Investigating novel gene regulatory networks for development of myeloid cells

ABSTRACT Myeloid cells in and around the brain are at the center of proper central nervous system (CNS) function by playing crucial roles in homeostatic surveillance and immunity1. A type of myeloid cell, microglia, are the resident immune cells of the CNS and they are vital for neuronal network architecture and injury response1. Conversely, microglia also play a key role in some diseases like autism2 and multiple sclerosis3. In some cases, microglia depletion is a promising therapy and relies on known genetic regulators of microglia production4,5. However, recent research in mice and zebrafish shows subpopulations of developmental microglia that are genetically distinguishable6,7. These data present a gap in knowledge of the complex gene regulatory networks that govern microglia production. To begin to understand the heterogeneity of microglia-producing genetic programs, we identified an undescribed cell in the brain that expresses canonical microglia markers, clears debris, and expands in injury7. These microglia are labeled with Mannose Receptor C, type 1a (mrc1a)7, a membrane receptor expressed in lymphatic and venous vasculature8. To uncover more about the genetic regulation of these cells in our supporting data, we interrogated the transcription factor sox17. Sox17 is expressed in endothelium9,10 and was recently shown to regulate the transdifferentiation of lymphatic vessels to blood vessels11. We found that genetic perturbation of sox17 significantly reduced mrc1a+ microglia abundance in the embryonic zebrafish brain. Given that sox17 functions in a vast array of embryogenic processes10,12,13, it is difficult to theorize specifically how the transcription factor could be regulating embryonic microglia production. Therefore, we carried out a CRISPR screen and identified 6 additional candidate genetic modifiers of microglia production. The aim in the F99 proposed study is to further investigate the effects of sox17 mutation on microglia and investigate genetic interactions between sox17 and other candidate genetic modifiers. I will accomplish this by using a combination of in vivo timelapse imaging, in situ hybridization of mRNA, and CRISPR gene editing. In the K00 phase of the application, the investigation of myeloid cells in and around the brain will be expanded. Aside from microglia, novel populations of other myeloid cells (monocytes and neutrophils) have also been identified in the brain14. These monocytes and neutrophils are skull bone-marrow derived, and are genetically distinct from previously-described populations of these cells14. However, it remains unknown if the skull bone marrow could be a source for CNS myeloid cells during development and if microglia are amongst these cells. The goal of the K00 proposed study is to investigate the postnatal skull bone marrow (P7, P10) as a source of myeloid cells during development. I will accomplish this by using a combination of fate mapping, cell tracking, intravital imaging, and immunofluorescence approaches. This work has the potential to inform the clinic of genes that impact myeloid cell development so that we may increase the efficacy of diagnostic testing and therapeutic strategies for treating neurodevelopmental and neurodegenerative disorders.