Cxcl8/PKC Signaling in the Endothelial Cell Niche Regulates Long-Term Hematopoietic Stem Cell Fate

The sinusoidal endothelial cell niche is a component of the hematopoietic microenvironment with an important role in regulating hematopoietic stem cell (HSC) fate. Mechanisms by which the niche control the long-term functional and phylogenetic diversity of the hematopoietic system are poorly understood. We performed gene expression profiling on FACS-purified endothelial cells from the zebrafish caudal hematopoietic territory (CHT), the sinusoidal endothelial niche equivalent to the mammalian fetal liver. Kdrl:GFP⁺lyve1b:DsRed⁺ CHT endothelial cells were compared to kdrl:GFP⁺lyve1b:DsRed^- non-CHT endothelial cells by bulk RNA-seq and 106 genes were downregulated in the CHT with q<0.05. Enforced expression of candidate genes in the CHT was achieved using a zebrafish E-selectin (sele) enhancer/promoter element. Microinjection of a sele:prkcda-2A-mCherry (protein kinase C (PKC), delta a) construct into Runx1+23:GFP HSPC reporter zebrafish embryos resulted in a significant increase in the number of HSPCs compared to empty vector control (5.4 ± 1.0 vs 2.5 ± 0.4, p = 0.007). The same construct was microinjected into hemizygous GESTALT zebrafish with barcoding sgRNAs and Cas9. Barcoded embryos were grown to adulthood, peripheral blood was sampled at 3, 6, 9 and 12 months post-fertilization (mpf), and phylogenetic diversity of the hematopoietic system was measured in a longitudinal manner by enumerating unique GESTALT clones. Transgenic animals microinjected with the sele:prkcda-2A-mCherry construct consistently showed greater HSC clonal diversity compared to empty vector control (9.5 ± 0.9 vs 6.4 ± 0.7 clones at 12 mpf, p = 0.024). Single cell RNA sequencing (scRNA-seq) of the marrow of these animals revealed expanded populations of macrophages and erythrocyte precursors in those with dysregulated expression of prkcda. PKC family members are signaling intermediates for cxcr1, the receptor for the chemokine cxcl8, and a factor we have previously shown to positively regulate CHT colonization. Treatment of human umbilical vein endothelial cells in vitro with recombinant cxcl8 showed no change in prkcd transcript levels by bulk RNAseq or quantitative RT-PCR, but did show enhanced accumulation of PKC-d protein after 2 hours consistent with induction of this pathway at the post-transcriptional level. To understand how cxcl8/PKC signaling might alter the niche in vivo, transgenic zebrafish with enforced expression of cxcl8 were generated by microinjecting embryos with a Runx1+23:cxcl8 expression construct. These were raised to 12 mpf and kidney marrow was harvested and subjected to scRNA-seq, demonstrating over 2-fold expansion of stromal and endothelial elements in Runx1:cxcl8 animals compared to controls. Hematopoietic cells were identified, a developmental trajectory was plotted using pseudotime analysis, and this was anchored using expression of genes characteristic of early (meis1b, tal1) and late (lyz, mpx) myeloid development. Runx1:cxcl8 mosaic transgenics had a relative expansion of early myeloid cells compared to control animals (61% vs 38% early myeloid cells, p = 0.0002). Our data support the following model: HSPCs or other myeloid cells may actively remodel the niche via cxcl8 expression, signaling through cxcr1 and PKC, increasing niche capacity and thereby expanding the number of HSCs that can contribute to hematopoiesis. The presence of reserve capacity within the niche may be relevant to conditions where loss of HSC clonal diversity carries a risk of myeloid neoplasia.