Figure 3.
Granulin expression is upregulated in vertebrate myeloid cells and is essential for myeloid cell differentiation during adult hematopoiesis. (A) t-Distributed stochastic neighbor embedding (t-SNE) analysis showing grna expression levels (red, high; orange and yellow, medium; blue, absent) of single cells sequenced from wild-type zebrafish kidney marrow using the online visualizer Single Cell inDrops RNA-Seq Visualization of Adult Zebrafish Whole Kidney Marrow (https://molpath.shinyapps.io/zebrafishblood/#pltly).45 The main tSNE clusters identified expressing grna are denoted by open circles. Yellow open circles represent clusters defined as “macrophages.” Open green circles are grna-expressing clusters whose cells were identified as “myeloid cells.” (B) Mouse hematopoietic model showing the dynamic expression of Grn derived from microarray data (Affymetrix Mouse Genome 430 2.0 Array). Notice that lymphocyte differentiation beyond common lymphoid progenitor (CLP) is not shown here. (C) Representative flow cytometric light scatter profile showing the different hematopoietic populations present in grna+/+ (left) and grna−/− (right) kidney marrow. (D) Representative pictures from grna+/+ and grna−/− whole kidney marrows cytospins stained with Wright-Giemsa stain showing increased early myeloid precursors (orange arrowheads) and decreased mature neutrophils (green arrowheads) in the absence of grna (bottom panel) compared with grna+/+ control siblings (upper panel). Magnification ×100. (E) Manual quantification of kidney marrow hematopoietic cells in grna−/− (green squares, n = 5) compared with control grna+/+ (black dots, n = 5) from 2 independent experiments. Horizontal lines and error bars indicate mean ± SEM. ***P < .001; ****P < .0001. Ery, erythrocytes; FSC, forward scatter; GMLP, granulocyte-monocyte-lymphoid progenitor; GMP, granulocyte-macrophage progenitor; Gra Gr+, granulocyte; HSC, hematopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; MkP, megakaryocyte progenitor; Mono, monocyte; MPP, multipotential progenitor; pCFU-e, pre-colony-forming unit-erythroid; pGMP, pre-granulocyte-macrophage progenitor; Plt, platelet; pMEP, pre-megakaryocyte-erythroid progenitor; SSC, side scatter; sCMP, strict common myeloid progenitor.

Granulin expression is upregulated in vertebrate myeloid cells and is essential for myeloid cell differentiation during adult hematopoiesis. (A) t-Distributed stochastic neighbor embedding (t-SNE) analysis showing grna expression levels (red, high; orange and yellow, medium; blue, absent) of single cells sequenced from wild-type zebrafish kidney marrow using the online visualizer Single Cell inDrops RNA-Seq Visualization of Adult Zebrafish Whole Kidney Marrow (https://molpath.shinyapps.io/zebrafishblood/#pltly).45  The main tSNE clusters identified expressing grna are denoted by open circles. Yellow open circles represent clusters defined as “macrophages.” Open green circles are grna-expressing clusters whose cells were identified as “myeloid cells.” (B) Mouse hematopoietic model showing the dynamic expression of Grn derived from microarray data (Affymetrix Mouse Genome 430 2.0 Array). Notice that lymphocyte differentiation beyond common lymphoid progenitor (CLP) is not shown here. (C) Representative flow cytometric light scatter profile showing the different hematopoietic populations present in grna+/+ (left) and grna−/− (right) kidney marrow. (D) Representative pictures from grna+/+ and grna−/− whole kidney marrows cytospins stained with Wright-Giemsa stain showing increased early myeloid precursors (orange arrowheads) and decreased mature neutrophils (green arrowheads) in the absence of grna (bottom panel) compared with grna+/+ control siblings (upper panel). Magnification ×100. (E) Manual quantification of kidney marrow hematopoietic cells in grna−/− (green squares, n = 5) compared with control grna+/+ (black dots, n = 5) from 2 independent experiments. Horizontal lines and error bars indicate mean ± SEM. ***P < .001; ****P < .0001. Ery, erythrocytes; FSC, forward scatter; GMLP, granulocyte-monocyte-lymphoid progenitor; GMP, granulocyte-macrophage progenitor; Gra Gr+, granulocyte; HSC, hematopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; MkP, megakaryocyte progenitor; Mono, monocyte; MPP, multipotential progenitor; pCFU-e, pre-colony-forming unit-erythroid; pGMP, pre-granulocyte-macrophage progenitor; Plt, platelet; pMEP, pre-megakaryocyte-erythroid progenitor; SSC, side scatter; sCMP, strict common myeloid progenitor.

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