Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures

Sousa, Carole; Golebiewska, Anna; Poovathingal, Suresh K; Kaoma, Tony; Pires-Afonso, Yolanda; Martina, Silvia; Coowar, Djalil; Azuaje, Francisco; Skupin, Alexander; Balling, Rudi; Biber, Knut; Niclou, Simone P; Michelucci, Alessandro

Regarding the first part of the comment, we agree with the reviewer's comment that it would be interesting to investigate the spatial distribution of "subset microglia". As suggested, we addressed this question by IHC using a gene marker that is upregulated in the "subset" microglia (Notch4). Notably, we were able to show that NOTCH4 positive microglia (IBA1 positive cells) were evenly distributed across different brain regions. We further quantified this pattern by FACS (5.4% NOTCH4 positive cells in saline-injected mice and 18.9% in LPS-treated mice). We added this information in the main text (page 14) and included the corresponding results in Fig. EV5 . For the second part of the remark, we believe that the comparison of the spatial distribution of "subset LPS" between male and female mice, although surely of high interest, goes beyond the scope of the current manuscript. As suggested, several additional genes have been included in the revised manuscript in Fig. 2E . We agree with the reviewer that a heatmap will be beneficial for a better visualization of the data. As suggested, in the revised manuscript the tSNE plots of single genes have been replaced with a heatmap. We amended the text and the figure legend accordingly. The addition of the headings has been done. We thank the reviewer to point out the inaccuracy regarding the number of states. Monocle 2 learns a principal trajectory for the centroids of the raw data cloud and afterwards puts the data points on this path. States are missing when cells in the transition state are very few and the corresponding points are projected to nearby branches. The correct number is actually 9 states for both Fig 5A and 5C . It has been corrected and specified accordingly.