Regulation of Postnatal Forebrain Amoeboid Microglial Cell Proliferation and Development by the Transcription Factor Runx1

Abstract

Microglia are the immune cells of the nervous system, where they act as resident macrophages during inflammatory events underlying many neuropathological conditions. Microglia derive from primitive myeloid precursors that colonize the nervous system during embryonic development. In the postnatal brain, microglia are initially mitotic, rounded in shape (amoeboid), and phagocytically active. As brain development proceeds, they gradually undergo a transition to a surveillant nonphagocytic state characterized by a highly branched (ramified) morphology. This ramification process is almost recapitulated in reverse during the process of microglia activation in the adult brain, when surveillant microglia undergo a ramified-to-amoeboid morphological transformation and become phagocytic in response to injury or disease. Little is known about the mechanisms controlling amoeboid microglial cell proliferation, activation, and ramification during brain development, despite the critical role of these processes in the establishment of the adult microglia pool and their relevance to microglia activation in the adult brain. Here we show that the mouse transcription factor Runx1, a key regulator of myeloid cell proliferation and differentiation, is expressed in forebrain amoeboid microglia during the first two postnatal weeks. Runx1 expression is then downregulated in ramified microglia. Runx1 inhibits mouse amoeboid microglia proliferation and promotes progression to the ramified state. We show further that Runx1 expression is upregulated in microglia following nerve injury in the adult mouse nervous system. These findings provide insight into the regulation of postnatal microglia activation and maturation to the ramified state and have implications for microglia biology in the developing and injured brain.

Neonatal Peripheral Immune Challenge Activates Microglia and Inhibits Neurogenesis in the Developing Murine Hippocampus

“…Common opinion holds that yolk sac-derived myeloid precursors colonize the developing murine CNS between embryonic days 7 and 7.5 [36], where they proliferate extensively during embryonic and early postnatal development, eventually giving rise to mature ramified microglia [29,37]. Proliferation is rapidly restricted in the first 2 postnatal weeks [29], with adult microglia displaying low turnover [38] and highly limited replacement by peripheral monocytes under non-pathological conditions [39].…”

“…These observations of similar microglial densities in LPS-administered P7, P21 and P74 animals could speculatively suggest that there may be a target or maximum density in the brain and that control of microglial proliferation may be juxtacrine in nature. Indeed microglia express both Notch-1 receptor and its ligands Jagged-1 and Delta-1 [41], and Notch activation induces expression of the Runx1 (Runt-related) transcription factor [42], which in turn inhibits amoeboid microglial progenitor proliferation and facilitates progression from amoeboid precursor to ramified microglia [37]. Having observed an LPS-mediated acute increase in microglial cell density at P7, we asked whether these additional microglia were derived from endogenous microglia/myeloid precursor cell proliferation or peripheral monocyte recruitment.…”

Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing Groucho/TLE:Hes1-mediated inhibition of neuronal differentiation

“…Primary neural progenitor cells from E13.5 mouse neocortices were obtained and cultured as described. 11,12,37 Double-label immunofluorescence analysis of endogenous proteins was performed as described 11,41 using the following primary antibodies: rat anti-Gro/TLE (1 : 10), rabbit anti-Pin1 (1 : 500), mouse anti-Pin1 (1 : 200; R&D Systems) or rabbit anti-HIPK2 42 (1 : 300). For differentiation assays, cells were transfected as described 11,12,37 after 48 h in vitro using the following plasmids in the combinations indicated in the appropriate figure legend: pEGFP (0.1 mg/well) alone or in combination with pCMV2-FLAG-Gro/TLE1 (0.4 mg/well), pCMV2-FLAGHes1 (0.4 mg/well), pFLAG-HIPK2 or pFLAG-HIPK2(K221R) (0.4 mg/well) and pcDNA3-Pin1-HA (0.4 mg/well).…”

“…11,12,15,37 Three days following transfection, cells were analyzed by immunofluorescence using antibodies against Ki67 (1 : 500; BD Pharmingen), nestin (1 : 100; Millipore), Sox2 (1 : 250; R&D Systems), type III b-tubulin (1 : 1000; Promega, Madison, WI, USA), MAP2 (1 : 1000; Sigma-Aldrich) and NeuN (1 : 250; Millipore), as described. 41,43 Cells were counterstained with Hoechst 33258 (Sigma-Aldrich) before examination by fluorescence microscopy. Grayscale images were digitally assigned to the appropriate red or green channel using Northern Eclipse software (Empix Imaging, Inc., Mississauga, ON, Canada).…”

Roles of Runx Genes in Nervous System Development

“…Finally, it is important to mention that Runx1 can contribute to nervous system development and repair in an additional manner, namely by modulating the activity of microglia, the resident immune cells of the nervous system (Zusso et al 2012). Microglia survey the nervous system for signs of infection, injury or disease and mediate immune responses during many neuropathological conditions (Ransohoff and El Khoury 2015;Shemer et al 2015).…”