Extended Data Fig. 1: In vivo characterization of Hoxb8FL-derived myeloid cells.
From: In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation
a, Representative flow cytometry plots of myeloid differentiation during the in vitro 48h treatment before i.v. injection. b, Percentage of myeloid Hoxb8FL-derived cells in vitro and in vivo (left) and quantification of the Hoxb8FL-derived myeloid cells in different tissue compartments in vivo. N = 4 animals c, Correlation of bulk RNA transcript counts in sorted Hoxb8FL-derived monocytes, compared to the same population of endogenous blood monocytes. N = 3 animals. d, Representative flow cytometry plots of the transferred Hoxb8FL-derived cells (bottom row, green) compared to endogenous cells (top row, gray) in the blood of the same mouse with EAE at the peak of disease, with two different panels (left) and quantification (right). N = 4 animals for the myeloid marker panel and three animals for the polarization marker panel. e-h, Percentage of Hoxb8FL-derived cells (green) and endogenous cells (gray) in the same animal in the spleen (h), femur bone marrow (i), vertebrae bone marrow (j) and inguinal draining lymph nodes (k), at the peak of EAE. N = 4 animals. i, Histological characterization of the distribution of Hoxb8FL-derived and endogenous myeloid cells in EAE lesions in the spinal cord pia matter and parenchyma, blue dashed line indicates pia-parenchyma separation. Right, percentage of total endogenous (gray) or Hoxb8FL (green) cells present in the pia (top) or parenchymal (bottom) compartments of the lesion. N = 3 animals, 3 sections per animal. Scale bar 100 μm. j, Representative image of the meningeal niche containing CD206+ resident macrophages but no infiltrated Hoxb8FL-derived myeloid cells. Dashed line indicates the dura. Scale bar 100 μm. k, Representative image of the perivascular niche containing Lyve+ resident macrophages but no infiltrated Hoxb8FL-derived myeloid cells. Dashed line indicates the vessel lumen. Scale bar 10 μm. l, Scheme of the experimental approach to study the migration phenotype of Ccr2-KO myeloid cells. m, Representative flow cytometry plots of control-tdTomato or Ccr2-KO GFP Hoxb8FL-derived myeloid cells in the bone marrow (left) or spinal cord (right) of a mouse with EAE at the peak of disease. n, Relative amounts of control-derived and Ccr2-KO-derived monocytes/macrophages or granulocytes across different tissues in mice with EAE at the peak of disease. N = 5 animals. o, EAE disease course of mice with (green) or without (gray) a Hoxb8FL cell transfer. N = 40 animals per group p-q, Correlation of bulk RNA transcript counts in sorted polarized Hoxb8FL-derived macrophages compared to the same population of endogenous spinal cord iNOS+ (p) or Arg1+ (q) macrophages at the peak of EAE. N = 3 animals. (a) Two-tailed paired t-test; (d-h) multiple two-tailed paired t-tests or Wilcoxon tests with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli for multiple comparisons; (n) ordinary one-way ANOVA left F = 87.58 P < 0.0001, right F = 1.242, P = 0.3274; Kolmogorov-Smirnov test used in (o); ns p-value > 0.05, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001; figures show mean ± sd.
