A single-cell transcriptomic atlas reveals resident dendritic-like cells in the zebrafish brain parenchyma

Reviewer #1 (Public Review):

Summary:
The authors used several zebrafish reporter lines to demonstrate the presence, regional distribution, and transcriptional profile of the immune cells in adult zebrafish brains. They identified DC-like cells distinct from microglia or other macrophages, resembling murine cDC1s. Analysis of different mutants further revealed that this DC population was dependent on Irf8, Batf3, and Csf1rb, but did not rely on Csf1ra.

Strengths:
It is an elegantly designed study providing compelling evidence for further heterogeneity among brain mononuclear phagocytes in zebrafish, consisting of microglia, macrophages, and DC-like cells. This will provide a better understanding of the immune landscape in the zebrafish brain and will help to better distinguish the different cell types from microglia, and to assign specific functions.

Weaknesses:
While scRNA-seq data clearly revealed different subsets of microglia, macrophages, and DCs in the brain, it remains somewhat challenging to distinguish DC-like cells from P2ry12- macrophages by immunohistochemistry or flow cytometry.

Reviewer #2 (Public Review):

The authors made an atlas of single-cell transcriptome of on a pure population of leukocytes isolated from the brain of adult Tg(cd45:DsRed) transgenic animals by flow cytometry. Seven major leukocyte populations were identified, comprising microglia, macrophages, dendritic-like cells, T cells, natural killer cells, innate lymphoid-like cells, and neutrophils. Each cluster was analyzed to characterize subclusters. Among lymphocytes, in addition to 2 subclusters expressing typical T cell markers, a group of il4+ il13+ gata3+ cells was identified as possible ILC2. This hypothesis is supported by the presence of this population in rag2KO fish, in which the frequency of lck and zap70+ cells is strongly reduced. The use of KO lines for such validations is a strength of this work (and the zebrafish model).

The subcluster analysis of mpeg1.1 + myeloid cells identified 4 groups of microglial cells, one novel group of macrophage-like cells (expressing s100a10b, sftpbb, icn, fthl27, anxa5b, f13a1b and spi1b), and several groups of DC like cells expressing the markers siglec15l, ccl19a.1, ccr7, id2a, xcr1a.1, batf3, flt3, chl1a and hepacam2. Combining these new markers and transgenic reporter fish lines, the authors then clarified the location of leukocyte subsets within the brain, showing for example that DC-like cells stand as a parenchymal population along with microglia. Reporter lines were also used to perform a detailed analysis of cell subsets, and cross with a batf3 mutant demonstrated that DC-like cells are batf3 dependent, which was similar to mouse and human cDC1. Finally, analysis of classical mononuclear phagocyte deficient zebrafish lines showed they have reduced numbers of microglia but exhibit distinct DC-like cell phenotypes. A weakness of this study is that it is mainly based on FACS sorting, which might modify the proportion of different subtypes.

This atlas of zebrafish brain leukocytes is an important new resource for scientists using the zebrafish models for neurology, immunology, and infectiology, and for those interested in the evolution of the brain and immune system.

Reviewer #3 (Public Review):

Rovira, et al., aim to characterize immune cells in the brain parenchyma and identify a novel macrophage population referred to as "dendritic-like cells". They use a combination of single-cell transcriptomics, immunohistochemistry, and genetic mutants to conclude the presence of this "dendritic-like cell" population in the brain. The strength of this manuscript is the identification of dendritic cells in the brain, which are typically found in the meningeal layers and choroid plexus. A weakness is the lack of specific reporters or labeling of this dendritic cell population using specific genes found in their single-cell dataset. Additionally, it is difficult to remove the meningeal layers from the brain samples and thus can lead to confounding conclusions. Overall, I believe this study should be accepted contingent on sufficient labeling of this population and addressing comments.