ZFIN Figure: Ulloa et al., 2021, Fig. 1

Fig. 1

Developmental hematopoietic heterogeneity identified in zebrafish with single-cell RNA sequencing (scRNA-seq)

(A) Schema of scRNA-seq experiment: (1) drl:mCherry⁺;gata1:GFP⁻ cells were isolated by fluorescence-activated cell sorting (FACS) from pools of 1 and 2 dpf embryos to enrich for HSPCs, (2) 10X Genomics single-cell RNA libraries were prepared and sequenced, and (3) downstream computational analysis showing that UMAP dimensional reduction and unsupervised clustering of drl:mCherry⁺;gata1:GFP⁻ cells at 1 and 2 dpf with Monocle3 gives 10 clusters.

(B) Cell-type identification of the 10 Monocle3 clusters performed using key hematopoietic marker genes supported by previous zebrafish, murine, and human literature. Cell types identified include erythroid progenitors (EryPs), clusters 1–3 (c1–c3); lympho-erythroid-primed progenitor (LEP), cluster 4 (c4); pre-hemogenic endothelial cells (pre-HE), cluster 5 (c5); hemogenic endothelial cells generating HSCs (HE/HSC), cluster 6 (c6); lymphoid progenitor (LyP), cluster 7 and 10 (c7,10); lymphomyeloid progenitor with higher macrophage marker expression (LMP-M), cluster 8 (c8); and lymphomyeloid progenitor with higher granulocyte marker expression (LMP-G), cluster 9 (c9). Expression bar: scaling is performed per gene where mean is close to 0 and standard deviation of 1. Only genes with the highest scaled expression value will show the brightest yellow color in any of the cells (standard deviation above −2), and those that have the lowest scaled expression will be purple (standard deviation below 2).

(C) The cell-type classifications in UMAP space as identified in (B).

(D) RNA velocities overlaid over UMAP clusters of drl:mCherry⁺gata1:GFP⁻ cells at 1 and 2 dpf. The different colors represent the Seurat clusters derived from reprocessing the data. The dotted lines encircling each cluster are cell types identified with previously used marker genes (refer to Figure S1D). Arrows indicate inferred differentiation trajectory as determined by RNA velocities.

(E and F) Enrichment pathway analysis of upregulated top marker genes in pre-HE (E) and HE/HSC (F) clusters, conducted using Metascape. Top 200 markers selected using Monocle3 with the following criteria: fraction expressing >10% and marker test p < 0.00005. The colored circles after each term in the significance bar plot (left) represent a colored dot on the network representation (right).