# Multifaceted microglia during brain development: Models and tools

Cécile Bridlance, Morgane Sonia Thion

<figure><img src="image_1.png" /></figure>

## To cite this version:

Cécile Bridlance, Morgane Sonia Thion. Multifaceted microglia during brain development: Models and tools. Frontiers in Neuroscience, 2023, 17, pp.1125729. 10.3389/fnins.2023.1125729. hal-04752037

HAL Id: hal-04752037

https://hal.science/hal-04752037v1

Submitted on 24 Oct 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

Check for updates

OPEN ACCESS

EDITED BY

Subashika Govindan,
DBT/Wellcome Trust India Alliance, India

REVIEWED BY

Deborah R. Winter,
Northwestern University, United States
Kathryn M. Lenz,
The Ohio State University, United States

*CORRESPONDENCE

Morgane Sonia Thion

<figure><img src="image_4.png" /></figure>

thion@biologie.ens.fr

SPECIALTY SECTION

This article was submitted to
Translational Neuroscience,
a section of the journal
Frontiers in Neuroscience

RECEIVED 16 December 2022

ACCEPTED 24 February 2023

PUBLISHED 23 March 2023

CITATION

Bridlance C and Thion MS (2023) Multifaceted microglia during brain development: Models and tools.

Front. Neurosci. 17:1125729.
doi: 10.3389/fnins.2023.1125729

COPYRIGHT

© 2023 Bridlance and Thion. This is an
open-access article distributed under the terms
of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/).
The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted which
does not comply with these terms.

# Multifaceted microglia during brain development: Models and tools

Cécile Bridlance¹,²,³ and Morgane Sonia Thion¹,²*

¹Institut de Biologie de l'École Normale Supérieure (IBENS), École Normale Supérieure, CNRS, INSERM, Université PSL, Paris, France, ²Center for Interdisciplinary Research in Biology, Collège de France, CNRS, INSERM, Université PSL, Paris, France, ³Collège Doctoral, Sorbonne Université, Paris, France

Microglia, the brain resident macrophages, are multifaceted glial cells that belong to the central nervous and immune systems. As part of the immune system, they mediate innate immune responses, regulate brain homeostasis and protect the brain in response to inflammation or injury. At the same time, they can perform a wide array of cellular functions that relate to the normal functioning of the brain. Importantly, microglia are key actors of brain development. Indeed, these early brain invaders originate outside of the central nervous system from yolk sac myeloid progenitors, and migrate into the neural folds during early embryogenesis. Before the generation of oligodendrocytes and astrocytes, microglia thus occupy a unique position, constituting the main glial population during early development and participating in a wide array of embryonic and postnatal processes. During this developmental time window, microglia display remarkable features, being highly heterogeneous in time, space, morphology and transcriptional states. Although tremendous progress has been made in our understanding of their ontogeny and roles, there are several limitations for the investigation of specific microglial functions as well as their heterogeneity during development. This review summarizes the current murine tools and models used in the field to study the development of these peculiar cells. In particular, we focus on the methodologies used to label and deplete microglia, monitor their behavior through live-imaging and also discuss the progress currently being made by the community to unravel microglial functions in brain development and disorders.

KEYWORDS

microglia, brain, development, models, tools

Introduction

Microglia, the central nervous system (CNS) resident macrophages remained poorly studied until an exponential growth in interest during the last two decades led to fascinating insights into the origin of microglia, their functions, as well as dysfunctions in pathological conditions (Mosser et al., 2017; Hammond et al., 2018; Hoeffel and Ginhoux, 2018; Li and Barres, 2018; Thion et al., 2018a; Prinz et al., 2019). Contrary to most brain cells, microglia were shown to originate from mesodermal yolk sac (YS) macrophage progenitors that travel to reach the CNS during early embryonic development, around Embryonic day (E)9 in mice and gestational week 4/5 in humans (Monier et al., 2007; Ginhoux et al., 2010; Verney et al., 2010; Menassa et al., 2022). As such, these brain invaders constitute the main glial population before the emergence of other glial cells such as oligodendrocytes and astrocytes. After closure of the blood brain barrier around E14 in mice, microglia are believed to be
enclosed in the brain under steady-state conditions. These pioneer microglia thus proliferate, seed the entire parenchyma and progressively mature in symbiosis with the neural tissue microenvironment (Matcovitch-Natan et al., 2016; Thion et al., 2018b; Kracht et al., 2020) before self-renewing throughout life. This situation is different in zebrafish where microglia are fully replaced by another source of microglia by adulthood (Xu et al., 2015; Ferrero et al., 2018). A key aspect of their development is the high heterogeneity in their colonization patterns, morphologies and molecular properties. In particular, microglial colonization of the brain parenchyma is a long-lasting process that spans embryogenesis until the end of the second postnatal week, following a very stereotypical and uneven spatiotemporal pattern (Swinnen et al., 2013; Squarzoni et al., 2014; Menassa et al., 2022). These cells transiently accumulate at specific hotspots such as the cortico-striatal-amygdalar boundary and are excluded from others regions such as the cortical plate. In addition, they display a variety of morphologies (ameboid, poorly ramified, and elongated) associated with different brain localizations. Finally, owing to high throughput approaches, microglia have been shown to exhibit different transcriptomic states, specifically during development, in both mice and humans (Hammond et al., 2019; Li et al., 2019; Sankowski et al., 2019; Kracht et al., 2020). This high developmental heterogeneity contrasts with a relatively uniform distribution in the whole parenchyma alongside homogeneously ramified morphologies and molecular signatures at adult stages. Finally, sex-specific microglial features have been highlighted in postnatal steady-state conditions but also in response to environmental challenges (Schwarz et al., 2012; Lenz et al., 2013; Rebuli et al., 2016; Hanamsagar et al., 2017; Guneykaya et al., 2018; Thion et al., 2018b; Villa et al., 2018; VanRyzin et al., 2019).

Several seminal studies have demonstrated that, beyond their immune functions, microglia also perform a wide array of cellular functions that relate to the normal functioning of the brain and importantly to its development. In particular, they interact with synapses to mediate remodeling, pruning and transmission but have also been involved in synaptogenesis (Andoh and Koyama, 2021). They further participate to neurogenesis and oligodendrogenesis, partly through their regulation of cell death and survival (Sierra et al., 2010; Cunningham et al., 2013; Hagemeyer et al., 2017; Wlodarczyk et al., 2017; Nemes-Baran et al., 2020; Sherafat et al., 2021; Cserep et al., 2022). They also contribute to the refinement of axonal tracts (Pont-Lezica et al., 2014; Squarzoni et al., 2014) and to the development of cortical inhibitory circuits (Squarzoni et al., 2014; Thion et al., 2019; Favuzzi et al., 2021; Yu et al., 2022). Besides, microglia express various pattern recognition, purinergic, chemokine and cytokine receptors, collectively described as the sensome (Hickman et al., 2013), enabling them to detect and integrate environmental changes. Importantly and consistently with their wide array of cellular functions, microglial dysfunction has been associated with the etiology of neurodevelopmental disorders, including autism spectrum disorders and schizophrenia in humans and mouse models (Lukens and Eyo, 2022). Therefore, during this crucial period of development, it is key to better grasp their functions, the regulatory mechanisms underpinning their heterogeneity and how their molecular states may regulate their roles. Furthermore, elucidating how external signals can impact on these fundamental processes is a major challenge. This will be crucial to illuminate specific and diverse microglial contributions to brain wiring as well

as shed light on pathological mechanisms of neurodevelopmental disorders.

Beside microglia, other non-parenchymal macrophages called Border-Associated Macrophages (BAMs) are present at the interfaces of the brain: the meninges, choroid plexus and perivascular space (Lee et al., 2021; Figure 1). Although microglia and BAMs originate from yolk-sac derived progenitors and seed the brain during embryogenesis, some of them are further replaced by monocyte-derived cells, arising from hematopoietic stem cells (Goldmann et al., 2016; Mrdjen et al., 2018; Van Hove et al., 2019; Utz et al., 2020; Masuda et al., 2022). Moreover, while generally referred to as BAMs, they display age- and tissue-specific signatures (Kierdorf et al., 2019; Mildenberger et al., 2022). Most of the well-recognized microglial markers, reporter mouse lines and models that currently exist to label and deplete microglia can also target a large part of macrophages such as the BAMs in the CNS but also populations of peripheral macrophages (Green et al., 2020). Consequently, despite intense research efforts, these limitations prevent the identification and characterization of specific microglia functions, especially during development. Herein, we discuss about the current murine tools and models available to label or deplete microglia and subsequently assess their developmental functions in physiological and disease conditions.

## From broad microglial targeting to specific states labeling

### Catch me if you can: Microglia and other brain macrophages

Historically, microglia were mainly identified either through Iba1 immunostaining or using the Cx3cr1<sup>GFP/+</sup> (Jung et al., 2000) that labels many macrophages, including microglia and BAMs. Similarly, the well-established Cx3cr1<sup>creERT2</sup> mouse lines have been very useful to inactivate genes in these cells (Parkhurst et al., 2013; Yona et al., 2013). Nevertheless, they are not specific to microglia and may trigger microglial reactivity in neonates upon tamoxifen administration (Sahasrabuddhe and Ghosh, 2022). Recent studies, including single cell RNA sequencing analyses, highlighted more specific homeostatic microglia markers including p2ry12, *Sall1*, *Tmem119*, *Hexb*, *Siglech* (Gautier et al., 2012; Buttgereit et al., 2016; Satoh et al., 2016; Cserep et al., 2020; Masuda et al., 2020) allowing the use of specific antibodies to label either microglia (P2ry12, *Tmem119*, and *Siglech*) or BAMs (CD206, Lyve1, and *Siglec1*) (Mrdjen et al., 2018; Figure 1). Fluorescent *In Situ* Hybridization (FISH) has also been used to circumvent the absence of specific antibodies, particularly by taking advantage of the RNAscope technique (Matcovitch-Natan et al., 2016; Hammond et al., 2019). To specifically visualize, manipulate and assess microglia functions, reporter and *creERT2*-expressing lines were generated with minor recombination in BAMs: *Tmem119<sup>eGFP</sup>* and *Tmem119<sup>creERT2</sup>* (Kaiser and Feng, 2019), *Tmem119<sup>tdTomato</sup>* (Ruan et al., 2020), *Hexb<sup>tdTomato</sup>* and *Hexb<sup>creERT2</sup>* (Masuda et al., 2020), *p2ry12<sup>creERT2</sup>* (McKinsey et al., 2020), *Sall1<sup>GFP</sup>* and *Sall1<sup>creERT2</sup>* (Buttgereit et al., 2016), the latter recombining in neurons and other glia (Chappell-Maor et al., 2020; Table 1). Nonetheless, since most of these genes start to be expressed as microglia mature (Bennett et al., 2016), it is important to stress that there are so far no
<figure><img src="image_5.png" /><figcaption>FIGURE 1</figcaption></figure>

Diversity of CNS macrophages in steady-state conditions. During steady-state, microglia are localized within the brain parenchyma and are highly heterogeneous in morphologies, localization and molecular signatures throughout brain development. In particular, axon tract-associated microglia (ATM)/proliferative-region-associated microglia (PAM)/Cd11c-positive microglia have been described during early postnatal development in the corpus callosum and other white matter regions. Border-associated macrophages (BAMs) reside at distinct interfaces of the CNS such as the meninges, choroid plexus and perivascular space (Goldmann et al., 2016; Mrdjen et al., 2018; Van Hove et al., 2019; Utz et al., 2020; Masuda et al., 2022). Non-parenchymal microglia-like Kolmer's epiplexus BAMs (CP<sup>epi</sup>) reside in the apical surface of the choroid plexus facing the cerebrospinal fluid (CSF) and share some transcriptional features with microglia and ATM signature (Van Hove et al., 2019). In particular, meningeal MHCII<sup>low</sup> and MHCII<sup>high</sup> macrophages are localized in the dura matter while subdural macrophages (SDM) are restrained to the subdural area. Finally, while perivascular macrophages (PVM) are found in the perivascular space, between the vascular basement membrane and the glia limitans of the parenchyma, choroid plexus contains MHCII<sup>low</sup> and MHCII<sup>high</sup> BAMs. The insets depict the most commonly used markers for microglia (green), BAMs (blue), and common markers to both populations (black). ATM, axon tract-associated microglia; BAMs, border-associated macrophages; CP<sup>epi</sup>, Kolmer's epiplexus; CSF, cerebrospinal fluid; MHCII, major histocompatibility complex class II; PVS, perivascular space; PVM, perivascular macrophage; SDM, subdural macrophage.

alternatives to the Cx3cr1<sup>GFP/+</sup> or Cx3cr1<sup>creERT2</sup> lines to efficiently
target early embryonic microglia.

Targeting microglia more specifically is crucial, since studies
often assign a variety of roles to microglia using depletion
models that target both microglia and BAMs. Kim et al. (2021)
took advantage of an elegant “split cre” binary genetic construct
to generate the Cx3cr1<sup>cre;Sall1ncre</sup> and Cx3cr1<sup>cre;Lyve1ncre</sup>, to
selectively target microglia and BAMs, respectively. Furthermore,
several mouse lines including the Siglec1<sup>cre</sup> (Utz et al., 2020), Pf4-
Cre (McKinsey et al., 2020), Lyve1<sup>creERT2</sup>, and Mrc1<sup>CreERT2</sup> lines
(Masuda et al., 2022) have been shown to specifically label BAMs
during development and thereby enable to follow their trajectory
(Table 1). In addition, while no circulating cells are thought to enter
in the brain parenchyma during steady-state after BBB closure,
monocyte infiltration can occur in disease, aging and injury, and
can be monitored using bone marrow chimeras (Mills et al.,
2022). These new tools will be important to decipher the relative
contributions of microglia, BAMs or infiltrating myeloid cells, but
still require thorough characterization with regards to efficiency
and rate of spontaneous recombination at different timepoints
along development.

Finally, cell-specific viral gene delivery has been extensively
used to target different CNS population but robust transduction
in microglia remained quite inefficient until recently (Maes
et al., 2019). In a ground-breaking study, Lin et al. (2022)

successfully targeted 80% of microglia *in vivo* using adeno-
associated viruses without inducing microglia reactivity or changes
in gene expression, although it remains elusive to what extend
BAMs were also affected. Such approach opens new avenues to
study microglia but also have tremendous translational potential.

Looking with new eyes

Along with new markers, mouse lines and viral approaches
to target microglia, technical advances in diverse fields shed new
light on ways to study microglia, in particular going from fixed
immobile imaging in brain slices to dynamic and global approaches.
First, tissue clearing methods are constantly improving, with some
of them perfectly preserving the signal from reporter lines and
antibodies [reviewed in Eme-Scolan and Dando (2020)]. This
allows visualization of microglia in whole intact brains. However,
while these techniques are becoming well-established and easier
to use routinely, the difficulty lies in the analysis of the generated
data. Annotated 3D atlases of the developing brain will offer
many exciting possibilities toward a more comprehensive study of
microglia development.

Another revolution in the field of microglia came with
two-photon live-imaging experiments, which revealed never
resting microglia with their processes constantly surveilling their
<table><caption>TABLE 1 Tools to label, target and deplete microglia and BAMs.</caption><thead><tr><th>Tagging models</th><th>Microglia</th><th>BAMs</th><th>Prenatal and early postnatal</th><th>Adulthood</th><th>Remarks</th><th>References</th></tr></thead><tbody><tr><td>Cx3cr1<sup>GFP/+</sup></td><td>Yes</td><td>Yes</td><td>GFP expressed by E9.5</td><td>&gt;99%</td><td>Targets peripheral immune cells and myeloid BM progenitors</td><td>Jung et al., 2000</td></tr><tr><td>Cx3cr1<sup>creERT2</sup> (1,2)</td><td>Yes</td><td>Yes</td><td>40H-Tam. E9.0: 99% efficiency at E10.5 (2)</td><td>&gt;99%</td><td>Spontaneous recombination reported</td><td>Parkhurst et al., 2013 (1); Yona et al., 2013 (2)</td></tr><tr><td>Tmem119<sup>GFP</sup></td><td>Yes</td><td>Few</td><td>GFP expressed by P1</td><td>&gt;99%</td><td>Transiently labels blood vessels at P1</td><td>Kaiser and Feng, 2019</td></tr><tr><td>Tmem119<sup>creERT2</sup></td><td>Yes</td><td>Few</td><td>Tam. P2, P3 and P4: 90% efficiency by P14</td><td>&gt;99%</td><td>Targets few CD45+ cells in blood</td><td>Ruan et al., 2020</td></tr><tr><td>Tmem119<sup>tdTomat</sup></td><td>Yes</td><td>Few</td><td>N.A.</td><td>&gt;95%</td><td></td><td></td></tr><tr><td>Hexb<sup>tdTomato</sup></td><td>Yes</td><td>Few</td><td>TdTomato expressed by E12.5</td><td>&gt;99%</td><td>Labels few meningeal and perivascular BAMs</td><td>Masuda et al., 2020</td></tr><tr><td>Hexb<sup>creERT2</sup></td><td>Yes</td><td>Few</td><td>Tam. P1 and P3: 90% efficiency at P42</td><td>&gt;80%</td><td>Targets peripheral macrophages in kidney</td><td></td></tr><tr><td>P2ry12<sup>creERT2</sup></td><td>Yes</td><td>Few</td><td>Tam. E13.5, E15.5 and E17.5: robust efficiency by E18.5 in microglia and subsets of BAMs (40% in choroid plexus, 10% in meninges, few PVM)</td><td>90–95%</td><td>Targets 20–25% of BAMs in choroid plexus and meninges</td><td>McKinsey et al., 2020</td></tr><tr><td>Sall1<sup>GFP</sup></td><td>Yes</td><td>No</td><td>GFP expressed in 20% microglia by E12.5, 69% by E14.5, 90% by P2</td><td>&gt;95%</td><td>Targets &lt;10% other CNS cells</td><td></td></tr><tr><td>Sall1<sup>creERT2</sup></td><td>Yes</td><td>No</td><td>Tam. E14.5 and E16.5: 75% efficiency at E18.5</td><td>&gt;95%</td><td>Targets &lt;10% other CNS cells</td><td>Buttgereit et al., 2016; Utz et al., 2020</td></tr><tr><td>Cx3cr1<sup>ccre</sup>:Sall1<sup>ncre</sup></td><td>Yes</td><td>No</td><td>N.A.</td><td>&gt;90%</td><td></td><td>Kim et al., 2021</td></tr><tr><td>Cx3cr1<sup>ccre</sup>:Lyve1<sup>ncre</sup></td><td>No</td><td>Yes</td><td>N.A.</td><td>20% of Lyve1+ cells</td><td></td><td></td></tr><tr><td>Lyve1<sup>CreERT2</sup></td><td>No</td><td>Yes</td><td>40H-Tam. E16.5: 50% efficiency in meningeal and perivascular macrophages at P14</td><td>N.A.</td><td>Targets lymphatic endothelial cells</td><td>Masuda et al., 2022</td></tr><tr><td>Siglec1<sup>cre</sup></td><td>No</td><td>Yes</td><td>Efficiently floxes gene in BAMs at E18.5</td><td>N.A.</td><td>Siglec1 expressed by 60% BAMs at E14.5, 100% at E18.5</td><td>Utz et al., 2020</td></tr><tr><td>Pf4-Cre</td><td>Few</td><td>Yes</td><td>N.A.</td><td>&gt;99%</td><td></td><td>McKinsey et al., 2020</td></tr><tr><td>Mrc1<sup>CreERT2</sup></td><td>No</td><td>Yes</td><td>40H-Tam. E9.0: 10% efficiency in BAMs and 5% in microglia by E18.5</td><td>&gt;95%</td><td></td><td>Masuda et al., 2022</td></tr><tr><td>Adenoviruses</td><td>Yes</td><td>N.A.</td><td>N.A.</td><td>80%</td><td></td><td>Lin et al., 2022</td></tr></tbody></table>

(Continued)
<table><caption>TABLE 1 (Continued)</caption><thead><tr><th>Depletion models</th><th>Microglia</th><th>BAMs</th><th>Prenatal and early postnatal</th><th>Adulthood</th><th>Remarks</th><th>References</th></tr></thead><tbody><tr><td colspan="7"><strong>Killing by numbers</strong></td></tr><tr><td>Cx3cr1<sup>CreER;R26<sup>DTR</sup></sup></td><td>Yes</td><td>Yes</td><td>N.A.</td><td>&gt;99%</td><td>Fast repopulation</td><td>Parkhurst et al., 2013</td></tr><tr><td>IBA1-tTA<sup>DTAtetO/tetO</sup></td><td>Yes</td><td>Yes</td><td>Withdrawal of doxycycline from maternal diet from P5: 50% depletion at P8</td><td>~90% in retina</td><td>Fast repopulation</td><td>Miyamoto et al., 2016; Takeda et al., 2018</td></tr><tr><td>Sglech<sup>DTR/DTR</sup></td><td>Yes</td><td>No</td><td>Injection at E10.5: 80% depletion at E12.5, 60% at E14.5</td><td>80-85%</td><td>Fast repopulation</td><td>Konishi et al., 2017; Li et al., 2021</td></tr><tr><td colspan="7"><strong>CSF-1R inhibitors</strong></td></tr><tr><td>Anti CSF-1R antibodies</td><td>Yes</td><td>Yes</td><td>Injections at E6 and E7: &gt;98% depletion at E14.5</td><td>No effect</td><td>Fast repopulation</td><td>MacDonald et al., 2010; Squarzoni et al., 2014</td></tr><tr><td>Anti CSF-1 antibodies</td><td>Yes</td><td>No</td><td>Injections at E6 and E7: &gt;50% depletion at P0.5</td><td>60% in white matter region</td><td>Dose-dependent efficiency</td><td>Easley-Neal et al., 2019</td></tr><tr><td>Anti IL-34 antibodies</td><td>Yes</td><td>No</td><td>Injections at E6 and E7: no effect at P0.5. Injection at P0.5: 30% depletion at P4</td><td>50% in grey matter region</td><td>Dose-dependent efficiency</td><td></td></tr><tr><td>PLX5622</td><td>Yes</td><td>Yes</td><td>Chow PLX from E3.5: 99% depletion at E15.5</td><td>&gt;95% within 7 days</td><td>Fast repopulation</td><td>Huang et al., 2018; Rosin et al., 2018; Marsters et al., 2020</td></tr><tr><td>PLX3397</td><td>Yes</td><td>Yes</td><td>Chow PLX from E14 followed by s.c. injections in pups: 90% depletion at P5 in spinal cord</td><td>&gt;99% within 7 days</td><td>Fast repopulation</td><td>Elmore et al., 2014; Li et al., 2020</td></tr><tr><td colspan="7"><strong>Genetic models</strong></td></tr><tr><td>Pu.1<sup>-/-</sup></td><td>Yes</td><td>Yes</td><td>100%</td><td>100%</td><td>Homozygotes die shortly after birth</td><td>McKercher et al., 1996</td></tr><tr><td>Csf1<sup>OP/op</sup></td><td>Yes</td><td>Yes</td><td>N.A.</td><td>0-30%</td><td>Abnormal brain development</td><td>Michaelson et al., 1996</td></tr><tr><td>Il34<sup>LacZ/LacZ</sup> (1,2)</td><td>Yes</td><td>N.A.</td><td>&gt;80% decrease at P2 (1); normal colonization of the brain from E10.5 to newborn (2)</td><td>50% (1; 2)</td><td></td><td>Wang et al., 2012 (1); Greter et al., 2012 (2)</td></tr><tr><td>Csf1<sup>r/-</sup></td><td>Yes</td><td>Yes</td><td>&gt;99%</td><td>100%</td><td>Shortened lifespan and abnormal brain development</td><td>Erblich et al., 2011</td></tr><tr><td>Sall1<sup>CreER;Csf1<sup>fl/fl</sup></sup></td><td>Yes</td><td>No</td><td>N.A.</td><td>70-90%</td><td>Spatial variability in efficiency</td><td>Buttgereit et al., 2016</td></tr><tr><td>Hexb<sup>CreERT2;Csf1<sup>fl/fl</sup></sup></td><td>Yes</td><td>No</td><td>N.A.</td><td>60%</td><td></td><td>Masuda et al., 2020</td></tr><tr><td>Csf1<sup>R</sup><sup>Δ FIRE/Δ FIRE</sup></td><td>Yes</td><td>Few</td><td>Absence of CPepi in the choroid plexus, other BAMs reduced</td><td>100%</td><td></td><td>Rojo et al., 2019; Munro et al., 2020</td></tr><tr><td>Nestin<sup>cre;Csf1<sup>fl/fl</sup></sup></td><td>Yes</td><td>N.A.</td><td>60% decrease in E17.5 cerebellum</td><td>~50% in cerebellum</td><td></td><td>Kana et al., 2019</td></tr><tr><td>Nestin<sup>cre;Il34<sup>fl/fl</sup></sup></td><td>Yes</td><td>N.A.</td><td>N.A.</td><td>~85% in striatum</td><td></td><td>Badimon et al., 2020</td></tr></tbody></table>

Table summarizing the main mouse lines and tools available to label, target and deplete microglia and BAMs, indicating their specificity and efficiency in development and adulthood. BAMs, border-associated macrophages; BM, bone marrow; CNS, central nervous system; CPepi, Kolmer's epiplexus; DTA, diphtheria toxin fragment A; DTR, diphtheria toxin receptor; iDTR, inducible diphtheria toxin receptor; i.p., intraperitoneal; N.A., non-applicable; PVM, perivascular macrophages; s.c., subcutaneous; Tam, tamoxifen; 4H-Tam, 4-hydroxytamoxifen.
environment as well as rapidly reacting in case of injury (Davalos
et al., 2005; Nimmerjahn et al., 2005). In combination with other
markers, it is thus possible to monitor microglial interaction with
blood vessels (Csaszar et al., 2022), radial glia (Rosin et al., 2021),
neuronal populations as well as track microglial processes and
their specific contact with synapses (Wake et al., 2009) or nodes
of Ranvier (Ronzano et al., 2021). While some experiments are
performed on brain slices, inducing tissue damage and possibly
altering microglial behavior, most studies use cranial windows
or skull thinning that allows microglial observation in their
homeostatic environment. For pups, adapted approaches are being
developed taking advantage of the thinness of the embryonic skull
to perform *ex utero* live-imaging of microglia and macrophages
in the brain of intact embryos (Hattori et al., 2020, 2022; Munz
et al., 2022), that will probably be critical to better characterize key
aspects of microglia development such as their entry in the brain
parenchyma (Stremmel et al., 2018).

As a self-renewing population arising from a restricted pool
of pioneer cells, the questions of microglial expansion during
development, their migration and turnover were raised—and still
retain some mystery. The “Microfetti” mice developed by Tay
et al. (2017) in which microglia express randomly one out of
four fluorophores after tamoxifen induction, highlighted clonal
expansion of adult microglia in pathology. In addition, Ratz et al.
(2022) developed the new TREX technique, which combines single-
cell and spatial transcriptomic coupled to early (E9.5) in vivo
barcoding, in order to analyze the lineage relationships between
mature cells and progenitors. Thereby, they highlighted drastic
microglial expansion from a limited pool of progenitors. Though
not extensively used yet, these tools for clonal analysis and lineage
tracing will be valuable in the context of development to better
understand microglial expansion, migration and final distribution
in the brain.

Targeting the different flavors of
microglia

Historically, microglia were classified as displaying either
a neurotoxic pro-inflammatory M1 or neuroprotective anti-
inflammatory M2 phenotype but this binary perspective has
been largely revisited (Paolicelli et al., 2022). The development
of high throughput technologies such as single cell and single
nucleus RNA sequencing (sc/snRNA-seq), cytometry by time-
of-flight (CyTOF), or multiplex error-robust fluorescent *in situ*
hybridization (MERFISH) revealed a richer heterogeneity in
microglial profiles. While microglia constitute a relatively
homogeneous population in adulthood, they display a striking
heterogeneity during prenatal/early postnatal development, aging
and neurodegeneration (Deczkowska et al., 2018; Hammond
et al., 2019; Li et al., 2019; Masuda et al., 2019; Sankowski et al.,
2019; Kracht et al., 2020; Marschallinger et al., 2020; Safaiyan
et al., 2021; Stogsdill et al., 2022). Nevertheless, we should not
underestimate the experimental bias introduced by cell dissociation
and sorting-strategies, sequencing technologies and subsequent
analyzes selected to characterize microglial heterogeneity (Marsh
et al., 2022; Paolicelli et al., 2022; Sankowski et al., 2022). To avoid
extensive confusion in the field, it remains crucial to be cautious
about the semantic implication of microglial heterogeneity and the

subsequent functional diversity associated with it (Paolicelli et al.,
2022).

Specific developmental microglial states are thus starting
to be described such as the Axon Tract-associated Microglia
(ATM), also known as Proliferative-region-Associated Microglia
(PAM), Cd11c-positive microglia, and Youth-Associated Microglia
(YAM). They are characterized by the expression of several
genes such as *Spp1*, *Itgax*, *Gpnmb* and were initially identified
in the postnatal white matter as regulators of the development
of oligodendrocyte precursors and of subsequent myelinogenesis
(Hagemeyer et al., 2017; Wlodarczyk et al., 2017; Hammond
et al., 2019; Li et al., 2019; Silvin et al., 2022). Interestingly,
there are remarkable similarities in transcriptomic signatures
between ATM and Disease Associated Microglia (DAM), initially
characterized in mouse models of Alzheimer's disease (Keren-
Shaul et al., 2017; Krasemann et al., 2017), raising the question
of their potential similarities, differences—and relationship. Apart
from the use of specific antibodies or *in situ* hybridization
probes, the field lacks genetic tools to specifically label and target
microglia states. So far, only a CD11c<sup>cre</sup> line has been used to
target ATM (Wlodarczyk et al., 2017), but was unsuccessful in
depleting ATM in combination with Diphtheria Toxin strategies.
In addition, a novel Spp1<sup>tdTomato</sup> mouse line has been generated
and used to monitor SPP1 from perivascular macrophages (De
Schepper et al., 2022), and could constitute an interesting tool
for ATM-lineage studies. Based on their emerging molecular
characterization, novel mouse lines or viral approaches will
enable specific depletion or inactivation of microglial states
to further assess their fates and functions. Moreover, in-depth
characterization of transcriptomic, epigenomic, and metabolomic
landscapes in microglia would enable to better understand their
regulatory mechanisms, the transientness of these states and to
which extent they can be induced by the microenvironment at
different stages of life. Along these lines, the progress of spatial
transcriptomics toward increased structural resolution should
lead to a tremendous breakthrough to characterize microglial
heterogeneity (Stogsdill et al., 2022), in particular at hotspots
of accumulating microglia during development. Altogether, the
field is moving onto a specific targeting of developmental
microglial states which should shed new light on their regulatory
mechanisms, their plasticity and functions in steady-state and
disease conditions.

En route for specific depletion
approaches

Microglial functions in brain development, homeostasis and
diseases were historically assessed using *in vivo* depletion strategies
summarized in Table 1. Though a lot of techniques have been
developed, their diversity illustrates the difficulty to obtain a
specific, efficient and long-lasting depletion of microglia while
limiting its off-target effects.

Killing by numbers

The first strategies employed were aiming to directly trigger
microglial apoptosis by administration of clodronate liposomes,
that are specifically phagocytosed by microglia and BAMs and induce cell death upon cytoplasmic release (Green et al., 2020). This approach is efficient in the early post-natal brain and in adults, but the inability of the clodronate liposomes to cross the blood brain barrier requires an intracerebral injection, inducing an injury and possible release of inflammatory cytokines (Han et al., 2019). Another approach uses the diphtheria-toxin (DT) based systems, either with direct expression of the DTA or with DT receptor, the latter needing administration of DT. The first studies were performed in Cx3cr1$^{CreER}$;R26$^{iDTR}$ and Iba1-tTA:$DTAtetO/tetO$ targeting both microglia and BAMs (Parkhurst et al., 2013; Miyamoto et al., 2016; Takeda et al., 2018). On the contrary, the use of the Siglech$^{dtr+}$ mice led to specific and transient depletion of embryonic microglia, without affecting BAMs (Li et al., 2021). Thus, the constitutive or inducible DTR/DTA expression, combined with specific microglial lines provides a better temporal control as well as selective effect, though their efficiency remains variable.

## CSF-1R inhibitors

The CSF-1 receptor (CSF-1R) is expressed by microglia, macrophages and their progenitors and its signaling is essential for their survival and proliferation. It thus became a preferential target in the quest for microglial depletion methods. Injections of an anti-CSF-1R antibody performed at E6.5 and E7.5 lead to a drastic depletion of myeloid progenitors, macrophages and microglia, while repopulation spans the first postnatal week (Squarzoni et al., 2014; Hoeffel and Ginhoux, 2015; Thion et al., 2019). On the other hand, embryonic targeting of CSF-1, one of the two CSF-1R ligands, through anti-CSF-1 antibody, leads to a drastic depletion of forebrain microglia (Easley-Neal et al., 2019), with possible off-target effects of the circulating antibodies. Pharmacological inhibitors of the CSF-1R injected or delivered non-invasively *via* food pellets have been broadly developed to deplete adult microglia, such as PLX3397 and PLX5622, the latter having a higher specificity and improved brain penetrance (Elmore et al., 2014; Green et al., 2020) while it efficiently depletes embryonic microglia, enabling a temporal control of the depletion during gestation (Rosin et al., 2018; Marsters et al., 2020). Nevertheless, as this treatment can affect lactation by depleting maternal macrophages essential for mammary gland development (O'Brien et al., 2012), it can impact pup survival following birth. For early postnatal depletion, direct subcutaneous injections of PLX3397 or PLX5622 during the first post-natal week allows for an efficient depletion of microglia (Li et al., 2020; Favuzzi et al., 2021; Gesuita et al., 2022). Although beyond the scope of this review, CSF-1R inhibitors are also used in other species including humans, bringing novel therapeutic approaches [reviewed in Han et al. (2022)]. Overall, targeting the CSF-1R pathway has proven to be efficient and convenient, but also affects peripheral macrophages and BAMs, preventing the identification of specific microglial functions.

## Genetic models

Different genetic models, including constitutive knock-outs of fundamental transcription or survival factors, like Pu.1$^{-/}$

(McKercher et al., 1996) and Csf1r$^{-/}$ (Erblich et al., 2011), fail to develop microglia as well as most macrophages (Green et al., 2020). This results in many off-target effects and early death in Pu.1$^{-/}$ and Csf1r$^{-/}$. On the other hand, the Csf1r$^{fl/fl}$ allows for a more specific targeting of microglia, thanks to the microglia specific mouse lines now available and their possible temporal induction. This strategy has been used with Sall1$^{CreER}$;Csf1r$^{fl/fl}$ (Buttgereit et al., 2016) and Hexb$^{CreERT2}$;Csf1r$^{fl/fl}$ mice (Masuda et al., 2020), with a respective efficiency varying across brain regions from 70% to 90% and of 60%.

An additional important model is the Csf1r$^{ΔFIRE/ΔFIRE}$ mice, in which a Csf1r enhancer is deleted (Rojo et al., 2019; Munro et al., 2020). This leads to the absence of microglia from the brain parenchyma, and of resident macrophages of the skin, kidney, heart and peritoneum; while other macrophages and monocytes are unaffected. Importantly, these mice are healthy and fertile in a mixed B6CBAF1/C57BL/6J background, and do not display strong developmental defects described in the Csf1r$^{-/}$ So far, they were used to investigate the role of resident microglia in Alzheimer disease mice (Kiani Shabestari et al., 2022) and they will provide a more specific and long-lasting model to explore microglial functions.

Finally, full inactivation of CSF-1R ligands, Csf-1 (Chang et al., 1994; Kondo and Duncan, 2009) or Il34 (Greter et al., 2012; Wang et al., 2012), expressed by the neural tissue, results in time- and region-dependent partial depletion of microglia albeit there are still some controversies amongst studies and major impact on peripheral macrophages (Green et al., 2020; Table 1). Nevertheless, under the pan neuronal driver Nestin$^{cre}$ that restricts depletion to the nervous system, thus mainly affecting microglia, the Nestin$^{cre}$;Csf1r$^{fl/fl}$ and Nestin$^{cre}$;Il34$^{fl/fl}$ are respectively deprived of microglia in the white matter and cerebellum or in the gray matter highlighting region-specific dependency (Kana et al., 2019; Badimon et al., 2020). On the other hand, macrophages are unaffected in the yolk sac, fetal liver and fetal limbs. Last, because the CSF-1R ligands are differentially expressed and required from microglia through development, these lines provide promising models to investigate local microglial functions.

## Concluding remarks

Microglia now appear as key actors of brain development, interacting with most brain cells and regulating several crucial developmental processes. Nevertheless, these exciting advances were done in models affecting both microglia and BAMs. Thus, dissecting their respective contributions thanks to new specific tools will be key to the field. In addition, further understanding microglial diversity during development and the functions played by specific states should bring new light on their importance in neurodevelopmental pathologies and open new avenues for therapeutic intervention.

## Author contributions

CB and MST wrote the review. Both authors contributed to the article and approved the submitted version.
## Funding

The laboratories acknowledge support from the INSERM, CNRS, École Normale Supérieure, Collège de France and Fondation du Collège de France. CB was supported by an AMX doctoral fellowship and an ARC fellowship. MST is a CNRS investigator supported by the Foundation for Brain Research (FRC).

## Acknowledgments

We are grateful to members of the Thion and Garel laboratories for stimulating discussions and comments on the manuscript.

## References

Andoh, M., and Koyama, R. (2021). Microglia regulate synaptic development and plasticity. *Dev. Neurobiol.* 81, 568–590. doi: 10.1002/dneu.22814

Badimon, A., Strasburger, H. J., Ayata, P., Chen, X., Nair, A., Ikegami, A., et al. (2020). Negative feedback control of neuronal activity by microglia. *Nature* 586, 417–423. doi: 10.1038/s41586-020-2777-8

Bennett, M. L., Bennett, F. C., Liddelow, S. A., Ajami, B., Zamanian, J. L., Fernhoff, N. B., et al. (2016). New tools for studying microglia in the mouse and human CNS. *Proc. Natl. Acad. Sci. U.S.A.* 113, E1738–E1746. doi: 10.1073/pnas.1525528113

Buttgereit, A., Lelios, I., Yu, X., Vrohlings, M., Krakoski, N. R., Gautier, E. L., et al. (2016). Sall1 is a transcriptional regulator defining microglia identity and function. *Nat. Immunol.* 17, 1397–1406. doi: 10.1038/ni.3585

Chang, Y., Albright, S., and Lee, F. (1994). Cytokines in the central nervous system: Expression of macrophage colony stimulating factor and its receptor during development. *J. Neuroimmunol.* 52, 9–17. doi: 10.1016/0165-5728(94)90156-2

Chappell-Maor, L., Kolesnikov, M., Kim, J. S., Shemer, A., Haimon, Z., Grozovski, J., et al. (2020). Comparative analysis of CreER transgenic mice for the study of brain macrophages: A case study. *Eur. J. Immunol.* 50, 353–362. doi: 10.1002/eji.201948342

Csaszar, E., Lenart, N., Cserep, C., Kornyei, Z., Fekete, R., Posfai, B., et al. (2022). Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions. *J. Exp. Med.* 219:e20211071. doi: 10.1084/jem.20211071

Cserep, C., Posfai, B., Lenart, N., Fekete, R., Laszlo, Z. I., Lele, Z., et al. (2020). Microglia monitor and protect neuronal function through specialized somatic purinergic junctions. *Science* 367, 528–537. doi: 10.1126/science.aax6752

Cserep, C., Schwarc, A. D., Posfai, B., Laszlo, Z. I., Kellermayer, A., Kornyei, Z., et al. (2022). Microglial control of neuronal development via somatic purinergic junctions. *Cell Rep.* 40:111369. doi: 10.1016/j.celrep.2022.111369

Cunningham, C. L., Martinez-Cerdeno, V., and Noctor, S. C. (2013). Microglia regulate the number of neural precursor cells in the developing cerebral cortex. *J. Neurosci.* 33, 4216–4233. doi: 10.1523/JNEUROSCI.3441-12.2013

Davalos, D., Grutzendler, J., Yang, G., Kim, J. V., Zuo, Y., Jung, S., et al. (2005). ATP mediates rapid microglial response to local brain injury in vivo. *Nat. Neurosci.* 8, 752–758. doi: 10.1038/nn1472

De Schepper, S., Ge, J. G., Crowley, G., Ferreira, L. S. S., Garceau, D., Toomey, C. E., et al. (2022). Perivascular SPP1 mediates microglial engulfment of synapses in Alzheimer's disease models. *bioRxiv* [Preprint]. doi: 10.1101/2022.04.04.486547

Deczkowska, A., Keren-Shaul, H., Weiner, A., Colonna, M., Schwartz, M., and Amit, I. (2018). Disease-associated microglia: A universal immune sensor of neurodegeneration. *Cell* 173, 1073–1081. doi: 10.1016/j.cell.2018.05.003

Easley-Neal, C., Foreman, O., Sharma, N., Zarrin, A. A., and Weimer, R. M. (2019). CSF1R ligands IL-34 and CSF1 are differentially required for microglia development and maintenance in white and gray matter brain regions. *Front. Immunol.* 10:2199. doi: 10.3389/fimmu.2019.02199

Elmore, M. R., Najafi, A. R., Koike, M. A., Dagher, N. N., Spangenberg, E. E., Rice, R. A., et al. (2014). Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. *Neuron* 82, 380–397. doi: 10.1016/j.neuron.2014.02.040

Eme-Scolan, E., and Dando, S. J. (2020). Tools and approaches for studying microglia in vivo. *Front. Immunol.* 11:583647. doi: 10.3389/fimmu.2020.583647

## Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Erblich, B., Zhu, L., Etgen, A. M., Dobrenis, K., and Pollard, J. W. (2011). Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits. *PLoS One* 6:e26317. doi: 10.1371/journal.pone.0026317

Favuzzi, E., Huang, S., Saldi, G. A., Binan, L., Ibrahim, L. A., Fernandez-Otero, M., et al. (2021). GABA-receptive microglia selectively sculpt developing inhibitory circuits. *Cell* 184, 4048–4063.e32. doi: 10.1016/j.cell.2021.06.018

Ferrero, G., Mahony, C. B., Dupuis, E., Yvernogeau, L., Di Ruggiero, E., Miserocchi, M., et al. (2018). Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish. *Cell Rep.* 24, 130–141. doi: 10.1016/j.celrep.2018.05.066

Gautier, E. L., Shay, T., Miller, J., Greter, M., Jakubzick, C., Ivanov, S., et al. (2012). Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. *Nat. Immunol.* 13, 1118–1128. doi: 10.1038/ni.2419

Gesuita, L., Cavaccini, A., Argunsah, A. O., Favuzzi, E., Ibrahim, L. A., Stachniak, T. J., et al. (2022). Microglia contribute to the postnatal development of cortical somatostatin-positive inhibitory cells and to whisker-evoked cortical activity. *Cell Rep.* 40:111209. doi: 10.1016/j.celrep.2022.111209

Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., et al. (2010). Fate mapping analysis reveals that adult microglia derive from primitive macrophages. *Science* 330, 841–845. doi: 10.1126/science.1194637

Goldmann, T., Wieghofer, P., Jordao, M. J., Prutek, F., Hagemeyer, N., Frenzel, K., et al. (2016). Origin, fate and dynamics of macrophages at central nervous system interfaces. *Nat. Immunol.* 17, 797–805. doi: 10.1038/ni.3423

Green, K. N., Craper, J. D., and Hohsfield, L. A. (2020). To kill a microglia: A case for CSF1R Inhibitors. *Trends Immunol.* 41, 771–784. doi: 10.1016/j.it.2020.07.001

Greter, M., Lelios, I., Pelczar, P., Hoeffel, G., Price, J., Leboeuf, M., et al. (2012). Stroma-derived interleukin-34 controls the development and maintenance of langerhans cells and the maintenance of microglia. *Immunity* 37, 1050–1060. doi: 10.1016/j.immuni.2012.11.001

Guneykaya, D., Ivanov, A., Hernandez, D. P., Haage, V., Wojtas, B., Meyer, N., et al. (2018). Transcriptional and translational differences of microglia from male and female brains. *Cell Rep.* 24, 2773–2783.e6. doi: 10.1016/j.celrep.2018.08.001

Hagemeyer, N., Hanft, K. M., Akriditou, M. A., Unger, N., Park, E. S., Stanley, E. R., et al. (2017). Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood. *Acta Neuropathol.* 134, 441–458. doi: 10.1007/s00401-017-1747-1

Hammond, T. R., Dufort, C., Dissing-Olesen, L., Giera, S., Young, A., Wysoker, A., et al. (2019). Single-Cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes. *Immunity* 50, 253–271.e6. doi: 10.1016/j.immuni.2018.11.004

Hammond, T. R., Robinton, D., and Stevens, B. (2018). Microglia and the brain: Complementary partners in development and disease. *Annu. Rev. Cell Dev. Biol.* 34, 523–544. doi: 10.1146/annurev-cellbio-100616-060509

Han, J., Chitu, V., Stanley, E. R., Wszolek, Z. K., Karrenbauer, V. D., and Harris, R. A. (2022). Inhibition of colony stimulating factor-1 receptor (CSF-1R) as a potential therapeutic strategy for neurodegenerative diseases: Opportunities and challenges. *Cell Mol. Life Sci.* 79, 219. doi: 10.1007/s00018-022-04225-1
Han, X., Li, Q., Lan, X., El-Mufti, L., Ren, H., and Wang, J. (2019). Microglial depletion with clodronate liposomes increases proinflammatory cytokine levels, induces astrocyte activation, and damages blood vessel integrity. *Mol. Neurobiol.* 56, 6184-6196. doi: 10.1007/s12035-019-1502-9

Hanamsagar, R, Alter, M. D., Block, C. S., Sullivan, H., Bolton, J. L., and Bilbo, S. D. (2017). Generation of a microglial developmental index in mice and in humans reveals a sex difference in maturation and immune reactivity. *Glia* 65, 1504-1520. doi: 10.1002/glia.23176

Hattori, Y., Kato, D., Murayama, F., Koike, S., Naito, Y., Kawaguchi, A., et al. (2022). Border-associated macrophages transventricularly infiltrate the early embryonic cerebral wall to differentiate into microglia. *bioRxiv* [Preprint]. doi: 10.1101/2022.07.27.501563

Hattori, Y., Naito, Y., Tsugawa, Y., Nonaka, S., Wake, H., Nagasawa, T., et al. (2020). Transient microglial absence assists postmigratory cortical neurons in proper differentiation. *Nat. Commun.* 11:1631. doi: 10.1038/s41467-020-15409-3

Hickman, S. E., Kingery, N. D., Ohsumi, T. K., Borowsky, M. L., Wang, L. C., Means, T. K., et al. (2013). The microglial sensome revealed by direct RNA sequencing. *Nat. Neurosci.* 16, 1896-1905. doi: 10.1038/nn.3554

Hoeffel, G., and Ginhoux, F. (2015). Ontogeny of tissue-resident macrophages. *Front Immunol.* 6:486. doi: 10.3389/fimmu.2015.00486

Hoeffel, G., and Ginhoux, F. (2018). Fetal monocytes and the origins of tissue-resident macrophages. *Cell Immunol.* 330, 5-15. doi: 10.1016/j.cellimm.2018.01.001

Huang, Y., Xu, Z., Xiong, S., Sun, F., Qin, G., Hu, G., et al. (2018). Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. *Nat Neurosci.* 21, 530-540. doi: 10.1038/s41593-018-0090-8

Jung, S., Aliberti, J., Graemmel, P., Sunshine, M. J., Kreutzberg, G. W., Sher, A., et al. (2000). Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. *Mol. Cell Biol.* 20, 4106-4114. doi: 10.1128/MCB.20.11.4106-4114.2000

Kaiser, T., and Feng, G. (2019). Tmem119-EGFP and tmem119-CreERT2 transgenic mice for labeling and manipulating microglia. *eNeuro* 6, ENEURO.0448-18.2019. doi: 10.1523/ENEURO.0448-18.2019

Kana, V., Desland, F. A., Casanova-Acebes, M., Ayata, P., Badimon, A., Nabel, E., et al. (2019). CSF-1 controls cerebellar microglia and is required for motor function and social interaction. *J. Exp. Med.* 216, 2265-2281. doi: 10.1084/jem.20182037

Keren-Shaul, H., Spinrad, A., Weiner, A., Matcovitch-Natan, O., Dvir-Szternfeld, R., Ulland, T. K., et al. (2017). A unique microglia type associated with restricting development of Alzheimer's disease. *Cell* 169, 1276-1290.e17. doi: 10.1016/j.cell.2017.05.018

Kiani Shabestari, S., Morabito, S., Danhash, E. P., McQuade, A., Sanchez, J. R., Miyoshi, E., et al. (2022). Absence of microglia promotes diverse pathologies and early lethality in Alzheimer's disease mice. *Cell Rep.* 39:110961. doi: 10.1016/j.celrep.2022.110961

Kierdorf, K., Masuda, T., Jordao, M. J. C., and Prinz, M. (2019). Macrophages at CNS interfaces: Ontogeny and function in health and disease. *Nat. Rev. Neurosci.* 20, 547-562. doi: 10.1038/s41583-019-0201-x

Kim, J. S., Kolesnikov, M., Peled-Hajaj, S., Scheyltjens, I., Xia, Y., Trzebanski, S., et al. (2021). A binary Cre transgenic approach dissects microglia and CNS border-associated macrophages. *Immunity* 54, 176-190.e7. doi: 10.1016/j.immuni.2020.11.007

Kondo, Y., and Duncan, I. D. (2009). Selective reduction in microglia density and function in the white matter of colony-stimulating factor-1-deficient mice. *J. Neurosci. Res.* 87, 2686-2695. doi: 10.1002/jnr.22096

Konishi, H., Kobayashi, M., Kunisawa, T., Imai, K., Sayo, A., Malissen, B., et al. (2017). Siglec-H is a microglia-specific marker that discriminates microglia from CNS-associated macrophages and CNS-infiltrating monocytes. *Glia* 65, 1927-1943. doi: 10.1002/glia.23204

Kracht, L., Borggrewe, M., Eskandar, S., Brouwer, N., Chuva de Sousa Lopes, S. M., Laman, J. D., et al. (2020). Human fetal microglia acquire homeostatic immunosensing properties early in development. *Science* 369, 530-537. doi: 10.1126/science.aba5906

Krasemann, S., Madore, C., Cialic, R., Baufeld, C., Calcagno, N., El Fatimy, R., et al. (2017). The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. *Immunity* 47, 566-581.e9. doi: 10.1016/j.immuni.2017.08.008

Lee, E., Eo, J. C., Lee, C., and Yu, J. W. (2021). Distinct features of brain-resident macrophages: Microglia and non-parenchymal brain macrophages. *Mol. Cells* 44, 281-291. doi: 10.14348/molcells.2021.0060

Lenz, K. M., Nugent, B. M., Haliyur, R., and McCarthy, M. M. (2013). Microglia are essential to masculinization of brain and behavior. *J. Neurosci.* 33, 2761-2772. doi: 10.1523/JNEUROSCI.1268-12.2013

Li, C., Konishi, H., Nishiwaki, K., Sato, K., Miyata, T., and Kiyama, H. (2021). A mouse model of microglia-specific ablation in the embryonic central nervous system. *Neurosci. Res.* 173, 54-61. doi: 10.1016/j.neures.2021.06.002

Li, Q., and Barres, B. A. (2018). Microglia and macrophages in brain homeostasis and disease. *Nat. Rev. Immunol.* 18, 225-242. doi: 10.1038/nri.2017.125

Li, Q., Cheng, Z., Zhou, L., Darmanis, S., Neff, N. F., Okamoto, J., et al. (2019). Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing. *Neuron* 101, 207-223.e10. doi: 10.1016/j.neuron.2018.12.006

Li, Y., He, X., Kawaguchi, R., Zhang, Y., Wang, Q., Monavarfeshani, A., et al. (2020). Microglia-organized scar-free spinal cord repair in neonatal mice. *Nature* 587, 613-618. doi: 10.1038/s41586-020-2795-6

Lin, R., Zhou, Y., Yan, T., Wang, R., Li, H., Wu, Z., et al. (2022). Directed evolution of adeno-associated virus for efficient gene delivery to microglia. *Nat. Methods* 19, 976-985. doi: 10.1038/s41592-022-01547-7

Lukens, J. R., and Eyo, U. B. (2022). Microglia and neurodevelopmental disorders. *Annu. Rev. Neurosci.* 45, 425-445. doi: 10.1146/annurev-neuro-110920-023056

MacDonald, K. P., Palmer, J. S., Cronau, S., Seppanen, E., Olver, S., Raffelt, N. C., et al. (2010). An antibody against the colony-stimulating factor 1 receptor depletes the resident subset of monocytes and tissue- and tumor-associated macrophages but does not inhibit inflammation. *Blood* 116, 3955-3963. doi: 10.1182/blood-2010-02-266296

Maes, M. E., Colombo, G., Schulz, R., and Siegert, S. (2019). Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges. *Neurosci. Lett.* 707:134310. doi: 10.1016/j.neulet.2019.134310

Marschallinger, J., Iram, T., Zardeneta, M., Lee, S. E., Lehallier, B., Haney, M. S., et al. (2020). Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. *Nat. Neurosci.* 23, 194-208. doi: 10.1038/s41593-019-0566-1

Marsh, S. E., Walker, A. J., Kamath, T., Dissing-Olesen, L., Hammond, T. R., de Soya, T. Y., et al. (2022). Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain. *Nat. Neurosci.* 25, 306-316. doi: 10.1038/s41593-022-01022-8

Marsters, C. M., Nesan, D., Far, R., Klenin, N., Pittman, Q. J., and Kurrasch, D. M. (2020). Embryonic microglia influence developing hypothalamic glial populations. *J. Neuroinflammation* 17:146. doi: 10.1186/s12974-020-01811-7

Masuda, T., Amann, L., Monaco, G., Sankowski, R., Staszewski, O., Krueger, M., et al. (2022). Specification of CNS macrophage subsets occurs postnatally in defined niches. *Nature* 604, 740-748. doi: 10.1038/s41586-022-04596-2

Masuda, T., Amann, L., Sankowski, R., Staszewski, O., Lenz, M., d'Errico, P., et al. (2020). Novel Hexb-based tools for studying microglia in the CNS. *Nat. Immunol.* 21, 802-815. doi: 10.1038/s41590-020-0707-4

Masuda, T., Sankowski, R., Staszewski, O., Bottcher, C., Amann, L., Sagar, et al. (2019). Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution. *Nature* 566, 388-392. doi: 10.1038/s41586-019-0924-x

Matcovitch-Natan, O., Winter, D. R., Giladi, A., Vargas Aguilar, S., Spinrad, A., Sarrazin, S., et al. (2016). Microglia development follows a stepwise program to regulate brain homeostasis. *Science* 353:aad8670. doi: 10.1126/science.aad8670

McKercher, S. R., Torbett, B. E., Anderson, K. L., Henkel, G. W., Vestal, D. J., Baribault, H., et al. (1996). Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. *EMBO J.* 15, 5647-5658.

McKinsey, G. L., Lizama, C. O., Keown-Lang, A. E., Niu, A., Santander, N., Larphaveesarp, A., et al. (2020). A new genetic strategy for targeting microglia in development and disease. *Elife* 9:e54590. doi: 10.7554/eLife.54590

Menassa, D. A., Muntslag, T. A. O., Martin-Estebane, M., Barry-Carroll, L., Chapman, M. A., Adorjan, I., et al. (2022). The spatiotemporal dynamics of microglia across the human lifespan. *Dev. Cell* 57, 2127-2139.e6. doi: 10.1016/j.devcel.2022.07.015

Michaelson, M. D., Bieri, P. L., Mehler, M. F., Xu, H., Arezzo, J. C., Pollard, J. W., et al. (1996). CSF-1 deficiency in mice results in abnormal brain development. *Development* 122, 2661-2672. doi: 10.1242/dev.122.9.2661

Mildenberger, W., Stifter, S. A., and Greter, M. (2022). Diversity and function of brain-associated macrophages. *Curr. Opin. Immunol.* 76:102181. doi: 10.1016/j.coi.2022.102181

Mills, J., Ladner, L., Soliman, E., Leonard, J., Morton, P. D., and Theus, M. H. (2022). Cross-talk and subset control of microglia and associated myeloid cells in neurological disorders. *Cells* 11:3364. doi: 10.3390/cells11213364

Miyamoto, A., Wake, H., Ishikawa, A. W., Eto, K., Shibata, K., Murakoshi, H., et al. (2016). Microglia contact induces synapse formation in developing somatosensory cortex. *Nat. Commun.* 7:12540. doi: 10.1038/ncomms12540

Monier, A., Adle-Biassette, H., Delezoide, A. L., Evrard, P., Gressens, P., and Verney, C. (2007). Entry and distribution of microglial cells in human embryonic and fetal cerebral cortex. *J. Neuropathol. Exp. Neurol.* 66, 372-382. doi: 10.1097/nen.0b013e3180517b46

Mosser, C. A., Baptista, S., Arnoux, I., and Audinat, E. (2017). Microglia in CNS development: Shaping the brain for the future. *Prog. Neurobiol.* 14, 1-20. doi: 10.1016/j.pneurobio.2017.01.002

Mrdjen, D., Pavlovic, A., Hartmann, F. J., Schreiner, B., Utz, S. G., Leung, B. P., et al. (2018). High-dimensional single-cell mapping of central nervous system immune
cells reveals distinct myeloid subsets in health, aging, and disease. *Immunity* 48:599. doi: 10.1016/j.immuni.2018.02.014

Munro, D. A. D., Bradford, B. M., Mariani, S. A., Hampton, D. W., Vink, C. S., Chandran, S., et al. (2020). CNS macrophages differentially rely on an intronic Csf1r enhancer for their development. *Development* 147:dev194449. doi: 10.1242/dev.194449

Munz, M., Bharioke, A., Kosche, G., Moreno-Juan, V., Brignall, A., Graff-Meyer, A., et al. (2022). Embryonic cortical layer 5 pyramidal neurons form an active, transient circuit motif perturbed by autism-associated mutations. *bioRxiv* [Preprint]. doi: 10.1101/2022.08.31.506080

Nemes-Baran, A. D., White, D. R., and DeSilva, T. M. (2020). Fractalkine-dependent microglial pruning of viable oligodendrocyte progenitor cells regulates myelination. *Cell Rep.* 32:108047. doi: 10.1016/j.celrep.2020.108047

Nimmerjahn, A., Kirchhoff, F., and Helmchen, F. (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. *Science* 308, 1314–1318. doi: 10.1126/science.1110647

O'Brien, J., Martinson, H., Durand-Rougely, C., and Schedin, P. (2012). Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution. *Development* 139, 269–275. doi: 10.1242/dev.071696

Paolicelli, R. C., Sierra, A., Stevens, B., Tremblay, M. E., Aguzzi, A., Ajami, B., et al. (2022). Microglia states and nomenclature: A field at its crossroads. *Neuron* 110, 3458–3483. doi: 10.1016/j.neuron.2022.10.020

Parkhurst, C. N., Yang, G., Ninan, I., Savas, J. N., Yates, J. R. III, Lafaille, J. J., et al. (2013). Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. *Cell* 155, 1596–1609. doi: 10.1016/j.cell.2013.11.030

Pont-Lezica, L., Beumer, W., Colasse, S., Drexhage, H., Versnel, M., and Bessis, A. (2014). Microglia shape corpus callosum axon tract fasciculation: Functional impact of prenatal inflammation. *Eur. J. Neurosci.* 39, 1551–1557. doi: 10.1111/ejn.12508

Prinz, M., Jung, S., and Priller, J. (2019). Microglia biology: One century of evolving concepts. *Cell* 179, 292–311. doi: 10.1016/j.cell.2019.08.053

Ratz, M., von Berlin, L., Larsson, L., Martin, M., Westholm, J. O., La Manno, G., et al. (2022). Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics. *Nat. Neurosci.* 25, 285–294. doi: 10.1038/s41593-022-01011-x

Rebuli, M. E., Gibson, P., Rhodes, C. L., Cushing, B. S., and Patisaul, H. B. (2016). Sex differences in microglial colonization and vulnerabilities to endocrine disruption in the social brain. *Gen. Comp. Endocrinol.* 238, 39–46. doi: 10.1016/jygcen.2016.04.018

Rojo, R., Raper, A., Ozdemir, D. D., Lefevre, L., Grabert, K., Wollcheid-Lengeling, E., et al. (2019). Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations. *Nat. Commun.* 10:3215. doi: 10.1038/s41467-019-11053-8

Ronzano, R., Roux, T., Thetiot, M., Aigrot, M. S., Richard, L., Lejeune, F. X., et al. (2021). Microglia-neuron interaction at nodes of Ranvier depends on neuronal activity through potassium release and contributes to remyelination. *Nat. Commun.* 12:5219. doi: 10.1038/s41467-021-25486-7

Rosin, J. M., Marsters, C. M., Malik, F., Far, R., Adnani, L., Schuurmans, C., et al. (2021). Embryonic microglia interact with hypothalamic radial glia during development and upregulate the TAM receptors MERTK and AXL following an insult. *Cell Rep.* 34:10858. doi: 10.1016/j.celrep.2020.10858

Rosin, J. M., Vora, S. R., and Kurrasch, D. M. (2018). Depletion of embryonic microglia using the CSF1R inhibitor PLX5622 has adverse sex-specific effects on mice, including accelerated weight gain, hyperactivity and anxiolytic-like behaviour. *Brain Behav. Immun.* 73, 682–697. doi: 10.1016/j.bbi.2018.07.023

Ruan, C., Sun, L., Kroshilina, A., Beckers, L., De Jager, P., Bradshaw, E. M., et al. (2020). A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system. *Brain Behav. Immun.* 83, 180–191. doi: 10.1016/j.bbi.2019.10.009

Safaiyan, S., Besson-Girard, S., Kaya, T., Cantuti-Castelvetri, L., Liu, L., Ji, H., et al. (2021). White matter aging drives microglial diversity. *Neuron* 109, 1100–1117.e10. doi: 10.1016/j.neuron.2021.01.027

Sahasrabuddhe, V., and Ghosh, H. S. (2022). Cx3Cr1-Cre induction leads to microglial activation and IFN-1 signaling caused by DNA damage in early postnatal brain. *Cell Rep.* 38:110252. doi: 10.1016/j.celrep.2021.110252

Sankowski, R., Bottcher, C., Masuda, T., Geirdsottir, L., Sagar, Sindram, E., et al. (2019). Mapping microglia states in the human brain through the integration of high-dimensional techniques. *Nat. Neurosci.* 22, 2098–2110. doi: 10.1038/s41593-019-0532-y

Sankowski, R., Monaco, G., and Prinz, M. (2022). Evaluating microglial phenotypes using single-cell technologies. *Trends Neurosci.* 45, 133–144. doi: 10.1016/j.tins.2021.11.001

Satoh, J., Kino, Y., Asahina, N., Takitani, M., Miyoshi, J., Ishida, T., et al. (2016). TMEM119 marks a subset of microglia in the human brain. *Neuropathology* 36, 39–49. doi: 10.1111/neup.12235

Schwarz, J. M., Sholar, P. W., and Bilbo, S. D. (2012). Sex differences in microglial colonization of the developing rat brain. *J. Neurochem.* 120, 948–963. doi: 10.1111/j.1471-4159.2011.07630.x

Sherafat, A., Pfeiffer, F., Reiss, A. M., Wood, W. M., and Nishiyama, A. (2021). Microglial neuropilin-1 promotes oligodendrocyte expansion during development and remyelination by trans-activating platelet-derived growth factor receptor. *Nat. Commun.* 12:2265. doi: 10.1038/s41467-021-22532-2

Sierra, A., Encinas, J. M., Deudero, J. J., Chancey, J. H., Enikolopov, G., Overstreet-Wadiche, L. S., et al. (2010). Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. *Cell Stem Cell* 7, 483–495. doi: 10.1016/j.stem.2010.08.014

Silvin, A., Uderhardt, S., Piot, C., Da Mesquita, S., Yang, K., Geirdsottir, L., et al. (2022). Dual ontogeny of disease-associated microglia and disease inflammatory macrophages in aging and neurodegeneration. *Immunity* 55, 1448–1465.e6. doi: 10.1016/j.immuni.2022.07.004

Squarzoni, P., Oller, G., Hoeffel, G., Pont-Lezica, L., Rostaing, P., Low, D., et al. (2014). Microglia modulate wiring of the embryonic forebrain. *Cell Rep.* 8, 1271–1279. doi: 10.1016/j.celrep.2014.07.042

Stogsdill, J. A., Kim, K., Binan, L., Farhi, S. L., Levin, J. Z., and Arlotta, P. (2022). Pyramidal neuron subtype diversity governs microglia states in the neocortex. *Nature* 608, 750–756. doi: 10.1038/s41586-022-05056-7

Stremmel, C., Schuchert, R., Wagner, F., Thaler, R., Weinberger, T., Pick, R., et al. (2018). Yolk sac macrophage progenitors traffic to the embryo during defined stages of *Nat. Commun.* 9:75. doi: 10.1038/s41467-017-02492-2

Swinnen, N., Smolders, S., Avila, A., Notelaers, K., Paesen, R., Ameloot, M., et al. (2013). Complex invasion pattern of the cerebral cortex by microglial cells during development of the mouse embryo. *Glia* 61, 150–163. doi: 10.1002/glia.22421

Takeda, A., Shinozaki, Y., Kashiwagi, K., Ohno, N., Eto, K., Wake, H., et al. (2018). Microglia mediate non-cell-autonomous cell death of retinal ganglion cells. *Glia* 66, 2366–2384. doi: 10.1002/glia.23475

Tay, T. L., Mai, D., Dautzenberg, J., Fernandez-Klett, F., Lin, G., Sagar, et al. (2017). A new fate mapping system reveals context-dependent random or clonal expansion of microglia. *Nat. Neurosci.* 20, 793–803. doi: 10.1038/nn.4547

Thion, M. S., Ginhoux, F., and Garel, S. (2018a). Microglia and early brain development: An intimate journey. *Science* 362, 185–189. doi: 10.1126/science.aat0474

Thion, M. S., Low, D., Silvin, A., Chen, J., Grisel, P., Schulte-Schrepping, J., et al. (2018b). Microbiome influences prenatal and adult microglia in a sex-specific manner. *Cell* 172, 500–516.e16. doi: 10.1016/j.cell.2017.11.042

Thion, M. S., Mosser, C. A., Ferezou, I., Grisel, P., Baptista, S., Low, D., et al. (2019). Biphasic impact of prenatal inflammation and macrophage depletion on the wiring of neocortical inhibitory circuits. *Cell Rep.* 28, 1119–1126.e14. doi: 10.1016/j.celrep.2019.06.086

Utz, S. G., See, P., Mildenberger, W., Thion, M. S., Silvin, A., Lutz, M., et al. (2020). Early fate defines microglia and non-parenchymal brain macrophage development. *Cell* 181, 557–573.e18. doi: 10.1016/j.cell.2020.03.021

Van Hove, H., Martens, L., Scheyltjens, I., De Vlaminck, K., Pombo Antunes, A. R., De Prijck, S., et al. (2019). A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment. *Nat. Neurosci.* 22, 1021–1035. doi: 10.1038/s41593-019-0393-4

VanRyzin, J. W., Marquardt, A. E., Argue, K. J., Vecchiarelli, H. A., Ashton, S. E., Arambula, S. E., et al. (2019). Microglial phagocytosis of newborn cells is induced by endocannabinoids and sculpts sex differences in juvenile rat social play. *Neuron* 102, 435–449.e6. doi: 10.1016/j.neuron.2019.02.006

Verney, C., Monier, A., Fallet-Bianco, C., and Gressens, P. (2010). Early microglial colonization of the human forebrain and possible involvement in periventricular white-matter injury of preterm infants. *J. Anat.* 217, 436–448. doi: 10.1111/j.1469-7580.2010.01245.x

Villa, A., Gelosa, P., Castiglioni, L., Cimino, M., Rizzi, N., Pepe, G., et al. (2018). Sex-specific features of microglia from adult mice. *Cell Rep.* 23, 3501–3511. doi: 10.1016/j.celrep.2018.05.048

Wake, H., Moorhouse, A. J., Jinno, S., Kohsaka, S., and Nabekura, J. (2009). Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. *J. Neurosci.* 29, 3974–3980. doi: 10.1523/JNEUROSCI.4363-08.2009

Wang, Y., Szretter, K. J., Vermi, W., Gilfillan, S., Rossini, C., Cella, M., et al. (2012). IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. *Nat. Immunol.* 13, 753–760. doi: 10.1038/ni.2360

Wlodarczyk, A., Holtman, I. R., Krueger, M., Yogev, N., Bruttger, J., Khorooshi, R., et al. (2017). A novel microglial subset plays a key role in myelinogenesis in developing brain. *EMBO J.* 36, 3292–3308. doi: 10.1525/embj.201696056

Xu, J., Zhu, L., He, S., Wu, Y., Jin, W., Yu, T., et al. (2015). Temporal-spatial resolution fate mapping reveals distinct origins for embryonic and adult microglia in zebrafish. *Dev. Cell* 34, 632–641. doi: 10.1016/j.devcel.2015.08.018

Yona, S., Kim, K. W., Wolf, Y., Mildner, A., Varol, D., Breker, M., et al. (2013). Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. *Immunity* 38, 79–91. doi: 10.1016/j.immuni.2012.12.001

Yu, D., Li, T., Delpech, J. C., Zhu, B., Kishore, P., Koshi, T., et al. (2022). Microglial GPR56 is the molecular target of maternal immune activation-induced parvalbumin-positive interneuron deficits. *Sci. Adv.* 8:eabm2545. doi: 10.1126/sciadv.abm2545