmonic generation microscopy to image the three-dimensional (3D) structure of decidual fibrillar collagen and present a procedure for quantification of the decidualization reaction. Briefly, to establish the 3DMOUSEneST method we collected and imaged intact, optically cleared E4.5-E6.5 mouse implantation sites with multiphoton microscopy using second-harmonic generation (SHG) and third-harmonic generation (THG). We systematically analyzed all 3D scans using Imaris software. Using SHG, we defined a novel 3D structure, the decidual nest—the result of rapid deposition and organized arrangement of fibrillar collagen by decidual cells around the implanted embryo. We demonstrate that volumetric analysis of the decidual nest can be used to quantify decidualization and present a standard growth pattern for the decidual nest during normal early pregnancy. We demonstrate that decidual nest volume is a measurable indicator of implantation efficacy and early pregnancy progression with 92% accuracy based on a logistic regression model developed using Smad1/5 and Smad2/3 conditional knockout mice with known implantation defects. Moreover, we show that THG microscopy can be used to visualize conceptuses and assess their growth. In combination, the data derived from SHG and THG microscopy provide unprecedented insights into the maternal–embryo dynamics during early pregnancy. Our study bridges extracellular matrix biology with reproductive biology, adding new knowledge of the 3D remodeling of extracellular matrix in decidualization and applying this knowledge to establish a unique 3D analysis method to study decidual development. 3DMOUSEneST has great potential to facilitate the characterization of the continuously growing number of mouse models developed for studying human diseases, particularly models exhibiting early embryonic lethality and fertility issues. 

Due to the confidentiality of the unpublished data, we cannot share the poster.

The EMBO Workshop ‘Imaging mouse development‘ took place from 2 – 5 September 2024 at EMBL Heidelberg and virtually.