Lyl-1 regulates primitive macrophages and microglia development

Wang, Shoutang; Ren, Deshan; Arkoun, Brahim; Kaushik, Anna-Lila; Matherat, Gabriel; Lécluse, Yann; Filipp, Dominik; Vainchenker, William; Raslova, Hana; Plo, Isabelle; Godin, Isabelle

doi:10.1038/s42003-021-02886-5

Download PDF

Article
Open access
Published: 09 December 2021

Lyl-1 regulates primitive macrophages and microglia development

Communications Biology volume 4, Article number: 1382 (2021) Cite this article

1772 Accesses
6 Citations
1 Altmetric
Metrics details

Subjects

Abstract

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/”early EMP” and transient-definitive/”late EMP” waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis.

Deciphering human macrophage development at single-cell resolution

Article 20 May 2020

Yolk sac-derived Pdcd11-positive cells modulate zebrafish microglia differentiation through the NF-κB-Tgfβ1 pathway

Article Open access 24 July 2020

Specification of CNS macrophage subsets occurs postnatally in defined niches

Article 20 April 2022

Introduction

Amongst the components of the transcription factor network that regulate hematopoietic cells features, Tal-1, Lmo2, Runx1, and Gata2 stand out as major regulators of hematopoietic progenitor development^1,2. Tal-1, Lmo2, and Gata-2 belong to a transcriptional complex, which also includes the basic helix–loop–helix (bHLH) transcription factor lymphoblastic leukemia-derived sequence 1 (Lyl-1). Unlike its paralog Tal-1, which is mandatory for the specification of all hematopoietic progenitors^3,4, Lyl-1 roles during developmental hematopoiesis remains poorly characterized. We analyzed these functions at the onset of YS hematopoietic development using Lyl-1^LacZ/LacZ mutant mice⁵.

During ontogeny, hematopoietic progenitors are generated in three successive and overlapping waves^6,7. The emergence of the Hematopoietic Stem Cells (HSC) that will maintain lifelong hematopoiesis in the adult occurs at mid-gestation in the third and definitive hematopoietic wave. HSC generated in the aorta region immediately migrate to the fetal liver (FL) where they mature and amplify before homing to the bone marrow before birth^8,9. Prior to HSC generation, the production of blood cells relies on two hematopoietic waves provided by the YS. This HSC-independent hematopoiesis comprises first the primitive hematopoietic wave, with the transient production of progenitors with embryonic specific features: From Embryonic day (E) 7.00, the YS produces monopotent progenitors for erythrocytes, megakaryocytes and macrophages (MΦ)^10,11, along with bipotent Erythro-Megakaryocytic progenitors¹², in a Myb-independent pathway^13,14. The second YS wave, called transient-definitive, provides for a limited duration progenitors (mostly erythro-myeloid) that seed the FL and produce a hematopoietic progeny that displays definitive/adult differentiation features. Erythro-myeloid cell production in this wave occurs in a Myb-dependent pathway, through the progressive differentiation of erythro-myeloid progenitors (EMP) in a pathway similar to the adult one⁶. As primitive and transient definitive YS waves both produce cells from erythro-myeloid lineages, they are also termed respectively “early EMP” and “late EMP”^15,16.

Considering the MΦ lineage, fate-mapping approaches aimed at determining the embryonic origin of resident-MΦs indicated that most tissues harbor resident-MΦs of diverse origins (YS, FL and adult bone marrow)^15,17,18, which complicates the characterization of wave-dependent functions of the various subsets. However, these fate-mapping analyses established that, contrary to others tissue, brain MΦs (microglia and Border Associated MΦ (BAM)) develop only from YS-derived MΦ-progenitors^{14,16,19,20,21}, confirming a model we previously put forward²². Due to the coexistence of two waves in the YS, the origin of microglia has been debated (reviewed in refs. ^6,15,18,23). An origin of microglia from MΦ-progenitors from the primitive/”early EMP” wave was supported by microglia labeling following an early (E7.0-E7.5) CRE-mediated induction of Runx1¹⁹ and by the intact microglia pool in Myb-deficient mice^14,21. The origin of microglia was also attributed to the primitive wave in zebrafish embryos, since primitive macrophage (MΦ^Prim) progenitors arise in this species from a location distinct from other hematopoietic progenitors^24,25,26. Finally, the normal microglia development in mice lacking Kit ligand, leading to an impaired EMP development and the depletion of resident-MΦs in the skin, lung, and liver supports this model²⁷.

We here show that, at the early stages of YS hematopoiesis, Lyl-1 expression characterizes primitive MΦ-progenitors. Through RNA-seq. analyses, it appears that these primitive MΦ-progenitors harbor an immune-modulatory phenotype, while those produce at a later stage favor the inflammatory signaling which promotes the emergence of HSC in the third and definitive hematopoietic wave²⁸.

Our results also indicate that in the brain, Lyl-1 is expressed in the entire microglia/BAM cell population at the onset of brain colonization and appeared to regulate microglia/BAM development.

Altogether, these data point to Lyl-1 as a major regulator of early embryonic MΦ-progenitors development and advocate for further analyses to more precisely delineate Lyl-1 function during the development of resident-MΦs in homeostatic and pathological contexts.

Results and discussion

Lyl-1 expression marks MΦ^Prim progenitors from the early YS

Lyl-1 being expressed in the YS from the onset of YS hematopoiesis²⁹, we first explored its function by characterizing the clonogenic potential of WT, Lyl-1^WT/LacZ and Lyl-1^LacZ/LacZ YS. E8-YS were maintained in organ culture for 1 day (E8 OrgD1-YS), allowing only the development of primitive and transient-definitive progenitors^30,31. Compared to WT, the production of MΦ progenitors was increased in Lyl-1^WT/LacZ and Lyl-1^LacZ/LacZ OrgD1-YS. Otherwise, the clonogenic potential and progenitor distribution were unmodified (Fig. 1a).

**Fig. 1: Lyl-1 expression marks MΦPrim progenitors in the early YS.**

Using FACS-Gal assay, we noticed that the entire MΦ-progenitor population (Kit⁺CD45⁺CD11b⁺) expressed Lyl-1 at E9. In contrast, two MΦ-progenitor subsets, discriminated by FDG/Lyl-1 expression, were present after E9.5 (Fig. 1b). Since after E9.5, the YS harbors both MΦ^Prim and transient-definitive (MΦ^T-Def) MΦ−progenitors, and as these progenitor subsets cannot be discriminated by phenotype¹¹, we investigated the known features discriminating these two waves: the origin from monopotent progenitors for MΦ^Prim progenitors^10,11 and the Myb-dependent^14,32 differentiation of MΦ^T-Def progenitors from EMP, via the production of granulo-monocytic (GM-), then granulocyte (G-) and MΦ-progenitors⁶.

At E8 (0-5 somites), when the YS only harbors MΦ^Prim progenitors, clonogenic assays also pointed to an increased production of MΦ-progenitors in mutant E8-YS compared to WT (Fig. 1c). At this stage, all MΦ-progenitors, which harbor a CD11b⁺CD31⁺ phenotype¹¹, expressed FDG/Lyl-1 (Fig. 1d). Most FDG⁺/Lyl-1⁺ CD11b⁺CD31⁺ cells (69.27%±0.33%) from Lyl-1^WT/LacZ E8-YS reliably produced MΦ colonies (72.78 ± 9.65%; n = 3) in clonogenic assays, amounting 1-4 MΦ progenitors per YS, a value consistent with previously published data^10,11.

Lyl-1 expression by MΦ^Prim progenitors was strengthened by RT-qPCR comparison of Myb expression: Both E9-YS MΦ^Prim progenitors and FDG⁺/Lyl-1⁺ progenitors from E10-YS expressed low Myb levels strengthening their primitive status, while FDG^-/Lyl-1^- progenitors from E10-YS progenitors displayed Myb levels similar to lineage-negative Sca1⁺Kit⁺ progenitors from E12-FL (Fig. 1e).

The differentiation potential of FDG⁺/Lyl-1⁺ and FDG^-/Lyl-1^- fractions of Ter119^-Kit⁺CD45⁺CD11b⁺ myeloid progenitors isolated from E10-YS also pointed to Lyl-1 expression by MΦ^Prim progenitors (Fig. 1f): Similar to WT E9-YS MΦ^Prim progenitors, E10 FDG⁺/Lyl-1⁺ progenitors appeared monopotent, as they nearly exclusively produced MΦ colonies. In contrast, E10 FDG^-/Lyl-1^- myeloid progenitors produced GM, G and MΦ colonies, a feature typical of transient-definitive progenitors⁶. Overall, these data together suggested that Lyl-1 may mark MΦ^Prim progenitors from the earliest wave.

Distinct features of WT MΦ-progenitors at E9 and E10

The distinction between E9 and E10 MΦ-progenitors was confirmed in RNA-seq. analysis of CD45⁺CD11b⁺Kit⁺ MΦ-progenitors sorted at E9 (MΦ^Prim progenitors) and E10 (MΦ^Prim and MΦ^T-Def progenitors). Principal Component Analysis separated E9 and E10 MΦ-progenitors according to stage and genotypes (Fig. 2A). E9 and E10 WT MΦ-progenitors differed by the expression of 726 genes, 176 being up-regulated at E9 and 550 at E10. Considering the coexistence of MΦ^Prim and MΦ^T-Def progenitors in E10-YS, differentially expressed genes found at E10 may reflect wave-specific differences or stage-dependent changes related to MΦ^Prim progenitor maturation.

Overlapping the identified differentially expressed genes to the EMP and E10.25-E10.5 MΦs signatures obtained by Mass et al.³³ confirmed that WT E9 MΦ^Prim progenitors were distinct from these two populations, since none of the 176 genes upregulated at E9 belonged to these signatures. Comparatively, about 5% of the genes up-regulated at E10 belonged to the EMP and MΦ signatures (Fig. 2B). A similar separation was observed in GSEA analyses (Supplementary Fig. 1b). These observations suggest that within E10 MΦ-progenitors some, likely the MΦ^T-Def ones, retain part of the EMPs signature.

In a WT context, E9 MΦ^Prim progenitors differed from E10 MΦ-progenitors by their transcription factors repertoire. Genes regulating erythroid development (Gata1, Gata2, Klf1) and globin genes, embryonic (Hbb-bh1, Hba-x, Hbb-y) and definitive (Hba-a2, Hba-a1, Hbb-bt), were enriched at E10 (Fig. 2B), while Spi1/PU.1 was highly expressed compared to Gata1 at both stages (Fig. 2C). The lower expression level at E9 of erythroid genes and of genes involved in granulo-monocytic (Mpo, Csf2r/GM-CSF receptors, Cebp, Jun) and megakaryocytic development (Pf4, TPO signaling) (Fig. 2B; Supplementary Table 1) sustains the monopotent/primitive status of E9 MΦ-progenitors, and suggests that MΦ^T-Def progenitors may retain the expression of genes that characterize their EMP ancestor.

Due to the simultaneous presence of MΦ^Prim and MΦ^T-Def progenitors in E10-YS, the differential expressions of markers that are wave-specific at these stages (Primitive: Lyl-1; transient-definitive: Myb and Tlr2³⁴) were not significant despite a tendency to decrease for Lyl-1 and increase for Myb and Tlr2 (Supplementary Fig. 1c).

IPA and GSEA analyses indicated that E9 MΦ^Prim progenitors were more active in Eicosanoid signaling than E10 progenitors (Fig. 2D). They were also enriched in type I interferon (IFN) β and type II IFNγ signaling (Fig. 2E) and in MHC-II related genes, especially Cd74 (top 1 IPA network) (Fig. 2F; Supplementary Fig. 1d). Cytometry analyses confirmed a low, but significant, enrichment of MHC-II expression at E9, compared to E10 (Supplementary Fig. 1e). Comparatively, E10 MΦ-progenitors were more active in inflammatory signaling (Fig. 2D, E; Supplementary Fig. 1d, f–h; Supplementary Table 1), and metabolically active (Supplementary Table 1). The complement cascade and phagocytosis also prevailed at E10 (Supplementary Fig. 1i, j).

Altogether, the signature for WT E9 MΦ^Prim progenitors points to an immuno-modulatory function, while E10 MΦ-progenitors appear involved in phagocytosis and inflammatory signaling. Interestingly, inflammatory signaling has been revealed as a key factor favoring embryonic HSC emergence (reviewed in ref. ³⁵). The source of inflammatory signals was further identified as MΦ-progenitors expressing Mrc1/CD206³⁶, a marker up-regulated in E10 WT MΦ-progenitors compared to E9 (Fig. 2B).

Lyl-1 deficiency impacts embryonic development

When evaluating the effect of Lyl-1-deficiency at the earliest stage of MΦ^Prim development, clonogenic assays pointed to an increased production of MΦ-progenitors in Lyl-1^LacZ/LacZ MΦ^Prim E8-YS compared to WT (Fig. 1c). The analysis of the RNA-seq. comparison of E9 WT and Lyl-1^LacZ/LacZ MΦ^Prim suggests that the increased size of the initial MΦ-progenitor pool could results from an elevated commitment of mesodermal/pre-hematopoietic cells to a MΦ fate, rather than from a defective differentiation (Supplementary Fig. 2a–d and Supplementary note 1). The high increase of Itga2b/CD41 expression level in E9 Lyl-1^LacZ/LacZ MΦ^Prim progenitors (Fig. 3a) may reflect this elevated commitment. Lyl-1 expression in YS mesoderm, where it cannot substitute for Tal-1 mandatory function for the generation of hematopoietic progenitors⁴, was already established^3,29. Recently, Lyl-1 was identified as a regulator of mesoderm cell fate³⁷ and of the maintenance of primitive erythroid progenitors³⁸.

**Fig. 3: Lyl-1 regulates the production of E8 MΦPrim progenitors.**

The transcription factors network that controls developmental hematopoiesis² was also modified (Fig. 3b): beside the expected reduction of Lyl-1 expression, the expression of Lmo2, a Lyl-1 target³⁹, was down-regulated, while Tal-1 up-regulation might reflect some compensatory function³. The consequences were apparent in GSEA analyses: both pathways and GO terms uncovered an up-regulation of signaling pathways involved in embryo patterning (Wnt, Hox and Smad) in Lyl-1^LacZ/LacZ MΦ-progenitors, as well as a highly modified collagen, integrin, and cadherin usage (Supplementary Table 2). Accordingly, developmental trajectories were affected (Fig. 3c), with the up-regulation in E9 Lyl-1^LacZ/LacZ MΦ-progenitors of gene sets related to “anterior-posterior pattern specification” and “anatomical structure formation involved in morphogenesis”, notably skeletal and nervous system development.

GSEA and KEGG comparison of Lyl-1^LacZ/LacZ and WT MΦ-progenitors at E10 highlighted another patterning modification, namely the down-regulation of gene sets involved in heart development (Supplementary Fig. 2e; Supplementary Table 3), which might stem from a defective MΦ development. The heart harbors three resident-MΦ subsets, two of which originate from the YS⁴⁰. Amongst the features that distinguish WT E9 MΦ^Prim progenitors from E10 MΦ-progenitors, the enriched expression of MHC-II (Fig. 2F) and poor expression of phagocytosis-related genes (Supplementary Fig. 1i) at E9 also characterize one of the two YS-derived CCR2^- resident-MΦ subsets in the heart^40,41. Therefore, a function for Lyl-1⁺ MΦ^Prim progenitors in heart development may be considered. This observation reinforces the need to better characterize the contributions of MΦ-progenitors from both primitive and transient-definitive waves to tissues harboring YS-derived resident-MΦs.

The patterning defects highlighted in defective MΦ^Prim, might be responsible, at least in part, for the significantly decreased litter size and increased perinatal lethality observed in Lyl-1^LacZ/LacZ mice compared to WT (Supplementary Fig. 2F), which indicates the requirement for Lyl-1 during various developmental processes. Whether this function is specific to mesodermal stage or related to a patterning function for MΦ^Prim progenitors remains to be determined. Other Lyl-1-expressing lineages^29,42 might also participate to impaired development processes in Lyl-1^LacZ/LacZ embryos, calling for the development of new mouse models that will allow lineage specific reports of Lyl-1 expression and function in endothelial or hematopoietic lineages during discrete steps of early YS development and beyond.

Defective MΦ^Prim development in Lyl-1 mutant YS

The analysis of Lyl-1 expression in A1-A2-A3 subsets from Cx3cr1^WT/GFP YS indicated that Lyl-1 is expressed throughout MΦ-progenitor differentiation, with levels decreasing from A1 to A3 (Supplementary Fig. 3a). We monitored the distribution of A1-A2 and A3 MΦ subsets (Supplementary Fig. 2a) at E10-YS, when all three subsets are present, using the Cx3cr1^WT/GFP:Lyl-1^LacZ/LacZ strain. While the size of the whole MΦ population was not overtly modified, Lyl-1-deficiency impacted the subset distribution, with increased A1 and reduced A2 and A3 pool sizes (Fig. 4A). Lyl-1 thus appears to regulate MΦ differentiation towards mature MΦs. A limited/delayed differentiation of E9 Lyl-1^LacZ/LacZ MΦ^Prim progenitors could result from an altered cytokine signaling: Signaling through Jak-Stat pathway (Jak1-Stat1/6), which underlays cytokine signaling in hematopoietic cells, was down-regulated In E9 mutant progenitors, (Fig. 4B; Supplementary Fig. 3b), with profoundly decreased expression of interleukins and their receptors (Il1a-b/Il1r1-2; Il4i1/Il4Ra; Il6st/Il6ra; Il10/ Il10ra). It was also supported by a down-regulated Spi1/PU.1 signaling pathway (Supplementary Fig. 3c; Supplementary Tables 4–5) and by the decreased expression of Ptprc/CD45, Csf1r, Itgam/CD11b and CD33 (Fig. 4C).

**Fig. 4: Defective differentiation of *Lyl-1*^LacZ/LacZ MΦ-progenitors from E10-YS.**

Within the MΦ lineage, Lyl-1 function during normal development would initially consist to restrict the size of the MΦ^Prim progenitor pool and/or the duration of its production, which is transient⁶, as indicated by the maintenance of the intermediate mesoderm to MΦ-progenitor pool observed in Lyl-1^LacZ/LacZ E8-YS. Indeed, the increased size of the MΦ-progenitor pool in E8-E9 YS appears independent from the defective/delayed differentiation of MΦ-progenitors observed at E10, since this process starts after E9.5^10,11,15. Subsequently, the increased size of MΦ^Prim progenitor pool in E10 Lyl-1^LacZ/LacZ YS likely results from a defective/delayed differentiation mediated by a defective cytokine signaling, implying that during normal development, Lyl-1 would promote the differentiation of MΦ^Prim progenitors.

Lyl-1^LacZ/LacZ MΦ-progenitors were also deficient in the IFN signaling that characterize E9 MΦ^Prim progenitors, notably Irf8, a factor involved in YS-MΦ and microglia development^21,43 (Fig. 4D). Compared to WT, MΦ-progenitors from Lyl-1^LacZ/LacZ E9-YS up-regulated the LXR/RXR activation pathway (Fig. 4E) and metabolic pathways, some enriched WT MΦ-progenitors at E10 (Butanoate, steroid) (Supplementary Table 1), and other not (Fructose/mannose, fatty acid) (Fig. 3c). They were also less active in inflammatory signaling pathways, particularly through NFκb, a factor known to interact with Lyl-1⁴⁴, and in TLR signaling (Fig. 4D; Supplementary Fig. 3b, d–e; Supplementary Table 5).

Overlapping the differentially expressed genes identified in Lyl-1^LacZ/LacZ MΦ-progenitors at E9 and E10 identified the core signature of Lyl-1-deficiency, independent of the maturation occurring between these stages (Fig. 4F). Unfortunately, the co-existence of MΦ^Prim and MΦ^T-Def progenitors in E10-YS complicates the attribution of gene expression changes to a stage-dependent maturation of MΦ^Prim progenitors or to a signature specific to MΦ^T-Def progenitors. However, most pathways favored by E10 progenitors were insensitive to Lyl-1-deficiency, except for a decreased expression of phagocytosis genes (Axl1, Mertk, Trem2, P2y6r/P2ry6) (Fig. 4G). Conversely, the TLR signaling pathway was increased in Lyl-1^LacZ/LacZ MΦ-progenitors at E10, compared to E9.

Lyl-1-expressing MΦ-progenitors contribute to the fetal liver and brain

The FL⁹ and brain^19,22 are colonized as early as E9 by YS-derived resident-MΦ progenitors. We evaluated the contribution of Lyl-1-expressing MΦ^Prim progenitors to these rudiments at E10 (Fig. 5A). While E10-YS comprised FDG⁺/Lyl-1⁺ and FDG^-/Lyl-1^- MΦ-progenitors and mature (F4/80⁺) MΦ subsets (Fig. 1b), the brain from the same embryos essentially harbored FDG⁺/Lyl-1⁺ MΦ-progenitors and MΦs. In contrast, both FDG⁺/Lyl-1⁺ and FDG^-/Lyl-1^- MΦ-progenitors were present in E10-FL, as in E10-YS, and MΦ-progenitors were more abundant in mutant FL than in WT (Fig. 5B).

**Fig. 5: Contribution of Lyl-1-expressing MΦ-progenitors to the fetal liver and brain.**

We next focused on brain MΦs during the colonization stage, which lasts until E11⁴⁵. At this stage, microglia and perivascular, meningeal and choroid plexus MΦs, collectively referred to as BAMs, are all located in the brain mesenchyme and therefore undistinguishable^16,46. FACS-Gal assay demonstrated that the whole F4/80⁺ microglia/BAM expressed Lyl-1 throughout the settlement period (Fig. 5C). The presence of FDG⁺/Lyl-1⁺F4/80⁺ microglia/BAM at early stage of brain colonization suggests that MΦs could participate to this step.

Lyl-1⁺ MΦ^Prim progenitors and early microglia/BAM shared similar features, such as an early appearance timing and low level of Myb expression (Fig. 5D), concordant with a Myb-independent development of microglia^14,21. Lyl-1 was also similarly expressed in A1-A2 and A3 MΦ subsets from the YS and brain (Fig. 5E; Supplementary Fig. 3a). Lyl-1-deficiency impacted the distribution of MΦs subsets in E10 Cx3cr1^WT/GFP:Lyl-1^LacZ/LacZ brain: an increased A1 and a reduced A3 pool size indicated that Lyl-1 regulates MΦ-progenitor differentiation in both YS and brain (Fig. 5F).

The proximity between YS MΦ^Prim progenitors and microglia was also apparent in RNA-seq. data: E9 WT MΦ^Prim progenitors expressed significantly lower Mrc1/CD206 and higher Sall3 levels than E10 MΦ-progenitors, and a slightly increased Sall1 level (Fig. 5G), a transcriptomic pattern that characterizes microglia^33,45,47. This partial bias toward a microglia signature suggests that the first stage of microglia development program is already initiated in MΦ^Prim /“early EMP”progenitors in E9-YS.

Lyl-1 inactivation impairs microglia development at two development stages

Having defined Lyl-1 implication during microglia/BAM settlement in the brain, we turned to later development stages. Cytometry and database analyses⁴⁵ confirmed the continuous expression of Lyl-1 in CD45^low microglia until adulthood (Supplementary Fig. 4a). LYL-1 expression was also reported in microglia from healthy murine and human adults^48,49,50,51. We examined the impact of Lyl-1-deficiency on microglia pool size during development. Microglia quantification pointed to E12 as the first step impacted. The arrested increase of microglia pool in Lyl-1^LacZ/LacZ brain at E12 (Fig. 6a) resulted from a reduced proliferation (Fig. 6b) rather than an increased apoptosis (Supplementary Fig. 4b). Moreover, Lyl-1-deficiency provoked morphological changes in E12 Cx3cr1^WT/GFP:Lyl-1^LacZ/LacZ, compared to Cx3cr1^WT/GFP microglia, namely a reduced number and extent of ramifications (Fig. 6c; Supplementary Fig. 4c, d). From E14, the microglia pool size returned to levels similar to WT (Fig. 6a), probably due to the highly reduced apoptosis level in Lyl-1^LacZ/LacZ microglia at E14 (Supplementary Fig. 4b).

**Fig. 6: *Lyl-1* deficiency leads to transient reductions of the microglia pool at E12 and P0-P3.**

P0-P3 was identified as a second stage altered in Lyl-1^LacZ/LacZ microglia. At birth, the cellularity of Lyl-1^LacZ/LacZ brain was significantly decreased compared to WT (Fig. 6d), which was not the case at earlier stages (Supplementary Fig. 4e). CD11b⁺ cells recovery was also reduced (WT: 140.96 ± 0.91 × 10³, n = 9; Lyl-1^LacZ/LacZ: 87.18 ± 0.37 × 10³, n = 9). Consequently, Lyl-1-deficiency triggered a nearly 2-fold reduction of the microglia population (Fig. 6d). This perinatal reduction of microglia appeared transient, since no difference with WT brain was observed in the adult (Supplementary Fig. 4f). Transient decreases of microglia pool size, such as those we observed at E12 and P0-P3 in Lyl-1^LacZ/LacZ mutant, have been reported to occur during normal development in postnatal weeks 2–3⁵², but also in Cx3cr1 mutant mice during the 1^st postnatal week⁵³. This indicates a highly dynamic control of the microglia pool size during key steps of neural development that seems preserved in Lyl-1 mutant, with the exception of the E12 and P0-P3 time-points. At this later stage, the reduction of brain cellularity in Lyl-1^LacZ/LacZ mice points to Lyl-1 as a possible regulator of the trophic function of microglia on brain cells^54,55.

The identification of E12 and P0-P3 as key stages for Lyl-1 function in microglia development was confirmed by RT-qPCR analyses of the expression of genes essential for MΦs (Spi1/PU.1, Csf1r, Mafb) and/or microglia (Runx1, Cx3cr1, Irf8) development and function, of known regulators of developmental hematopoiesis (Tal-1, Lmo2, Runx1) and related factors (Tcf3/E2A, Tcf4/E2.2) (Fig. 6e, f; Supplementary Fig. 4g). Time-course analyses highlighted the down-regulation of Csf1r, Irf8 and Lmo2 in Lyl-1^LacZ/LacZ microglia at E12 and P0-P3, while Cx3cr1 was only decreased at E12 (Fig. 6g). Note that Lyl-1 expression was unmodified in Cx3cr1^GFP/GFP mutants (Fig. 6g). Interestingly, Cx3cr1, as well as Irf8 and Lmo2, belong to potential Lyl-1 target genes².

Mafb expression levels in Lyl-1^LacZ/LacZ microglia transiently decreased at P0-P3 and later returned back to WT expression levels (Fig. 6h). As Mafb represses resident-MΦ self-renewal⁵⁶, the recovery of a normal amount of microglia after birth may stem from this transient decrease. Spi1/PU.1, Tcf3/E2A and Tcf4/E2.2 expression levels were unmodified in Lyl-1^LacZ/LacZ microglia, while Runx1 expression was only affected after birth. Tal-1 expression was decreased at E14 and increased after birth, suggesting that this Lyl-1 paralog³ does not compensate Lyl-1-deficiency during embryonic stages, but may do so at postnatal stages (Supplementary Fig. 4g). Remarkably, RNA-seq. results indicated that some genes deregulated in Lyl-1^LacZ/LacZ microglia at E12 and P0-P3 were also down-regulated in Lyl-1^LacZ/LacZ MΦ-progenitors at E9 (Csf1r: Supplementary Fig. 3c, Lmo2: Fig. 3b; Irf8: Fig. 4D; Cx3cr1: Fig. 6i). These deregulations were transient, however in both locations and stages they coincided with a defective MΦ/microglia differentiation.

Other genes enriched in microglia (Fcrls, Mef2c, Maf)⁵⁷ or involved in the maintenance of microglia homeostasis (P2ry12)⁵⁸ were also expressed in E9 Lyl-1^LacZ/LacZ MΦ-progenitors at a lower level than in the WT, except Lpr8 and Aif-1/Iba1 (Fig. 6i). Interestingly, the lower expression of phagocytosis genes (Axl1, Mertk, Trem2, P2y6r/P2ry6) uncovered in Lyl-1^LacZ/LacZ MΦ-progenitors from E10-YS (Fig. 4G), was present and stronger in the microglia from Lyl-1^LacZ/LacZ newborn, except for Trem2 which expression was unmodified (Fig. 6j).

These deregulations highlight again shared features between MΦ^Prim progenitors and microglia/neural development which became apparent upon Lyl-1 inactivation considering the large number of neural signaling pathways up-regulated in E9 Lyl-1^LacZ/LacZ MΦ-progenitors (Fig. 3c) and the relationship of the differentially expressed genes enriched in E9 MΦ^Prim progenitors with brain formation and neuro-development uncovered in IPA analysis (Supplementary Fig. 4h).

Based on the gene expression pattern of Lyl-1-deficient microglia and the signature of MΦ^Prim progenitors in the early YS, a contribution of Lyl-1-deficiency to neurodevelopmental disorders may be considered. Synaptic pruning and neural maturation, which characterize the perinatal phase of microglia development⁴⁵, might be impaired in Lyl-1^LacZ/LacZ embryos considering the defects observed at P0-P3, the later key developmental stage regulated by Lyl-1. Indeed, Lyl-1 deregulation has been observed in datasets reporting pathological models of brain myeloid cells (http://research-pub.gene.com/BrainMyeloidLandscape/#Mouse-gene/Mouse-gene/17095/geneReport.html)⁵⁹, as well as in human neuropathies^60,61, including the 19p13.13 micro-deletion neuro-developmental disabilities⁶². However, since Lyl-1 is expressed in endothelial cells, including in the brain⁴², a contribution of LYL-1-deficient endothelial cells to these diseases must be considered.

Conclusions

Altogether, our findings reveal Lyl-1 as a key factor regulating the production and differentiation of YS MΦ-progenitors and the development of microglia. Lyl-1 is the least studied partners of the transcription factor complex that regulates developmental hematopoiesis. The development of more appropriate models is required to precise Lyl-1 functions in microglia and determine its role in the development of other resident-MΦs populations.

Methods

Mice

The following mouse strains, housed in Gustave Roussy Institute animal facility (License #H94-076-11) were used (See Table 1 for a summary of the breeding schemes): 1- C57BL/6 mice (Harlan or Charles Rivers Laboratories), referred to as wild type (WT); 2- Lyl-1^LacZ mice⁵. To avoid the possible detection of FDG/Lyl-1 expression from maternally-derived MΦ¹¹ in heterozygous embryos, Lyl-1^LacZ/LacZ males were crossed with WT females; 3- Cx3cr1^GFP mice⁶³. Cx3cr1^GFP/GFP males were crossed with WT females to generate Cx3cr1^WT/GFP mice/embryos or with Lyl-1^LacZ/LacZ females to generate Cx3cr1^WT/GFP:Lyl-1^WT/LacZ mice/embryos. 4-Cx3cr1^GFP/GFP:Lyl-1^LacZ/LacZ double mutant strain developed from Cx3cr1^WT/GFP:Lyl-1^WT/LacZ crosses. Cx3cr1^WT/GFP:Lyl-1^WT/LacZ and Cx3cr1^WT/GFP:Lyl-1^LacZ/LacZ mice/embryos were obtained by crossing Cx3cr1^GFP/GFP:Lyl-1^LacZ/LacZ males respectively to C57BL/6 or Lyl-1^LacZ/LacZ females. Experiments were conducted in compliance with French/European laws, under authorized project #2016-030-5798, approved by officially accredited local institutional animal (committee n°26) and French “Ministère de la Recherche” ethics boards..

Table 1 Breeding crosses used throughout this study.

Full size table

The day of vaginal plug observation was considered as E0.5. Pregnant females were sacrificed by cervical dislocation. Pre-somite embryos were staged according to Downs et al.⁶⁴, by somite counting from E8 to E10.5 and thereafter by morphological landmarks.

Tissues

Cells from the yolk sac (E7.5-E10.5), from E10 FL or whole brain (E9-E14) were obtained after mechanical disruption^9,65. For cytometry analyses, the dissected region of the brain comprises the di-, mes- and metencephalon at E9-E10, the ectoderm was carefully removed²². After E12, microglia were recovered from the midbrain following Percoll (P1644, Sigma) separation⁶⁶. Cells were filtered through a 70 μm cell strainer and centrifuged at 300 g for 10 min. Cells were resuspended in Fc block BD solution and incubated at room temperature for 10 min. After washing in PBS + 10% FCS and centrifugation, cells were resuspended in 50 μl + 10% FCS containing the fluorochrome coupled antibodies and incubated at 4 °C for 20 min. After washing in PBS + 10%FCS and centrifugation, cells were resuspended in 50ul + 10% FCS. Dead cells were excluded by adding 1 μg/mL DAPI (Sigma) before acquisition.

The number of microglia per brain was estimated by reporting the percentage of CD11b⁺CD45^lowF4/80⁺ microglia to the cellularity of the corresponding sample.

Tissue culture

YS explants, maintained for 1 day in organ culture in plates containing OptiMEM+Glutamax, 1% Penicillin-streptomycin, 0.1% β-mercaptoethanol (ThermoFisher) and 10% fetal calf serum (Hyclone), are referred to as OrgD1-YS.

In clonogenic assays, YS suspension or sorted cells were plated in triplicate (3 × 10³ or 100-150 cells/mL) in Methocult^® M3234 (StemCell Technologies) always supplemented the cytokines listed in Table 2. Colonies were scored at day 5 for primitive erythroblasts and day 7 for other progenitors.

Table 2 Cytokines used in this study.

Full size table

Flow cytometry

Cells, stained with antibodies listed in Table 3, for 30 min. on ice, were acquired (Canto II) or sorted (FACS-Aria III or Influx, BD Biosciences) and analyzed using FlowJo (Treestar) software.

Table 3 Antibodies and fluorescent stains used throughout this study.

Full size table

The β-Galactosidase substrate fluorescein di-β-galactopyranoside (FDG; Molecular probe), was used as reporter for Lyl-1 expression in FACS-Gal assay^67,68. For apoptosis analysis, microglia were immune-stained and incubated with Annexin V-FITC. 7AAD was added before acquisition. For proliferation assays, pregnant females (12 gestational days) were injected with BrdU (10μM) and sacrificed 2 h later. Microglia were isolated, immune-stained and prepared according to kit instruction (BD Pharmingen 552598). BrdU incorporation was revealed using anti-BrdU-APC.

Brain imaging

To assess microglia morphology in E12 embryos, the midbrain was dissected from Cx3cr1^WT/GFP:Lyl-1^WT/WT and Cx3cr1^WT/GFP:Lyl-1^LacZ/LacZ embryos and sectioned through the midline. After fixation (4% paraformaldehyde) overnight at 4 °C, whole midbrains were washed in phosphate-buffered saline (PBS)/0.1 M glycine and incubated overnight in PBS/15% sucrose at 4 °C. Midbrains were washed with PBS + 0.1% Tween and incubated 90 min. in blocking buffer (PBS + 10% FCS) at room temperature (RT). Midbrains were subsequently immune-labeled with F4/80-APC overnight at 4 °C. After washing, they were incubated 3 min. in PBS + DAPI (1 μg/mL) at RT and washed. Finally, midbrains were placed in the central well of glass-bottom culture dishes (P35G-1.5-10-C; MatTek, USA) filled with PBS + 10% FCS. After appropriate orientation of the sample, the well was covered with a 12 μm ∅ glass coverslip. Image stacks were collected using a Leica SP8 confocal microscope. To unsure an unbiased choice of the cells imaged, taking into account possible changes in cell distribution induced by Lyl-1 deficiency, we always acquired cells in similar positions regarding the landmark set in the midbrain flat mount, as shown in Supplementary data and Fig. 4C. Images were processed using Imaris x64 (version 7.7.2; Bitplane) and Photoshop 8.0 (Adobe Systems, San Jose, CA) softwares.

RT-qPCR analyses

RNA was extracted using Trizol. After cDNA synthesis (SuperScript™ VILO™ Master-Mix reverse transcriptase, ThermoFisher), Real Time (RT)-PCR was performed (SYBR Premix Ex TaqII, Takara Bio). Reference genes were Actin, Hprt and Tubulin. Gene expressions were normalized to the values obtained for E10-YS MΦ-progenitors, E12 WT Lin^-Sca⁺cKit⁺ (LSK) progenitors or E12 WT microglia. Primers are listed in Table 4.

Table 4 Primers used in this study.

Full size table

RNA-seq

For samples preparation, MΦ-progenitors (Kit⁺CD45⁺CD11b⁺) were sorted from E9 (MΦ^Prim progenitor) and E10 (MΦ^Prim + MΦ^T-Def progenitors) YS pools from WT or Lyl-1^LacZ/LacZ embryos (Four biological replicates). RNA was extracted as described above.

The RNA integrity (RNA Integrity Score ≥ 7.0) was checked on the Agilent 2100 Bioanalyzer (Agilent) and quantity was determined using Qubit (Invitrogen). SureSelect Automated Strand Specific RNA Library Preparation Kit was used according to manufacturer’s instructions with the Bravo Platform. Briefly, 50 ng of total RNA sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. Fragmented mRNA samples were subjected to cDNA synthesis and converted into double stranded DNA using reagents supplied in the kit. The resulting dsDNA was used for library preparation. The final libraries were bar-coded, purified, pooled together in equal concentrations and subjected to paired-end sequencing (2 × 100) on Novaseq-6000 sequencer (Illumina) at Gustave Roussy genomic facility.

The quality of RNA-seq. reads were assessed with FastQC 0.11.7 and MultiQC 1.5⁶⁹. Low quality reads were trimmed with Trimmomatic 0.33⁷⁰. Salmon 0.9.0 tool⁷¹ was used for quantifying the expression of transcripts using geneset annotation from Gencode project release M17 for mouse⁷². The version of transcriptome reference sequences used was GRCm38.p6.

Statistical analysis was performed using R with the method proposed by Anders and Huber implemented in the DESeq2 Bioconductor package⁷³. The differential expression analysis in DESeq2 uses a generalized linear model (GLM) where counts are modeled using a negative binomial distribution. Counts were normalized from the estimated size factors using the median ratio method and a Wald test was used to test the significance of GLM coefficients. Genes were considered differentially expressed when adjusted p-value < 0.05 and fold-change > 2.

Data were analyzed through the use of Ingenuity^® Pathway Analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis)⁷⁴, Gene set enrichment analysis (GSEA; https://www.gsea-msigdb.org/gsea/index.jsp)^75,76, Morpheus (https://software.broadinstitute.org/morpheus/) and Venny (https://bioinfogp.cnb.csic.es/tools/venny/) softwares.

Statistics and reproducibility

Statistical analyses including mean, SEM and p values were performed using using Prism 7 software (GraphPad). Statistical significance between two groups was assessed by unpaired two-tailed Student’s t tests or Mann–Whitney tests. Data are presented as mean ± SEM or by Box and whisker plots, min to max, with the median shown. Statistical significances were indicated by p-values and/or as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, ns: not significant). P-values below 0.05 were considered as significantly different. The number of biological replicates (n) and independent experiments is reported in the corresponding figure legend.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files or from the corresponding author upon reasonable request. RNAseq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession code E-MTAB-9618. The source data underlying figures and supplementary figures are provided as a Supplementary Data 1.

References

Pina, C. & Enver, T. Differential contributions of haematopoietic stem cells to foetal and adult haematopoiesis: insights from functional analysis of transcriptional regulators. Oncogene 26, 6750–6765 (2007).
Article CAS PubMed Google Scholar
Wilson, N. K. et al. Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. Cell Stem Cell 7, 532–544 (2010).
Article CAS PubMed Google Scholar
Curtis, D. J., Salmon, J. M. & Pimanda, J. E. Concise review: blood relatives: formation and regulation of hematopoietic stem cells by the basic helix-loop-helix transcription factors stem cell leukemia and lymphoblastic leukemia-derived sequence 1. Stem Cells 30, 1053–1058 (2012).
Article CAS PubMed Google Scholar
Porcher, C., Chagraoui, H. & Kristiansen, M. S. SCL/TAL1: a multifaceted regulator from blood development to disease. Blood 129, 2051–2060 (2017).
Article CAS PubMed Google Scholar
Capron, C. et al. The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell function and B-cell differentiation. Blood 107, 4678–4686 (2006).
Article CAS PubMed Google Scholar
McGrath, K. E., Frame, J. M. & Palis, J. Early hematopoiesis and macrophage development. Semin. Immunol. 27, 379–387 (2015).
Article CAS PubMed Google Scholar
Palis, J. Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo. FEBS Lett. 590, 3965–3974 (2016).
Article CAS PubMed Google Scholar
Cumano, A. & Godin, I. Ontogeny of the hematopoietic system. Annu Rev. Immunol. 25, 745–785 (2007).
Article CAS PubMed Google Scholar
Kieusseian, A., Brunet de la Grange, P., Burlen-Defranoux, O., Godin, I. & Cumano, A. Immature hematopoietic stem cells undergo maturation in the fetal liver. Development 139, 3521–3530 (2012).
Article CAS PubMed Google Scholar
Palis, J., Robertson, S., Kennedy, M., Wall, C. & Keller, G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073–5084 (1999).
Article CAS PubMed Google Scholar
Bertrand, J. Y. et al. Three pathways to mature macrophages in the early mouse yolk sac. Blood 106, 3004–3011 (2005).
Article CAS PubMed Google Scholar
Tober, J. et al. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis. Blood 109, 1433–1441 (2007).
Article CAS PubMed PubMed Central Google Scholar
Sumner, R., Crawford, A., Mucenski, M. & Frampton, J. Initiation of adult myelopoiesis can occur in the absence of c-Myb whereas subsequent development is strictly dependent on the transcription factor. Oncogene 19, 3335–3342 (2000).
Article CAS PubMed Google Scholar
Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012).
Article CAS PubMed Google Scholar
Hoeffel, G. & Ginhoux, F. Fetal monocytes and the origins of tissue-resident macrophages. Cell Immunol. 330, 5–15 (2018).
Article CAS PubMed Google Scholar
Utz, S. G. et al. Early fate defines microglia and non-parenchymal brain macrophage development. Cell 181, 557–573 e518 (2020).
Article CAS PubMed Google Scholar
Ginhoux, F. & Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 44, 439–449 (2016).
Article CAS PubMed Google Scholar
Mass, E. Delineating the origins, developmental programs and homeostatic functions of tissue-resident macrophages. Int Immunol. 30, 493–501 (2018).
Article CAS PubMed Google Scholar
Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).
Article CAS PubMed PubMed Central Google Scholar
Gomez Perdiguero, E. et al. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature 518, 547–551 (2015).
Article PubMed Google Scholar
Kierdorf, K. et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 16, 273–280 (2013).
Article CAS PubMed Google Scholar
Alliot, F., Godin, I. & Pessac, B. Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain. Brain Res. Dev. Brain Res. 117, 145–152 (1999).
Article CAS PubMed Google Scholar
Wu, Y. & Hirschi, K. K. Tissue-resident macrophage development and function. Front. Cell Dev. Biol. 8, 617879 (2021).
Herbomel, P., Thisse, B. & Thisse, C. Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process. Dev. Biol. 238, 274–288 (2001).
Article CAS PubMed Google Scholar
Ferrero, G. et al. Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish. Cell Rep. 24, 130–141 (2018).
Article CAS PubMed Google Scholar
Wittamer, V. & Bertrand, J. Y. Yolk sac hematopoiesis: does it contribute to the adult hematopoietic system? Cell Mol. Life Sci. 77, 4081–4091 (2020).
Azzoni, E. et al. Kit ligand has a critical role in mouse yolk sac and aorta-gonad-mesonephros hematopoiesis. EMBO Rep. 19, e45477 (2018).
Espin-Palazon, R. et al. Proinflammatory signaling regulates hematopoietic stem cell emergence. Cell 159, 1070–1085 (2014).
Article CAS PubMed PubMed Central Google Scholar
Giroux, S. et al. lyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny. Gene Expr. Patterns 7, 215–226 (2007).
Article CAS PubMed Google Scholar
Cumano, A., Dieterlen-Lievre, F. & Godin, I. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86, 907–916 (1996).
Article CAS PubMed Google Scholar
Cumano, A., Ferraz, J. C., Klaine, M., Di Santo, J. P. & Godin, I. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution. Immunity 15, 477–485 (2001).
Article CAS PubMed Google Scholar
Hoeffel, G. et al. C-Myb+ erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages. Immunity 42, 665–678 (2015).
Article CAS PubMed PubMed Central Google Scholar
Mass, E. et al. Specification of tissue-resident macrophages during organogenesis. Science 353, aaf4238 (2016).
Balounova, J. et al. Toll-like receptor 2 expression on c-kit(+) cells tracks the emergence of embryonic definitive hematopoietic progenitors. Nat. Commun. 10, 5176 (2019).
Article PubMed PubMed Central Google Scholar
Espin-Palazon, R., Weijts, B., Mulero, V. & Traver, D. Proinflammatory signals as fuel for the fire of hematopoietic stem cell emergence. Trends Cell Biol. 28, 58–66 (2018).
Article CAS PubMed Google Scholar
Mariani, S. A. et al. Pro-inflammatory aorta-associated macrophages are involved in embryonic development of hematopoietic stem cells. Immunity 50, 1439–1452.e5 (2019).
Palpant, N. J. et al. Chromatin and transcriptional analysis of mesoderm progenitor cells identifies HOPX as a regulator of primitive hematopoiesis. Cell Rep. 20, 1597–1608 (2017).
Article CAS PubMed PubMed Central Google Scholar
Chiu, S. K. et al. Shared roles for Scl and Lyl1 in murine platelet production and function. Blood 134, 826–835 (2019).
Article CAS PubMed Google Scholar
McCormack, M. P. et al. Requirement for Lyl1 in a model of Lmo2-driven early T-cell precursor ALL. Blood 122, 2093–2103 (2013).
Article CAS PubMed Google Scholar
Epelman, S. et al. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity 40, 91–104 (2014).
Article CAS PubMed PubMed Central Google Scholar
Leid, J. et al. Primitive embryonic macrophages are required for coronary development and maturation. Circulation Res. 118, 1498 (2016).
Article CAS PubMed Google Scholar
Pirot, N. et al. LYL1 activity is required for the maturation of newly formed blood vessels in adulthood. Blood 115, 5270–5279 (2010).
Article CAS PubMed Google Scholar
Hagemeyer, N. et al. Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation. EMBO J. 35, 1730–1744 (2016).
Article CAS PubMed PubMed Central Google Scholar
Ferrier, R. et al. Physical interaction of the bHLH LYL1 protein and NF-kappaB1 p105. Oncogene 18, 995–1005 (1999).
Article CAS PubMed Google Scholar
Matcovitch-Natan, O. et al. Microglia development follows a stepwise program to regulate brain homeostasis. Science 353, aad8670 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79812 (2016). Data sets.
Article PubMed Google Scholar
Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).
Article CAS PubMed PubMed Central Google Scholar
Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312–1326 (2014).
Article CAS PubMed PubMed Central Google Scholar
Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).
Article CAS PubMed PubMed Central Google Scholar
Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015).
Article CAS PubMed Google Scholar
Wehrspaun, C. C., Haerty, W. & Ponting, C. P. Microglia recapitulate a hematopoietic master regulator network in the aging human frontal cortex. Neurobiol. Aging 36, 2443 e2449–2443 e2420 (2015).
Article Google Scholar
Bennett, M. L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl Acad. Sci. 113, E1738–E1746 (2016).
Article CAS PubMed PubMed Central Google Scholar
Zhan, Y. et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nat. Neurosci. 17, 400–406 (2014).
Article CAS PubMed Google Scholar
Paolicelli, R. C. et al. Synaptic pruning by microglia is necessary for normal brain development. Science 333, 1456–1458 (2011).
Article CAS PubMed Google Scholar
Antony, J. M., Paquin, A., Nutt, S. L., Kaplan, D. R. & Miller, F. D. Endogenous microglia regulate development of embryonic cortical precursor cells. J. Neurosci. Res. 89, 286–298 (2011).
Article CAS PubMed Google Scholar
Ueno, M. et al. Layer V cortical neurons require microglial support for survival during postnatal development. Nat. Neurosci. 16, 543–551 (2013).
Article CAS PubMed Google Scholar
Soucie, E. L. et al. Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells. Science 351, aad5510 (2016).
Crotti, A. & Ransohoff, R. M. Microglial physiology and pathophysiology: insights from genome-wide transcriptional profiling. Immunity 44, 505–515 (2016).
Article CAS PubMed Google Scholar
Bisht, K., Sharma, K. & Tremblay, M.-È. Chronic stress as a risk factor for Alzheimer’s disease: Roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress. Neurobiol. Stress 9, 9–21 (2018).
Article PubMed PubMed Central Google Scholar
Friedman, B. A. et al. Diverse brain myeloid expression profiles reveal distinct microglial activation states and aspects of Alzheimer’s disease not evident in mouse models. Cell Rep. 22, 832–847 (2018).
Article CAS PubMed Google Scholar
Colangelo, V. et al. Gene expression profiling of 12633 genes in Alzheimer hippocampal CA1: transcription and neurotrophic factor down-regulation and up-regulation of apoptotic and pro-inflammatory signaling. J. Neurosci. Res. 70, 462–473 (2002).
Article CAS PubMed Google Scholar
Thomas, D. M., Francescutti-Verbeem, D. M. & Kuhn, D. M. Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage. Faseb J. 20, 515–517 (2006).
Article CAS PubMed Google Scholar
Nimmakayalu, M. et al. Apparent germline mosaicism for a novel 19p13.13 deletion disrupting NFIX and CACNA1A. Am. J. Med. Genet. A 161A, 1105–1109 (2013).
Article PubMed Google Scholar
Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell Biol. 20, 4106–4114 (2000).
Article CAS PubMed PubMed Central Google Scholar
Downs, K. M. & Davies, T. Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. Development 118, 1255–1266 (1993).
Article CAS PubMed Google Scholar
Bertrand, J. Y., Giroux, S., Cumano, A. & Godin, I. Hematopoietic stem cell development during mouse embryogenesis. Methods Mol. Med. 105, 273–288 (2005).
PubMed Google Scholar
Mildner, A. et al. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat. Neurosci. 10, 1544–1553 (2007).
Article CAS PubMed Google Scholar
Fiering, S. N. et al. Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs. Cytometry 12, 291–301 (1991).
Article CAS PubMed Google Scholar
Guo, W. & Wu, H. Detection of LacZ expression by FACS-Gal analysis. Nat. Protoc. Exch. (2008).
Ewels, P., Magnusson, M., Lundin, S. & Käller, M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics 32, 3047–3048 (2016).
Article CAS PubMed PubMed Central Google Scholar
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Article CAS PubMed PubMed Central Google Scholar
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Article CAS PubMed PubMed Central Google Scholar
Frankish, A. et al. GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 47, D766–d773 (2019).
Article CAS PubMed Google Scholar
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Article PubMed PubMed Central Google Scholar
Krämer, A., Green, J., Pollard, J. Jr. & Tugendreich, S. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics 30, 523–530 (2014).
Article PubMed Google Scholar
Mootha, V. K. et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat. Genet. 34, 267–273 (2003).
Article CAS PubMed Google Scholar
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

The authors thank Julien Bertrand for critical reading of the manuscript. We are grateful to the staff of the facilities at Gustave Roussy, the animal facility (PFEP, UMS AMMICa UMS 3655/US23, directed by P. Gonin), the imaging facility (PFIC, UMS AMMICa UMS 3655/US23, directed by C. Laplace-Builhe), the genomic facility directed by N. Droin, the bioinformatics facility (G. Meurice), directed by M. Deloger. This work was supported by fundings from Institut National de la Santé et de la Recherche Médicale to W. Vainchenker, I. Plo and H. Raslova, from Centre National de la Recherche Scientifique and Université de Paris-Saclay to I. Godin, from grants INCA PLBio to I. Plo, “Ligue Nationale contre le Cancer” Certified Team to H. Raslova, “Association pour la Recherche sur le Cancer” (n°4878) to I. Godin, Gustave Roussy (TA DERE 17) and National Natural Science foundation of China (N°32000669) to D. Ren, Grant Agency of the Czech Republic (GACR n°19–23154 S) to D. Filipp and from fellowships from “Association pour la Recherche sur le Cancer” to A.-L. Kaushik; “Société Française d’Hématologie” to S. Wang and Chinese Scholarship Council fellowships to S. Wang and D. Ren.

Author information

Shoutang Wang
Present address: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, 63110, USA
Deshan Ren
Present address: Ministry of Education Key Laboratory of Model Animal for Disease study; Model Animal Research Center, Medical school of Nanjing University, Chemistry and Biomedicine Innovation center, Nanjing University, Nanjing, 210093, China
Anna-Lila Kaushik
Present address: Plasseraud IP, 33064, Bordeaux, France
Gabriel Matherat
Present address: Agence Nationale pour la Recherche, Paris, France
These authors contributed equally: Shoutang Wang, Deshan Ren.

Authors and Affiliations

Gustave Roussy, INSERM UMR1287, Université Paris-Saclay, Villejuif, France
Shoutang Wang, Deshan Ren, Brahim Arkoun, Anna-Lila Kaushik, Gabriel Matherat, William Vainchenker, Hana Raslova, Isabelle Plo & Isabelle Godin
PFIC, lUMS AMMICa (US 23 INSERM/UMS 3655 CNRS; Gustave Roussy, Villejuif, France
Yann Lécluse
Laboratory of Immunobiology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic
Dominik Filipp

Authors

Shoutang Wang
View author publications
You can also search for this author in PubMed Google Scholar
Deshan Ren
View author publications
You can also search for this author in PubMed Google Scholar
Brahim Arkoun
View author publications
You can also search for this author in PubMed Google Scholar
Anna-Lila Kaushik
View author publications
You can also search for this author in PubMed Google Scholar
Gabriel Matherat
View author publications
You can also search for this author in PubMed Google Scholar
Yann Lécluse
View author publications
You can also search for this author in PubMed Google Scholar
Dominik Filipp
View author publications
You can also search for this author in PubMed Google Scholar
William Vainchenker
View author publications
You can also search for this author in PubMed Google Scholar
Hana Raslova
View author publications
You can also search for this author in PubMed Google Scholar
Isabelle Plo
View author publications
You can also search for this author in PubMed Google Scholar
Isabelle Godin
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

SW: Conceptualization; Methodology; Validation; Formal analysis; Investigation; Data curation; Writing – original draft preparation; Visualization; DR: Formal: analysis; Investigation; Data curation; Writing – original draft preparation; Visualization; A-LK; BA: Investigation, methodology; Formal analysis; Visualization; GM: Formal analysis; Investigation; methodology; YL: Investigation; methodology; DF: Writing—review & editing: critical review, commentary and revision; WV, HR, IP: Funding acquisition; Writing—review & editing: critical review, commentary and revision; IG: Conceptualization; Project administration; Supervision; Methodology; Validation; Formal analysis; Resources; Data curation; Writing – original draft preparation; Writing—review & editing; Visualization; Funding acquisition.

Corresponding author

Correspondence to Isabelle Godin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review information

Communications Biology thanks Ki-Wook Kim, Noelia Alonso Gonzalez and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editors: Eirini Trompouki and George Inglis. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Peer Review File

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data 1

Reporting Summary

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Wang, S., Ren, D., Arkoun, B. et al. Lyl-1 regulates primitive macrophages and microglia development. Commun Biol 4, 1382 (2021). https://doi.org/10.1038/s42003-021-02886-5

Download citation

Received: 07 May 2021
Accepted: 10 November 2021
Published: 09 December 2021
DOI: https://doi.org/10.1038/s42003-021-02886-5

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Subjects

Abstract

Similar content being viewed by others

Introduction

Results and discussion

Lyl-1 expression marks MΦPrim progenitors from the early YS

Distinct features of WT MΦ-progenitors at E9 and E10

Lyl-1 deficiency impacts embryonic development

Defective MΦPrim development in Lyl-1 mutant YS

Lyl-1-expressing MΦ-progenitors contribute to the fetal liver and brain

Lyl-1 inactivation impairs microglia development at two development stages

Conclusions

Methods

Mice

Tissues

Tissue culture

Flow cytometry

Brain imaging

RT-qPCR analyses

RNA-seq

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review information

Additional information

Supplementary information

Rights and permissions

About this article

Cite this article

Share this article

Comments

Search

Quick links

Lyl-1 expression marks MΦ^Prim progenitors from the early YS

Defective MΦ^Prim development in Lyl-1 mutant YS