Heterogeneity of reactive astrocytes might be caused by a complication of reactive microgliosis in Mlc1-ErbB2V664E brain. In addition to effects on astrocytes, reactive astrogliosis in diseased or injured brains is always accompanied by recruitment of reactive microglia . Indeed, we observed an increased prevalence of reactive microglia (Iba1+) in the brains of Mlc1-ErbB2V664E mice comparable to that of GFAP+ cells (Supplementary Figure S9a and b). These results showed that active ErbB signaling in mature astrocytes induced reactive responses with a molecular and cellular profile similar to that observed in reactive astrogliosis in injured or diseased brains. Considering the lack of ectopic ErbB activation in microglia or leukocytes that were infiltrated through the blood–brain barrier (Supplementary Figure S9c), our findings suggest that reactive responses of microglia in Mlc1-ErbB2V664E mice were induced by immunogenic factors released from reactive astrocytes. To confirm this idea, we examined Mlc1-ErbB2V664E mouse brains 3 days after Dox withdrawal. At this early stage, GFAP-expressing astrocytes started to appear in the cortices of Mlc1-ErbB2V664E mice, with some of them possessing Ki67+ nuclei. In contrast, Iba1+ cells in the same cortices did not increase, and did not have Ki67+ nuclei (Supplementary Figure S10a–d). These results were consistent with previous reports that reactive astrocytes release many factors including cytokines , and astrocytic gene targeting-induced astrogliosis results in reactive microglia responses . Indeed, we detected increase of cytokines including TGF-β2, interleukin-6 (IL-6), IL-1β, C-C motif chemokine ligand 2 (CCL2), and Ciliary neurotrophic factor (CNTF) in primary Mlc1-ErbB2V664E astrocytes (Supplementary Figure S10e). Noticeably, astrogliosis and microgliosis did not result in cell apoptosis. There were no terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)+ cells revealed in Mlc1-ErbB2V664E brain or littermate controls on either 3 or 20 days after Dox withdrawal (Supplementary Figure S10h).