Single-embryo and single-blastomere immunoblotting reports protein expression heterogeneity in early-stage preimplantation embryos

Abstract

Understanding how a zygote develops from a single cell into a multicellular organism has benefitted from single-cell tools, including RNA sequencing (RNA-Seq) and immunofluorescence (IF). However, scrutinizing inter-and intra-embryonic phenotypic variation is hindered by two fundamental limitations; the loose correlation between transcription and translation and the cross-reactivity of immunoreagents. To address these challenges, we describe a high-specificity microfluidic immunoblot optimized to quantify protein expression from all stages of mouse preimplantation development. Despite limited availability of isoform-specific immunoreagents, the immunoblot resolves inter-embryonic heterogeneity of embryo-specific isoforms (i.e., DICER-1). We observed significantly higher DICER-1 isoform expression in oocytes when compared to two-cell embryos, and further find that protein expression levels follow the same trend as mRNA for both the full-length and truncated DICER-1 isoforms. At the morula stage, we assayed both whole and disaggregated embryos for loading controls (-tubulin, GAPDH) and markers that regulate cell fate decisions (CDX-2, SOX-2). In disaggregated morula, we found that cell volume showed positive, linear correlation with expression of -tubulin and SOX-2. In dissociated twocell and four-cell blastomeres, we detect significant inter-blastomeric variation in GADD45a expression, corroborating suspected cellular heterogeneity even in the earliest multicellular stage of preimplantation embryos. As RNA-Seq and other transcript-centric approaches continue to further probe preimplantation development, the demand for companion protein-based techniques rises. The reported microfluidic immunoblot serves as an essential tool for understanding mammalian development by providing high-specificity and direct measurements of protein targets at single-embryo and single-blastomere resolution.

Totipotency continuity from zygote to early blastomeres: a model under revision

“…Being aware that linear correlation is generally poor between transcript and protein level (Schwanhausser et al 2011) and that there is a two-fold variation in the amount of protein and nucleic acid contained in the different blastomeres of an individual eight-cell mouse embryo (Cheung et al 2013), the single-cell proteome analysis of sister blastomeres is likely to be a key experimental approach to capture the molecular determinants of blastomere totipotency (Virant-Klun et al 2016). Older and more recent studies contribute data supporting interblastomere differences at the protein level, for example, SNAI1/SNAI2 (Bell & Watson 2009) and GADD45a (Rosas et al 2018). It will be necessary to analyze the composition of each blastomere at the twocell stage and identify critical proteome requirements supporting totipotency, in order to make further progress, but, of course, it is impossible to combine a destructive assay with a proof of function for the same cells.…”