No abstract

The fetal to adult switch in hematopoietic stem cell (HSC) behavior is characterized by alterations in lineage output and entry into deep quiescence. Here we identify the emergence of megakaryocyte (Mk) biased HSCs as an event coinciding with this developmental switch. Single-cell chromatin accessibility analysis reveals a ubiquitous acquisition of Mk lineage priming signatures in HSCs during the fetal to adult transition. These molecular changes functionally coincide with an increased amplitude of early Mk differentiation events following acute inflammatory insult. Importantly, we identify LIN28B - known for its role in promoting fetal-like self-renewal, as an insulator against the establishment of a Mk biased HSC pool. LIN28B protein is developmentally silenced in the third week of life and its prolonged expression delays emergency platelet output in young adult mice. We propose that developmental regulation of Mk priming may represent a switch for HSCs to toggle between prioritizing self-renewal in the fetus and increased host protection in postnatal life.

Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.