Fig. 2: Cx3cr1 haplo-insufficient HSPCs show a qualitative maturation advantage towards MLCs as compared to WT cells.

A Representative reconstruction of a brain slice from a competitively transplanted mouse where the engrafted BFP⁺Cx3cr1^−/+ (in green) and the Cherry⁺Cx3cr1^+/+ (in red) MLCs are visualized. Nuclei were stained with DAPI (blue signal). On top, a magnification (40X) showing the morphology of the BFP⁺Cx3cr1^−/+ and the Cherry⁺Cx3cr1^+/+ MLCs in the cortex. B Scheme displaying macro workflow for branching analysis. Confocal images, after maximum intensity projection, were analyzed with a standardized macro through the ImageJ software. Morphological criteria adopted to characterize donor-derived microglia cells were applied to each cell, which was thresholded, skeletonized, and deprived of the cell body. C–E Branching analysis on the BFP⁺Cx3cr1^−/+ and the Cherry⁺Cx3cr1^+/+ MLCs in the brain of IV or ICV competitively transplanted mice. The cumulative length of branches (C), complexity index (CI) (D), and covered environment area (CEA) (E) are shown. Mean and individual values ± SEM are shown. n > 50 cells per group, n = 3 mice/group, 3 independent experiments. An unpaired t-test with Welch’s correction, two-tailed, was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. F Example of Sholl analysis on an MLC analyzed via the ImageJ software. G, H Correlation between intersection radii and sum intersection parameters obtained from the Sholl analysis on BFP⁺Cx3cr1^−/+ and the Cherry⁺Cx3cr1^+/+ MLCs in the brain of IV (G) and ICV (H) competitively transplanted mice. The vertical and horizontal lines divide the graphs into four quadrants, to describe the cells according to different grades of morphologic complexity, i.e., UR upper right quadrant, for very complex cells characterized by a high sum of intersections and high number of intersecting radii; LL lower left quadrant, for cells with lower complexity; UL (upper left) and LR (lower right) quadrants for cells displaying intermediate complexity between the LL and the UR quadrants. I Histograms representing the percentage of cells retrieved in each of the four quadrants displayed in Fig. G and (H) to quantify the data. Images were acquired via Leica SPE confocal in the cortex region of the brain at 40X magnification. n > 50 cells per group, n = 3 mice/group. J Gene expression of microglia enriched genes and transcription factors in MLCs sorted by fluorescent marker expression from the brain of ICV competitively transplanted mice. Data were shown as fold change (calculated as 2 − ΔΔCT) on adult control microglia cells (dotted line, as reference equal to 1). Neonatal microglia (referred to as pup) are used as examples of immature myeloid/microglia cells. n = 3 mice/group. Individual values are shown. Source data are provided as a Source Data file.