Single-cell qPCR delineates four major blood cell lineages from adult zebrafish marrow. (A) Unsupervised hierarchical clustering after gene expression analysis of single blood cells isolated from the whole kidney marrow. Heat map shows high transcript expression in red and low/absent expression in blue. Four major clusters were identified, including the following: erythroid (red), myeloid (green), B cells (light blue), and T cells (dark blue). Select lineage-specific genes are shown on the right. n = 166 WKM cells combined from two animals. (B) WGCNA-identified genes that were expressed together in single cells. Each row and column corresponds to a specific gene primer set. The heat map represents WCGNA adjacency dissimilarity values in the range between 0 (low topological overlap, light yellow) and 1 (high topological overlap, dark red). (C) The genes in each lineage module from WGCNA are noted. (D) Violin plots show the distribution of gene expression of single cells. Cells types were assigned based on hierarchical clustering and assessed for transcript expression of well-known blood cell lineage genes. By ANOVA, ba1 (P = 5.67 × 10−168), slc4a1a/band3 (P = 8.88 × 10−64), sptb (P = 1.57 × 10−43), mpx (P = 4.31 × 10−73), lyz (P = 2.70 × 10−71), cpa5 (P = 1.67 × 10−62), pax5 (P = 1.45 × 10−43), cd37 (P = 1.19 × 10−82), cd79a (P = 1.30 × 10−79), tcra (P = 3.24 × 10−116, lck (P = 2.64 × 10−30), and il7r/cd127 (P = 4.48 × 10−24).
