(251n) Following Lineage Commitment of Pre-implantation Embryos Through Single-Embryo Western Blotting

Before implantation into the uterine wall, embryos undergo multiple cell divisions and transitions from zygote (1-cell) to 2-cell, 4-cell, 8-cell, morula and finally blastocyst (3). The first cell lineage specification occurs in these pre-implantation stages, with blastomeres dividing to form the inner cell mass (ICM) and the trophectoderm (TE) at the blastocyst stage. Debates remain whether this first asymmetry is due to different molecular phenotypes in early blastomeres, or whether cells are assigned to the ICM or TE depending on their position and polarity at the 32-cell stage (2). To answer these questions, research on pre-implantation development has typically focused on gene expression levels. However, the fluctuations in RNA caused by transcriptional bursts does not often correlate well with protein levels, given the different half-lives of RNA and protein (2). The need to perform protein measurements on single embryos and single blastomeres remains.

 

To enable detection of proteins from embryos, we have advanced the single cell western blot (scWestern), recently developed in the Herr Lab, to analysis of whole embryos. The scWestern is a thin polyacrylamide gel layer, grafted onto a standard microscope slide, and stippled with thousands of microwells (1). Cells are settled into microwells, lysed in-situ, and solubilized proteins are electrophoresed across the polyacrylamide layer. Proteins are then covalently bound to the polyacrylamide, by activation of photoactive molecules (benzophenone moieties) incorporated into the polyacrylamide matrix. Fluorescently-labeled antibodies are then diffused into the polyacrylamide for detection of protein targets. By increasing the microwell dimensions and polyacrylamide thickness to accommodate embryos (200 um in diameter), modulating the pore size to avoid clogging at the gel interface, and introducing lysis through a lid system, we have achieved simultaneous analysis of all pre-implantation stages, from zygote to blastocyst. We have detected bands of loading controls and are now focusing on markers associated with totipotency and early differentiation. Furthermore, to help answer whether single blastomeres show signs of lineage commitment before the 32-cell stage, embryos will be dissociated into individual blastomeres and analyzed individually for markers of TE and ICM. Protein analysis of embryos and single blastomeres will result in detailed temporal tracking of the emergence of cell asymmetry, helping to confirm or challenge current evidence supported by transcriptomic data.

References:

1. Hughes et al. â??Single cell western blottingâ?. Nature Methods. 2014. 11(7): 749-55.

2. Nimmo et al. â??Primed and ready: understanding lineage commitment through single cell analysisâ?. Trends in Cell Biology. 2015. 25: 8; 459-467.

3. Welling et al. â??Symmetry breaking in the early mammalian embryo: the case for quantitative single-cell imaging analysisâ?. Mol. Hum. Reprod. (2016) 22 (3): 172-181.