Functional analysis of two uncharacterized genes, C130074G19Rik and I830077J02Rik , during early hematopoietic development

Complex transcriptional programs and signaling pathways control early hematopoietic lineage specification. Many key regulators have been identified; however, a substantial portion of the genome remains functionally uncharacterized. Here, we investigated six uncharacterized 'Riken' genes identified through transcriptomic profiling of Flk-1+ (also known as Kdr)/Pdgfrα- (hematoendothelial-enriched) and Flk-1+/Pdgfrα+ (cardiac mesoderm-enriched) populations at day 4 of embryoid body (EB) differentiation. We generated knockouts in mouse embryonic stem cells and performed bulk RNA-sequencing at day 4. Three of these genes (C130074G19Rik, I830077J02Rik and A530016L24Rik) were selected for further investigation by single-cell RNA-sequencing at day 7 of differentiation, which provided novel insight for two of these genes. Knockout of C130074G19Rik (hereafter G19Rik) increased the abundance of megakaryocyte progenitors and reduced endothelial populations, with differentially expressed genes enriched for hemostasis and membrane trafficking pathways. I830077J02Rik (hereafter J02Rik)-knockout cells showed subtle changes in extracellular matrix and cell adhesion genes, with a shift toward hematoendothelial lineages. Both G19Rik and J02Rik genes encode (predicted) transmembrane proteins that modulate membrane-associated processes in early hematopoietic development. This work establishes a framework for the study of uncharacterized genes with potential roles in cell fate determination.