Shoutang Wang

Deshan Ren

Anna-Lila Kaushik

Gabriel Matherat

Yann Lécluse

Dominik Filipp

William Vainchenker

Hana Raslova

Isabelle Plo

Isabelle Godin
email: isabelle.godin@gustaveroussy.fr

Lyl-1 marks and regulates primitive macrophages and microglia development

Key points:

1-Lyl-1 expression marks yolk sac macrophages and brain macrophage/microglia/BAM. 2-Lyl-1 deficiency impairs primitive macrophage development and leads to the up-regulation of genes involved in embryo patterning.

3-Lyl-1-expressing primitive macrophages have an

INTRODUCTION

Amongst the components of the transcription factor network that regulate the various features of haematopoietic cells, Tal-1, Lmo2, Runx1 and Gata2 stand out as the major regulators of haematopoietic progenitor development during ontogeny [START_REF] Pina | Differential contributions of haematopoietic stem cells to foetal and adult haematopoiesis: insights from functional analysis of transcriptional regulators[END_REF][START_REF] Wilson | Combinatorial Transcriptional Control In Blood Stem/Progenitor Cells: Genome-wide Analysis of Ten Major Transcriptional Regulators[END_REF]. Tal-1, Lmo2 and Gata-2 belong to a transcriptional complex, which also includes the basic helix-loop-helix (bHLH) transcription factor lymphoblastic leukaemia-derived sequence 1 (Lyl-1).

Contrary to its paralog Tal-1, which is mandatory for the specification of all haematopoietic progenitors [START_REF] Curtis | 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[END_REF][START_REF] Porcher | SCL/TAL1: a multifaceted regulator from blood development to disease[END_REF], the function of Lyl-1 during developmental haematopoiesis remains largely unknown. We therefore analysed this function using Lyl-1 LacZ/LacZ mutant mice [START_REF] Capron | The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell function and B-cell differentiation[END_REF], focusing on the initial steps of haematopoietic cells development in the yolk sac (YS).

During ontogeny, haematopoietic progenitors are generated in three successive waves [START_REF] Palis | Hematopoietic stem cell-independent hematopoiesis: emergence of erythroid, megakaryocyte, and myeloid potential in the mammalian embryo[END_REF]. The two first occur in the YS prior to Haematopoietic Stem Cell (HSC) generation. This HSC-independent haematopoiesis comprises first the primitive haematopoietic wave, with the transient production of monopotent progenitors with embryonic specific features. The second YS wave, called transient-definitive, provides for a limited duration progenitors (mostly erythromyeloid) that seed the foetal liver (FL) and produce a haematopoietic progeny that displays definitive/adult differentiation features. Finally, HSC, generated in the aorta region in the third and definitive haematopoietic wave, immediately migrate to the FL where they mature and amplify to ultimately provide the population that will maintain lifelong haematopoiesis in the adult [START_REF] Cumano | Ontogeny of the hematopoietic system[END_REF][START_REF] Kieusseian | Immature hematopoietic stem cells undergo maturation in the fetal liver[END_REF].

During YS haematopoiesis, MΦ arising from the two waves derive either from a monopotent MΦ progenitor in the primitive wave [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF][START_REF] Palis | Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse[END_REF] or from the progressive differentiation of EMP via the production of GM progenitors, and ultimately of preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint granulocytes (G) and MΦ progenitors in the transient-definitive wave in a cMyb-dependent pathway [START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF].

In the recent years, fate-mapping approaches aimed at determining the embryonic origin of tissue resident MΦs indicated that most tissues harbour resident MΦs of various origins, such as YS, FL and adult bone marrow (reviewed in [START_REF] Ginhoux | Tissue-Resident Macrophage Ontogeny and Homeostasis[END_REF][START_REF] Hoeffel | Fetal monocytes and the origins of tissue-resident macrophages[END_REF]), which complicates the characterisation of the functions of the various MΦ subsets. In the case of brain resident MΦs, fate-mapping analyses established that, contrary to others tissue, these resident MΦs (microglia and Border Associated MΦ (BAM)) develop only from MΦ progenitors originating from the YS [START_REF] Ginhoux | Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages[END_REF][START_REF] Gomez Perdiguero | Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors[END_REF][START_REF] Kierdorf | Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways[END_REF][START_REF] Schulz | A lineage of myeloid cells independent of Myb and hematopoietic stem cells[END_REF][START_REF] Utz | Early Fate Defines Microglia and Non-parenchymal Brain Macrophage Development[END_REF], confirming a developmental model we had previously put forward [START_REF] Alliot | Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain[END_REF]. However, the wave (or waves) of origin remained unclear due to the limited criteria that discriminate progenitors from the two YS waves.

We here show that, at the early stages of YS haematopoiesis, Lyl-1 expression discriminates primitive (MΦ Prim ) from transient-definitive (MΦ T-Def ) MΦ progenitors. In the brain, Lyl-1 marked the entire microglia/BAM population at the onset of brain colonisation 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 MΦ in homeostatic and pathological contexts.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint

5

RESULTS

Lyl-1 expression discriminates MΦ Prim from MΦ T-Def progenitors in the early YS

Since Lyl-1 is expressed in the YS from the onset of haematopoiesis [START_REF] Giroux | lyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny[END_REF], we first explored its function by characterizing the progenitors produced by WT, Lyl-1 WT/LacZ and Lyl-1 LacZ/LacZ YS in clonogenic assay. To do so, YS at embryonic day (E) 8 were maintained in organ culture for 1 day (E8 OrgD1-YS), allowing only the development of progenitors from both primitive and transient-definitive waves [START_REF] Cumano | Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura[END_REF][START_REF] Cumano | Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution[END_REF]. Compared to WT, the production of MΦ colonies was increased in Lyl-1 WT/LacZ and Lyl-1 LacZ/LacZ OrgD1-YS.

Otherwise, the clonogenic potential and colony distribution were unmodified (Fig. 1a).

Using FACS-Gal assay (Sup. fig 1a), we noticed that the entire MΦ progenitor population (cKit + CD45 + CD11b + ) expressed Lyl-1 at E9. In contrast, from E9.5 (as in E8-OrgD1-YS), two MΦ progenitor subsets discriminated by FDG/Lyl-1 expression were present (Fig. 1b), suggesting that Lyl-1 may mark MΦ Prim progenitors from the earliest wave. After E9.5, the YS harbours both MΦ Prim and MΦ T-Def progenitors from the two YS waves and these two progenitors subsets cannot be discriminated by phenotype [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF]. We therefore investigated the known features that discriminate the two YS waves such as the origin from monopotent progenitors beginning at E7.25 for primitive progenitors [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF][START_REF] Palis | Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse[END_REF] or, for the transient-definitive progenitors, their progressive differentiation from EMP via the production of GM progenitors, and ultimately of granulocytes (G) and MΦ progenitors [START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF] and the dependence on cMyb expression for their production [START_REF] Hoeffel | C-Myb+ Erythro-Myeloid Progenitor-Derived Fetal Monocytes Give Rise to Adult Tissue-Resident Macrophages[END_REF][START_REF] Schulz | A lineage of myeloid cells independent of Myb and hematopoietic stem cells[END_REF].

FACS-Gal assay performed at E8 (0-5S), when only MΦ Prim are present, demonstrated that all early MΦ Prim progenitors, characterised by a CD11b + CD31 + phenotype [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF], displayed FDG/Lyl-1 expression (Fig. 1c). Most FDG + /Lyl-1 + cells (69.27%±0.33%) from Lyl-1 WT/LacZ preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 6 E8-YS co-expressed CD11b and CD31 and consistently 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 [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF][START_REF] Palis | Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse[END_REF].

As MΦ T-Def arise from EMPs that also generate GM progenitors and granulocytes [START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF], we characterized the differentiation potential of FDG + /Lyl-1 + and FDG -/Lyl-1 -fractions of myeloid progenitors (Ter119 -cKit + CD45 + CD11b + ) from E10 YS in clonogenic assay (Fig. 1d). All samples produced few non-myeloid contaminants, such as EMk and EMP in similar, nonsignificant amounts. Similar to WT E9-YS MΦ Prim progenitors, E10 FDG + /Lyl-1 + progenitor subset nearly exclusively produced MΦ colonies, confirming the restriction of Lyl-1 expression to MΦ Prim progenitors. In contrast, E10 FDG -/Lyl-1 -myeloid progenitors produced GM, G and MΦ colonies, confirming their multipotent/transient-definitive status.

The restriction of Lyl-1 expression to MΦ Prim progenitors was also strengthened by RT-qPCR comparison of cMyb expression: FDG -/Lyl-1 -progenitors displayed cMyb levels similar to Lineage negative Sca1 + cKit + progenitors from E12-FL, whereas, as E9-YS MΦ Prim progenitors, FDG + /Lyl-1 + YS progenitors expressed low cMyb levels, strengthening their primitive status (Fig. 1e).

A distinct separation of E9 and E10 MΦ progenitors was also confirmed in RNA-seq analysis of MΦ progenitors (CD45 + CD11b + cKit + ) sorted at E9, when the YS contains only MΦ Prim progenitors, and at E10, when it contains both MΦ Prim and MΦ T-Def progenitors. Principal Component Analysis pointed to a separation between E9 and E10 samples in both WT and Lyl-1 LacZ/LacZ YS (PC1) and a separation between WT and Lyl-1 LacZ/LacZ samples (PC2) clearer at E9 in frame with the restriction to MΦ Prim progenitors at this stage (Fig. 1f).

E9 and E10 WT MΦ progenitors differed by the expression of 726 genes. Amongst the differentially expressed genes (DEGs), 176 were up-regulated at E9 and 550 at E10. Considering the presence of both MΦ Prim and MΦ T-Def progenitors in E10-YS, the DEGs found at this stage may preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint reflect either waves-specific differences or stage-dependent changes related to the maturation of MΦ Prim progenitors.

Overlapping the identified DEGs to those obtained by Mass et al. (Mass et al., 2016) to characterise EMP and E10.25-E10.5 MΦs confirmed that E9 MΦ Prim progenitors were clearly distinct from these two populations. While about 5% of E10 WT up-regulated genes belonged to the EMP and MΦ signatures, these genes represented respectively 27.5% and 35.6% of these signatures (Fig. 1g). A similar separation was also observed in Gene Set Enrichment Analysis (GSEA) (Sup. fig. 1b). These observations suggest that within E10 MΦ progenitors some, likely the MΦ T-Def ones, retain part of the EMPs signature.

Focusing on the differences with E10 MΦ progenitors in a WT context, E9 MΦ Prim progenitors were found to differ from E10 progenitors by their transcription factors (TF) repertoire. E10 WT MΦ progenitors were enriched in genes associated with erythroid development (Gata1, Gata2, Klf1), as well as globin genes, both embryonic (Hbb-bh1, Hba-x, Hbb-y) and definitive (Hba-a2, Hba-a1, Hbb-bt) (Fig. 1g). E9 and E10 MΦ progenitors both exhibited a high expression of Spi1/PU.1 compared to Gata1 (Fig. 1h). The higher expression level of erythroid genes and of genes involved in granulo-monocytic (Mpo, Csf2r/GM-CSF receptors, Cebp, Jun) and megakaryocytic development (Pf4, TPO signalling) (Fig. 1g, Sup. table 1) at E10 sustains the notion that MΦ T-Def progenitors retain the expression of genes that characterize their EMP ancestor, as well as the monopotent status of E9 progenitors (Fig. 1d).

Altogether, these data validate the monopotent/primitive status of E9 MΦ progenitors.

However, due to the simultaneous presence of MΦ Prim and MΦ T-Def progenitors in E10-YS, the differential expressions of makers that are wave-specific at this stage, such as Lyl-1 for the primitive wave and cMyb and Tlr2 [START_REF] Balounova | Toll-like receptor 2 expression on c-kit(+) cells tracks the emergence of embryonic definitive hematopoietic progenitors[END_REF] for the transient-definitive one, were preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 8 not significant despite a net tendency to a decrease for Lyl-1 and an increase for cMyb and Tlr2 (Sup. fig. 1c). Through QIAGEN's Ingenuity® Pathway (IPA) and GSEA analyses, it appeared that MΦ Prim progenitors were more active in Eicosanoid signalling than E10 progenitors (Fig. 1i). E9 MΦ Prim progenitors were also enriched in type I interferon (IFN) β and type II IFNγ signalling (Fig. 1j) and, as a consequence, in MHC-II related genes, especially Cd74 (top 1 IPA network) (Fig. 1k; Sup. fig. 1d). Flow cytometry analyses confirmed a low, but significant, enrichment of MHC-II expression in E9 MΦ compared to E10 progenitors (Sup. fig. 1e). For their part, E10 MΦ progenitors were more active in inflammatory signalling (Fig. 1i,l; Sup. fig. 1d, f-h; Sup. table 1). They were also more metabolically active than E9 MΦ progenitors (Sup. table 1). In addition, the complement cascade and phagocytosis also prevailed at E10 (Sup. fig. 1i,j).

Lyl-1 deficiency in MΦ Prim leads to patterning defects

During YS development, both MΦ Prim and MΦ T-Def progenitors originate from cKit + CD31 + CD45 - progenitors (C subset), which differentiate into MΦσ via 3 subsets (A1 to A3) [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF]) (Sup. fig. 2a). When evaluating the effect of Lyl-1 deficiency at the earliest stage of MΦ Prim development, both FACS-Gal and clonogenic assays pointed to an increased production of MΦ progenitors in E8 Lyl-1 WT/LacZ compared to WT-YS (Fig. 2a), concordant with our first observation (Fig. 1a). The high increase of the expression level of Itga2b/CD41 in Lyl-1 lacZ/lacZ MΦ Prim progenitors (Fig. 2b) may reflect the elevated commitment of mesodermal/prehaematopoietic cells to a MΦ fate (Sup. data and sup fig. 2b-d).

We noticed a clear-cut modification of the TF network (Fig. 2c) that controls developmental haematopoiesis [START_REF] Wilson | Combinatorial Transcriptional Control In Blood Stem/Progenitor Cells: Genome-wide Analysis of Ten Major Transcriptional Regulators[END_REF]: beside the expected reduction of Lyl-1 expression, the expression of Lmo2, a target of Lyl-1 [START_REF] Mccormack | Requirement for Lyl1 in a model of Lmo2-driven early T-cell precursor ALL[END_REF], was also down-regulated, while preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint Tal-1 up-regulation might reflect a compensatory role [START_REF] Curtis | 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[END_REF]. The consequence of these changes on early stages of YS development were apparent in GSEA analyses: both pathways and GO terms uncovered a general up-regulation of signalling pathways involved in early stages of embryo patterning (Wnt, Hox and Smad) in Lyl-1 LacZ/LacZ MΦ progenitors, as well as deep changes in collagen, integrin and cadherin usage (Sup. table 2). Accordingly, various developmental trajectories were affected (Fig. 2d), with an 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.

Another patterning modification was observed when comparing Lyl-1 LacZ/LacZ to WT MΦ progenitors at E10: at this stage, GSEA and KEGG analyses pointed to the down-regulation of gene sets involved in heart development (Sup. fig. 2e; Sup. table 3). The heart harbours three resident MΦ subsets, two of which originate from the YS. Some of the features that differentiate the two CCR2 -YS-derived subsets [START_REF] Epelman | Embryonic and Adult-Derived Resident Cardiac Macrophages Are Maintained through Distinct Mechanisms at Steady State and during Inflammation[END_REF] also characterize E9 MΦ Prim progenitors (high MHC-II expression (Fig. 1k) and low phagocytosis ability (Sup. fig. 1j). Therefore, a function for Lyl-1 in heart development may be considered. These patterning defects, which appear triggered by defective MΦ Prim may be responsible, at least in part, for the significant decreased of litter size and increased perinatal lethality (Sup. fig. 2f) observed in Lyl-1 LacZ/LacZ mice compared to WT, which indicates the requirement for functional Lyl-1 during various developmental processes.

Defective MΦ Prim development in Lyl-1 mutants

We next monitored the distribution of A1-A2 and A3 MΦ subsets (Sup. fig. 2a) at E10-YS, when all three subsets are present, using the Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ double knock-in strain. The preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint analysis of Lyl-1 expression in A1-A2-A3 subsets from Cx3cr1 WT/GFP YS at E10 indicated that Lyl-1 is expressed throughout MΦ differentiation, with a level decreasing from A1 to A3 (Sup. fig. 3a).

The subset distribution was highly impacted by Lyl-1 deficiency, leading to an increased A1 and a reduced A2 and A3 pool sizes (Fig. 3a). Thus, Lyl-1 appears to regulate MΦ progenitors differentiation towards mature MΦ. This defective differentiation could originate from the altered cytokine signalling uncovered in E9 mutant progenitors through GSEA and IPA analyses (Fig. 3b; Sup. fig. 3b). A limited or delayed differentiation of E9 MΦ Prim progenitors was supported by the decreased expression of Ptprc/CD45, Csfr1, Itgam/CD11b and CD33 (Fig. 3c).

The PU.1 signalling pathway was also decreased in E9 Lyl-1 LacZ/LacZ progenitors (Sup. fig. 3c; Sup.

table 4b).

Lyl-1 LacZ/LacZ MΦ progenitors were also deficient in the IFN signalling that characterize E9 MΦ Prim progenitors, notably Irf8, a factor involved in the development of YS-MΦ and microglia (Hagemeyer et al., 2016;[START_REF] Kierdorf | Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways[END_REF] (Fig. 3d). Compared to WT progenitors, progenitors from Lyl-1 lacZ/lacZ E9-YS up-regulated the LXR/RXR activation pathway (Fig. 3e), as well as various metabolic pathways, including some enriched in E10 WT progenitors (Butanoate and steroid) (Sup. table 1), and other which were not (Fructose/mannose and Fatty acid) (Fig. 2d).

They were also less active in inflammatory signalling pathways, particularly through NFkB, a factor known to interact with Lyl-1 [START_REF] Ferrier | Physical interaction of the bHLH LYL1 protein and NF-kappaB1 p105[END_REF], and in TLR signalling (Tlr4, Tlr7, Tlr8, Tlr9) (Fig. 3d; Sup. fig. 3b, d-e

; Sup. table 4b).

To better identify the core signature of Lyl-1 deregulation taking into account the maturation that occur between E9 and E10, we overlapped DEGs identified in Lyl-1 LacZ/LacZ MΦ progenitors at both stages. From the 16 DEGs common to both stages (Fig. 3f), only one, Fcgr2b, showed an opposite deregulation during development, being first down-regulated in the mutant at E9, then expressed at higher levels than in the WT at E10. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint

Lyl-1 + MΦ progenitors contribute to the early brain and foetal liver.

The foetal liver [START_REF] Kieusseian | Immature hematopoietic stem cells undergo maturation in the fetal liver[END_REF] and brain [START_REF] Alliot | Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain[END_REF][START_REF] Ginhoux | Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages[END_REF] are both colonised as early as E9 by YS-derived resident MΦ progenitors. We therefore implemented a FACS-Gal assay to evaluate the contribution of Lyl-1-expressing MΦ Prim progenitors to these rudiments at E10 (Fig. 4a). While E10-YS comprised both FDG + /Lyl-1 + and FDG -/Lyl-1 -MΦ progenitor and mature (F4/80 + ) MΦ subsets (Fig. 1b), the brain from the same embryos essentially harboured FDG + /Lyl-1 + MΦ progenitors and mature MΦ. In contrast, MΦ progenitors of both phenotypes (FDG + /Lyl-1 + and FDG -/Lyl-1 -) were present in the FL, as in E10-YS. MΦ progenitors were more abundant in mutant FL than in WT (Fig. 4b), as in the YS, but the amount of mature MΦ was unmodified (Data not shown).

We next focused on brain MΦ during the colonisation stage, which occurs until E11 (Matcovitch-Natan et al., 2016). At this stage, microglia and perivascular, meningeal and choroid plexus MΦ, collectively refered to as BAMs, are all located in the brain mesenchyme and are therefore undistinguishable [START_REF] Goldmann | Origin, fate and dynamics of macrophages at central nervous system interfaces[END_REF][START_REF] Utz | Early Fate Defines Microglia and Non-parenchymal Brain Macrophage Development[END_REF]. FACS-Gal assay demonstrated that the whole F4/80 + microglia/BAM population expresses Lyl-1 throughout the settlement period (Fig. 4c). The presence of Lyl-1 + F4/80 + microglia/BAM at the earliest stage of brain colonisation suggests that already differentiated MΦ could participate to the colonization step.

A lineage relationship between Lyl-1 + MΦ Prim progenitors and early microglia/BAM is therefore strongly considered, based on the timing of their appearance and on similar features, such as the low level of cMyb expression (Fig. 4d), concordant with the cMyb-independent development of microglia [START_REF] Kierdorf | Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways[END_REF][START_REF] Schulz | A lineage of myeloid cells independent of Myb and hematopoietic stem cells[END_REF]. Such a putative lineage relationship is also supported by our RNA-seq data. WT E9 MΦ Prim progenitors expressed preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint significantly lower levels of Mrc1/CD206 than E10 MΦ progenitors, as well as a significantly higher level of Sall3 and a slight increase of Sall1 (Fig. 4e), a transcriptomic pattern that characterises microglia [START_REF] Lavin | Tissue-Resident Macrophage Enhancer Landscapes Are Shaped by the Local Microenvironment[END_REF]Mass et al., 2016;Matcovitch-Natan et al., 2016). This pattern suggests that E9-YS MΦ Prim progenitors show a partial bias toward a microglia signature.

These observations point to E9 MΦ Prim progenitors as a likely source of embryonic microglia with the first stage of microglia development program already initiated in YS MΦ Prim progenitors at E9.

We next examined the distribution of A1-A2 and A3 MΦ subsets in the brain of E10 Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ embryos to assess the impact of Lyl-1 deficiency at the onset of brain colonisation. Lyl-1 expression levels in the brain MΦ subsets were similar to those observed in the YS (Fig. 4f, Sup. fig. 3a). However, Lyl-1 deficiency led to an increased A1 and a reduced A3 pool size (Fig. 4g), indicating that in the brain, as in the YS, Lyl-1 regulates the differentiation of MΦ progenitors towards mature microglia/BAM.

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, focusing on CD45 lo microglia. Cytometry and database analyses (Matcovitch-Natan et al., 2016) confirmed the continuous expression of Lyl-1 in microglia until adulthood (Sup. fig. 4a). We therefore examined the impact of Lyl-1 inactivation on microglia pool size during development. Microglia quantification pointed to E12 as the first step impacted by the mutation. The arrested increase of the microglia pool in Lyl-1 LacZ/LacZ brain at E12 (Fig. 5a) resulted from a reduced proliferation (Fig. 5b) rather than an increased apoptosis (Sup. fig. 4b).

Moreover, Lyl-1 deficiency also provoked morphological changes in E12 Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ microglia, which displayed a reduced number and extent of ramifications compared to Cx3cr1 WT/GFP microglia (Fig. 5c; Sup. fig. 4c,d). Interestingly, from E14, the microglia pool size preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint returned to levels similar to WT embryos (Fig. 5a), a recovery that may result from the sharp reduction of apoptosis in Lyl-1 LacZ/LacZ microglia at E14 (Sup. fig. 4b).

P0-P3 was identified as a second development 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. 5d), which was not the case at earlier stages (Sup. fig. 4e). The recovery of CD11b + cells was also reduced (WT: 140.96±0.91x10 3 ; n=9; Lyl-1 LacZ/LacZ : 87.18±0.37x10 3 ; n=9). Consequently, Lyl-1 deficiency triggered a nearly 2-fold reduction of the microglia population (Fig. 5d). This perinatal reduction of the microglia pool appeared transient, since no difference with WT brain was observed in the adult (Sup. fig. 4f). At perinatal stages, 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 [START_REF] Antony | Endogenous microglia regulate development of embryonic cortical precursor cells[END_REF][START_REF] Ueno | Layer V cortical neurons require microglial support for survival during postnatal development[END_REF].

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 MΦ (Spi1/PU.1, Csf1r, Mafb) and/or microglia (Runx1, Cx3cr1, Irf8) development and function, as well as known regulators of developmental haematopoiesis (Tal-1, Lmo2, Runx1) and related factors (Tcf3/E2A, Tcf4/E2.2) (Fig. 5e,f; Sup. fig. 4g). Time course analyses highlighted the down-regulation of Csf1r, Irf8 and Lmo2 in Lyl-1 LacZ/LacZ microglia at both E12 and P0-P3, while Cx3cr1 was only decreased at E12 (Fig. 5g). Note that Lyl-1 expression was unmodified in Cx3cr1 GFP/GFP mutants (Fig. 5g). Interestingly, Cx3cr1, as well as Irf8 and Lmo2, belong to potential Lyl-1 target genes [START_REF] Wilson | Combinatorial Transcriptional Control In Blood Stem/Progenitor Cells: Genome-wide Analysis of Ten Major Transcriptional Regulators[END_REF].

Mafb expression levels in Lyl-1 LacZ/LacZ microglia transiently decreased at P0-P3 and later returned back to WT expression levels (Fig. 5h). As Mafb represses resident MΦ self-renewal [START_REF] Soucie | Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells[END_REF], this transient decrease may be linked to the recovery of a normal amount of microglia after perinatal stages. Spi1/PU.1, Tcf3/E2A and Tcf4/E2.2 expression levels were preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint unmodified in Lyl-1 LacZ/LacZ microglia at any stage, while Runx1 expression was only affected after birth. The expression of Tal-1 was decreased at E14 and increased after birth, suggesting that this Lyl-1 paralog [START_REF] Curtis | 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[END_REF] does not compensate for Lyl-1 deficiency during embryonic stages of microglia development, but may do so during postnatal stages (Sup. fig. 4g).

Remarkably, our RNA-seq results indicate that the expression level of some of the genes deregulated in Lyl-1 LacZ/LacZ microglia at E12 and in the new-born were also down-regulated in Lyl-1 LacZ/LacZ MΦ progenitors at E9 (Csfr1: Sup. fig. 3c, Lmo2: Fig. 2c; Irf8: Fig. 3d; Cx3cr1: Fig. 5i). The de-regulation of these genes in microglia was transient, however in both locations and stages they coincided with a defective Lyl-1 LacZ/LacZ MΦ/microglia differentiation.

Other genes enriched in microglia (Fcrls, Mef2c, Maf) [START_REF] Crotti | Microglial Physiology and Pathophysiology: Insights from Genome-wide Transcriptional Profiling[END_REF] or involved in the maintenance of microglia homeostasis (P2ry12) [START_REF] Bisht | Chronic stress as a risk factor for Alzheimer's disease: Roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress[END_REF] were also expressed in E9 Lyl-1 LacZ/LacZ MΦ progenitors at a lower level than in the WT, with exception of Lpr8 and Aif-1/Iba1 which expression levels were increased (Fig. 5i). The deregulation of these genes might underlie the relationship between MΦ Prim progenitors and microglia/neural development which became apparent upon Lyl-1 inactivation considering the large number of neural signalling pathways up-regulated in E9 Lyl-1 LacZ/LacZ MΦ progenitors (Fig. 2d) and the relationship of the DEGs enriched in E9 MΦ Prim progenitors with brain formation and neuro-development uncovered in IPA analysis (Sup. fig. 4h).

Altogether, these data suggest that Lyl-1 deficiency in microglia may lead to neurodevelopmental defects.

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15

DISCUSSION

We here identified Lyl-1 as a marker for YS-derived MΦ Prim progenitors and found that, at the earliest stage of YS development, this expression discriminated MΦ Prim progenitors from those produced in the transient-definitive waves. The early expression of Lyl-1 in YS mesoderm [START_REF] Giroux | lyl-1 and tal-1/scl, two genes encoding closely related bHLH transcription factors, display highly overlapping expression patterns during cardiovascular and hematopoietic ontogeny[END_REF], where it cannot substitute for the mandatory function of its paralog Tal-1 for the generation of haematopoietic progenitors [START_REF] Porcher | SCL/TAL1: a multifaceted regulator from blood development to disease[END_REF], has already been established (reviewed in [START_REF] Curtis | 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[END_REF]). More recently, Lyl-1 was also identified as a regulator of mesoderm cell fate in human induced pluripotent stem cells (iPSC) [START_REF] Palpant | Chromatin and Transcriptional Analysis of Mesoderm Progenitor Cells Identifies HOPX as a Regulator of Primitive Hematopoiesis[END_REF], and its function in the maintenance of primitive erythroid progenitors has been described [START_REF] Chiu | Shared roles for Scl and Lyl1 in murine platelet production and function[END_REF]. Parallel to Lyl-1 expression in the earliest wave of MΦ progenitor generation, our RNA-seq data now reveal Lyl-1 as an essential regulator in the specification of YS mesoderm to a haematopoietic fate since its invalidation affects major sets of genes involved in embryo patterning. Whether Lyl-1 role during the onset of organogenesis is specific to mesodermal stage or related to a patterning function for MΦ Prim progenitors remains to be determined. Lyl-1 involvement in two discrete developmental steps of embryogenesis would constitute yet another similarity with its paralog Tal-1, which regulates both YS mesoderm determination to a haematopoietic fate and the differentiation of primitive erythroid cells [START_REF] Curtis | 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[END_REF].

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 thought to be transient [START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF], as shown by the increased size of this pool observed in Lyl-1 LacZ/LacZ E8-E9 YS. Lyl-1 would also promote MΦ differentiation, when this process starts at E9.5, as the maintenance of an increased size of MΦ Prim progenitor pool in Lyl-1 LacZ/LacZ E10-YS appears to result from a defective cytokine signalling.

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16

We also defined a signature for WT MΦ Prim progenitors at E9, which comprises the Eicosanoid, MHC-II and IFN signalling pathways, pointing to an immuno-modulatory function for these MΦ Prim progenitors. Comparatively, MΦ progenitors at E10 were rather involved in the response to bacteria and viruses, through phagocytosis and inflammatory signalling, with the preferential expression of TNF, TLR and TGFβ signalling pathways. Unfortunately, the simultaneous presence of MΦ Prim and MΦ T-Def progenitors at E10 makes it difficult to attribute the changes of gene expression to a stage dependent maturation of MΦ Prim progenitors or to a signature specific to MΦ T-Def progenitors. However it indicates that most pathways favoured by MΦ progenitors at E10 were insensitive to Lyl-1 invalidation, except TLR signalling pathway that was downregulated in E9 mutant MΦ progenitors, compared to WT.

The limitations of the current Lyl-1 model call for the development of new mouse models that will allow lineage specific reports of Lyl-1 expression and function in endothelial or haematopoietic lineages during discrete steps of early YS development and beyond. These extended investigations would probably improve our knowledge of other HSC-independent lineages, and benefit to the Embryonic Stem Cells/iPSC field in which the production of definitive cells types is complicated by the poor discrimination between primitive and definitive populations.

When analysing the contribution of Lyl-1 + MΦ progenitors to resident MΦ, we detected their presence in FL and brain rudiments right from the onset of their colonisation. In the developing brain, microglia/BAM maintained Lyl-1 expression and shared with MΦ Prim progenitors a low level of cMyb expression. While fate-mapping analyses were instrumental to firmly ascribe a YS origin to microglia [START_REF] Ginhoux | Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages[END_REF] [START_REF] Kierdorf | Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways[END_REF][START_REF] Schulz | A lineage of myeloid cells independent of Myb and hematopoietic stem cells[END_REF] [START_REF] Gomez Perdiguero | Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors[END_REF], this strategy could not ascertain which YS progenitor wave contributes to microglia [START_REF] Mass | Delineating the origins, developmental programs and homeostatic functions of tissueresident macrophages[END_REF][START_REF] Mcgrath | Early hematopoiesis and macrophage development[END_REF][START_REF] Wittamer | Yolk sac hematopoiesis: does it contribute to the adult hematopoietic system?[END_REF]. Indeed, an preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint independent/specific targeting of only one MΦ progenitors wave was prevented by: 1-the similar phenotype of MΦ Prim and MΦ T-Def progenitors [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF]; 2-the short timespan separating the two YS waves (E7.25 and E8.25); 3-the high variability of development stages of individual embryos within a litter at these stage [START_REF] Downs | Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope[END_REF] and 4-the duration of TAM induction, now admitted to last up to 72h [START_REF] Senserrich | Analysis of Runx1 Using Induced Gene Ablation Reveals Its Essential Role in Pre-liver HSC Development and Limitations of an In Vivo Approach[END_REF]. Using an ex vivo approaches whereby expression and potential can be examined in individually staged embryos, we found that, at least between E8 and E10, Lyl-1 expression discriminates MΦ Prim from EMP-derived MΦ T-Def progenitors, and also marks the entire embryonic microglia/BAM populations.

We therefore favour a model ascribing microglia origin to Lyl-1 MΦ Prim progenitors, as they preferentially expressed genes belonging to the microglia signature (Sall1 and Sall3, P2ry12, Mef2c, Fcrls), while E10 MΦ progenitors favoured the expression of genes (Id3, Runx3), belonging to resident MΦ signatures in the lung and skin (Mass et al., 2016). Even though a later contribution of MΦ T-Def progenitors could be considered, provided they up-regulate Lyl-1 expression at a later stage, such model is supported by the low level of cMyb expressed by both Lyl-1 + MΦ Prim progenitors and Lyl-1 + microglia, in frame with the intact microglia pool in cMybdeficient mice [START_REF] Kierdorf | Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways[END_REF][START_REF] Schulz | A lineage of myeloid cells independent of Myb and hematopoietic stem cells[END_REF] arguing for an origin of microglia from MΦ Prim rather than MΦ T-Def progenitors. Another argument comes from the zebrafish model where the fate of MΦ Prim progenitors is more easily traced than in mammals as they arise from a location distinct from other haematopoietic progenitors [START_REF] Ferrero | Embryonic Microglia Derive from Primitive Macrophages and Are Replaced by cmyb-Dependent Definitive Microglia in Zebrafish[END_REF][START_REF] Herbomel | Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process[END_REF]. Finally, this model is supported by the recent report that in mice lacking the ligand for cKit, the impaired development of EMPs leads to the depletion of resident MΦs in the skin, lung and liver, but does not affect the development of microglia [START_REF] Azzoni | Kit ligand has a critical role in mouse yolk sac and aorta-gonadmesonephros hematopoiesis[END_REF].

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The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 18 Some confusion in the identification of the YS progenitors that gives rise to microglia stems from the fact that both YS waves produce cells from erythro-myeloid lineages, hence the "early EMP" and "late EMP" terminology used to qualify the brain-seeding MΦ progenitor. The monopotent/ primitive nature of E9 MΦ progenitors was confirmed by our RNA-seq data, so that the MΦ Prim progenitors terminology seems more appropriate than "early EMP" to qualify the primitive progenitors that give rise to microglia/BAM [START_REF] Ginhoux | Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages[END_REF][START_REF] Utz | Early Fate Defines Microglia and Non-parenchymal Brain Macrophage Development[END_REF], particularly because MΦ Prim and MΦ T-Def progenitors clearly constitute distinct entities that need to be better discriminated in order to investigate their respective contribution and function in the tissues resident MΦ contributed by the YS.

Amongst the features that distinguish WT E9 MΦ Prim progenitors from those present at E10, the enriched expression of MHC-II and the poor expression at E9 of genes related to phagocytosis also characterise one of the two YS-derived CCR2 -resident MΦ subsets identified in the heart [START_REF] Epelman | Embryonic and Adult-Derived Resident Cardiac Macrophages Are Maintained through Distinct Mechanisms at Steady State and during Inflammation[END_REF][START_REF] Leid | Primitive Embryonic Macrophages are Required for Coronary Development and Maturation[END_REF]. A function for Lyl-1-expressing resident MΦ in cardiac development thus appears probable considering also the down-regulation of gene sets related to heart development and function in E10 Lyl-1 LacZ/LacZ MΦ progenitors. This observation reinforces the need to better characterise the respective contributions of both YS-waves to resident MΦ in the tissues known to harbours YS-derived resident MΦ.

By investigating Lyl-1 function at later stages of microglia development, we identified E12 and P0-P3 as key stages impacted by Lyl-1 deficiency. These time points correspond respectively to the end of "early-" (E9-E12) and "pre"-microglia" (E14-P9) stages of the microglia development program (Matcovitch-Natan et al., 2016). Based on the gene expression pattern of Lyl-1-deficient microglia and the signature of MΦ Prim progenitors in the early YS, a major contribution of Lyl-1deficiency to neurodevelopmental disorders may be considered. Synaptic pruning and neural maturation, which characterise the perinatal phase of microglia development (Matcovitch-Natan preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint et al., 2016), 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.

Considering the sustained expression of Lyl-1 in microglia during adulthood, and the downregulation of genes essential for microglia function in Lyl-1 LacZ/LacZ microglia at E12 and P0-P3, Lyl-1 deregulation might contribute to various neuro-degenerative/neuro-inflammatory diseases and possibly play a role in the development of brain tumours. Such an involvement of Lyl-1 in the development of neuropathies is suggested by its deregulation in various pathological models of brain myeloid cells, uncovered through the analyses of brain myeloid cells datasets [START_REF] Friedman | Diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse Models[END_REF] (http://research-pub.gene.com/BrainMyeloidLandscape/#Mouse-gene/Mousegene/17095/geneReport.html). Moreover, LYL-1 expression was also reported in human microglia from healthy adult frontal cortex [START_REF] Wehrspaun | Microglia recapitulate a hematopoietic master regulator network in the aging human frontal cortex[END_REF] and some reports highlight the relevance of LYL-1 deregulation in human neuro-developmental and neuro-degenerative diseases [START_REF] Colangelo | Gene expression profiling of 12633 genes in Alzheimer hippocampal CA1: transcription and neurotrophic factor downregulation and up-regulation of apoptotic and pro-inflammatory signaling[END_REF][START_REF] Thomas | Gene expression profile of activated microglia under conditions associated with dopamine neuronal damage[END_REF]. Finally, LYL-1 belongs to the 5 genes commonly deleted in patients suffering from 19p13.13 micro-deletions, which might contribute to the neuro-developmental disabilities [START_REF] Nimmakayalu | Apparent germline mosaicism for a novel 19p13.13 deletion disrupting NFIX and CACNA1A[END_REF]. However, since Lyl-1 is expressed in endothelial cells, including in the brain [START_REF] Pirot | LYL1 activity is required for the maturation of newly formed blood vessels in adulthood[END_REF], a possible contribution of LYL-1-deficient endothelial cells to these diseases should be considered.

Altogether, our findings reveal Lyl-1 as a key factor regulating the production and differentiation of YS MΦ progenitors, as well as microglial development. Lyl-1 is one partners of the haematopoietic transcription factor complex which function during developmental haematopoiesis was the least studied. The development of new/more appropriate mouse models should be considered in order to carefully delineate its diverse functions in other resident MΦ populations and possibly other haematopoietic and cardiovascular lineages.

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20

MATERIALS AND METHODS

Ethic statement. Mice were housed in the animal facility of Gustave Roussy Institute ("Plateforme d'évaluation préclinique", licence #H94-076-11). All animal experiments were conducted in compliance with French and European laws and regulations, under authorized project #2016-030-5798, approved by officially accredited local institutional animal (committee n°26) and

French "Ministère de la Recherche" ethics boards.

Mice and embryos. We used the following mouse strains: 1-C57BL/6 mice from Harlan or Charles Rivers Laboratories, France, referred to as wild type (WT); 2-Lyl-1 LacZ mice, in which an in-frame insertion of the β-Galactosidase reporter encoding gene LacZ replaced the HLH domain and the entire 3' end of the Lyl-1 gene. Lyl-1 mice were genotyped as described before [START_REF] Capron | The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell function and B-cell differentiation[END_REF]. Lyl-1 LacZ/LacZ males were crossed with WT or Lyl-1 LacZ/LacZ females to respectively generate Lyl-1 WT/LacZ or Lyl-1 LacZ/LacZ embryos. This breeding scheme avoided the possible detection of FDG/Lyl-1 expression in maternally derived MΦ [START_REF] Bertrand | Three pathways to mature macrophages in the early mouse yolk sac[END_REF] in early Lyl-1 WT/LacZ embryos; 3-Cx3cr1 GFP mice [START_REF] Jung | Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion[END_REF]. Cx3cr1 GFP/GFP males were crossed with C57BL/6 to generate Cx3cr1 WT/GFP mice/embryos or to Lyl-1 LacZ/LacZ females to generate Cx3cr1 WT/GFP :Lyl-1 WT/LacZ mice/embryos. 4-The Cx3cr1 GFP/GFP :Lyl-1 LacZ/LacZ double mutant strain was 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 to C57BL/6 or Lyl-1 LacZ/LacZ females, respectively. To exclude maternal/placental cell contamination, the Cx3cr1 transgene was always inherited from the male parent. 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 [START_REF] Downs | Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope[END_REF]. E8 to E10.5 embryos were staged by somite counting and thereafter according to morphological landmarks.

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The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 21 Tissues preparation and cell counts. YS (E7.5-E10.5) and E10-FL were dissected as described before (Bertrand et al., 2005a;[START_REF] Kieusseian | Immature hematopoietic stem cells undergo maturation in the fetal liver[END_REF]. To perform cytometry analyses of the developing brain, two different protocols were applied:

1-For analyses performed at early stages (E9-E11), the whole brain were dissected and dissociated as previously described [START_REF] Alliot | Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain[END_REF] and further cleaned from surrounding tissues.

2-From E12 to adult stages, microglia were recovered following Percoll (P1644, Sigma) separation, according to [START_REF] Mildner | Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions[END_REF]. After Percoll purification, 100 µl of the cell suspension was used for cell counting and the remaining cells for flow cytometry analyses. To estimate the number of microglia per brain, the percentage of CD11b + CD45 lo F4/80 + microglia was reported to the cell count recorded for the corresponding sample.

In vitro culture.

Organ culture: YS explants were placed into plates containing "Complete OptiMEM medium", i.e.

OptiMEM with Glutamax (51985-042), 1% Penicillin-streptomycin, 0.1% β-mercaptoethanol (all from ThermoFisher) and 10% foetal calf serum (FCS; Hyclone). YS explants were maintained in organ culture at 37°C, 5% CO 2 for 1 day and are referred to as OrgD1-YS.

Clonogenic assay: Whole YS suspension or sorted cells were plated in triplicate at respectively 3x10 3 or 100-150 cells/mL in Methocult® M3234 (StemCell Technologies Inc.) always supplemented with Stem Cell factor (50ng/mL), EPO (3U/mL), IL-3 (10 ng/mL), all from Peprotech, IL-6 (10 ng/mL, a gift from Sam Burstein, Maryville IL, USA), CSF-1 (10 ng/mL) and TPO (10ng/mL, provided by Kirin Brewery, Tokyo, Japan). Cultures were maintained in a humidified incubator at 37°C, 5% CO 2 and colonies were scored at day 5 for primitive erythrocytes and day 7 for the other progenitor types.

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22

Flow cytometry, FACS-Gal, proliferation and apoptosis assays. See Sup. table 5 for the list of antibodies and dyes used throughout this study. CD31, F4/80, CD45, CD11b and cKit antibodies were used to characterize myeloid cells. Dead cells were excluded by adding 1 µg/mL 7-amino actinomycin or DAPI (Sigma) before acquisition. Acquisitions were performed on a Canto II cytometer and cell sorting using FACS-Aria III or Influx (All from BD Biosciences). Data were analysed using FlowJo (Treestar) software.

FACS-Gal assay:

This assay [START_REF] Fiering | Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs[END_REF][START_REF] Guo | Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation[END_REF], used as a reporter for Lyl-1 expression, allows the flow cytometry characterization of cells that display a β-Gal activity, using its fluorescent substrate (Fluorescein di-β-galactopyranoside (FDG) F1179; Molecular probe; Thermo Fisher).

Apoptosis assays:

For apoptosis analysis, microglia were stained with anti-CD45-PECy7, anti-CD11b-APC-eFluor®780 and anti-F4/80-APC, washed and incubated with Annexin V-FITC. 7AAD was added before acquisition.

Proliferation assay (BrdU incorporation):

Pregnant WT and Lyl-1 LacZ/LacZ females were injected with BrdU (10µM) 12 days after plug detection and sacrificed 2 hours later. Microglia were isolated and stained with CD45-PECy7, CD11b-APC-eFluor®780 and F4/80-PE antibodies. Cells were fixed, permeabilised and treated with DNase (1 hour at 37°C) according to kit instruction (BD Pharmingen No. 552598) and 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 in 4% paraformaldehyde overnight at 4°C, whole midbrains were washed in phosphate-buffered saline (PBS)/0.1M 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 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 23 (PBS+10% FCS) at room temperature (RT). Midbrains were subsequently immuno-labelled 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 mm ∅ glass coverslip. Image stacks were collected using a Leica SP8 confocal microscope. Images were processed using Imaris x 64 (version 7.7.2; Bitplane) and Photoshop 8.0 (Adobe Systems, San Jose, CA) softwares. 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 Sup. fig. 4c.

RT-qPCR analyses. Total RNA was extracted from sorted CD11b + F4/80 + CD45 low microglia using Trizol (ThermoFisher). After cDNA synthesis using a SuperScript™ VILO™ Master Mix reverse transcriptase (ThermoFisher), quantitative PCR was performed using SYBR Premix Ex TaqII (Tli RNase H Plus, Takara Bio). Reference genes were Actin, Hprt and Tubulin. Gene expressions were normalized to the value obtained from E10-YS MΦ progenitors, E12 WT Lin -Sca + cKit + (LSK) progenitors or E12 WT microglia, and relative gene expression levels were determined by the ΔΔCt method. Gene expression was considered undetectable if Ct values were >35 cycles. The sequences of the primers used are provided in Sup. table 6.

RNA-sequencing.

Sample preparation: MΦ progenitors (CD45 + CD11b + cKit + ) were sorted from E9 (MΦ Prim progenitor) and E10 (MΦ Prim + MΦ T-Def progenitors) YS pools from either WT or Lyl-1 LacZ/LacZ preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint embryos. Four biological replicates were prepared for each sample. RNA was extracted as described above.

Sample processing:

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, 50ng of total RNA sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The fragmented mRNA samples were subjected to cDNA synthesis and further converted into double stranded DNA using the reagents supplied in the kit, and 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 x 100) on Novaseq-6000 sequencer (Illumina) at Gustave Roussy genomic facility. RNA-seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-9618.

RNA-sequencing analysis:

Quality of RNA-seq reads was assessed with FastQC 0.11.7 and MultiQC 1.5 [START_REF] Ewels | MultiQC: summarize analysis results for multiple tools and samples in a single report[END_REF]. Low quality reads were trimmed with Trimmomatic 0.33 [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]. Salmon 0.9.0 tool [START_REF] Patro | Salmon provides fast and biasaware quantification of transcript expression[END_REF] was used for quantifying the expression of transcripts using geneset annotation from Gencode project release M17 for mouse [START_REF] Frankish | GENCODE reference annotation for the human and mouse genomes[END_REF]. 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 package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. The differential expression analysis in DESeq2 uses a generalized linear model (GLM) where counts are modelled 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.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint Genes have been considered differentially expressed when adjusted p-value < 0.05 and foldchange > 2.

Data were analysed through the use of Ingenuity® Pathway Analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) [START_REF] Krämer | Causal analysis approaches in Ingenuity Pathway Analysis[END_REF], Gene set enrichment analysis (GSEA; https://www.gsea-msigdb.org/gsea/index.jsp) [START_REF] Mootha | PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[END_REF][START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles[END_REF] OrgD1-YS (n=3-5, each sample containing 3-6 YS; mean ± s.e.m.; Unpaired, two-tailed t-Test). The size of the MΦ colonies and the cell morphology were similar for the 3 genotypes (data not shown). Right: The distribution of other progenitors with a myeloid potential (EMP and GM) was similar in WT, Lyl-1 WT/LacZ and Lyl-1 LacZ/LacZ E8 OrgD1-YS.

b. While all MΦ progenitors in E9-YS (left panel) expressed FDG/Lyl-1, E9.5 and E10-YS (middle panels) harboured two MΦ progenitors subsets discriminated by their FDG/Lyl-1 expression. FDG + /Lyl-1 + and FDG - /Lyl-1 -mature MΦ (CD11b + F4/80 + ) also coexisted in E10-YS (right). Lyl-1 expression in MΦ progenitors was analysed by FACS-Gal assay, using the β-Gal fluorescent substrate FDG as a reporter for Lyl-1 expression. The contour plots in WT samples indicate the level of non-specific background β-Gal activity/FDG labelling in WT samples. Representative profiles of 3 independent samples, each consisting of 3-4 YS (See the gating strategy in Sup. fig. 1a). c. MΦ Prim progenitors express Lyl-1. Upper panel: Flow cytometry profiles of WT (left) and Lyl-1 WT/LacZ (middle left) E8-YS (0-3S). CD11b + CD31 -MΦs (top gate) correspond to maternal MΦ present at this early stage (Bertrand et al., 2005). All CD11b + CD31 + MΦ progenitors (lower gate) displayed FDG/Lyl-1 expression. d. FDG/Lyl-1 positive and negative myeloid progenitors produce a distinct progeny: The type of progenitors produced by sorted Ter119-cKit + CD45 + CD11b + myeloid progenitors was determined by clonogenic assays using E9 WT and Lyl-1 WT/LacZ YS (<18 S; n=7), and E10 WT YS (n=15) in 3 independent experiments. At E10, myeloid progenitors from Lyl-1 WT/LacZ YS were subdivided into FDG/Lyl-1 negative (n=15) and positive (n=12) fractions (5 independent experiments). Samples were biological replicates comprising 6-8 YS. 100 to 150 cKit + CD45 + CD11b + cells per condition were platted in triplicate. FDG + /Lyl-1 + progenitors essentially produced MΦ colonies, while FDG -/Lyl-1 -progenitors produced also GM and G colonies, thus belonging to the transient definitive wave. e. RT-qPCR quantification of cMyb expression levels in cKit + CD45 + CD11b + MΦ progenitors sorted from WT E9-YS, WT and Lyl-1 WT/LacZ E10-YS, as well as from the FDG/Lyl-1 positive and negative fractions of MΦ progenitors from Lyl-1 WT/LacZ E10-YS. Lin -Sca + cKit + (LSK) progenitors from WT E12 FL were used as positive control. FDG + /Lyl-1 + MΦ progenitors from E10-YS expressed cMyb Low/Neg levels similar to E9-YS, which characterize the primitive YS wave. The FDG -/Lyl-1 -fraction expressed significantly higher cMyb levels, similar to LSK cells from E12-FL. cMyb expression levels, shown on a Log 2 scale, were normalized to the mean expression value obtained for WT E10-YS, considered as 1 (Unpaired, two-tailed t-Test).

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The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint f. Differentially expressed genes in MΦ progenitors (CD45 + CD11b + cKit + ) sorted from WT and Lyl-1 LacZ/LacZ YS at E9 and E10. Upper panel: Unsupervised principal component analysis (PCA) plot positioned E9 and E10 MΦ progenitors in two distinct groups, followed by segregation of WT and Lyl-1 LacZ/LacZ samples. Lower Left panel: Flow cytometry quantification of FDG + /Lyl-1 + CD11b + CD31 + MΦ progenitors from WT and Lyl-1 WT/LacZ E8-YS (0-3S). The MΦ progenitor subset was enlarged in Lyl-1 WT/LacZ YS (right; n=3). Right panel: In clonogenic assays, less than one EMP and/or GM progenitor per E8-YS was detected in WT and mutant samples, confirming that the assay was performed at a time when EMP-derived-MΦ progenitors were absent. The majority of the 25-30 colonies per YS were Ery P (60 to 80% in the 3 genotypes). The number of MΦ colonies obtained from E8-YS (0-3S) was increased in Lyl-1 WT/LacZ and Lyl-1 LacZ/LacZ compared to WT (left). Other progenitors were occasionally and randomly found in the Lyl-1 WT/LacZ preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 34 FDG + /Lyl-1 + fraction including EMPs (0.81%±0.66; n=3). (n=3-5, each sample contained 5-10 YS; plots show mean ± s.e.m.; Unpaired, two-tailed t-Test).

b. The relative expression levels (read counts) of the CD41 coding gene Itg2b is increased in Lyl-1 lacZ/lacZ MΦ progenitors compared to WT at E9 (unpaired t-Test, two-tailed).

c. Relative expression levels of transcriptions factors regulating haematopoietic progenitor emergence in

Lyl-1 LacZ/LacZ MΦ progenitors compared to WT at E9 (p-values were determined by unpaired, two-tailed t-Test).

d. GSEA pathways (Top; FDR q-Value <0.29) and GO terms (Bottom; FDR q-value <0.01) enriched in E9 Lyl-1 lacZ/lacZ compared to E9 WT MΦ progenitors. Highlighted are the pathways specifically related to embryo patterning (blue) and to the development of skeletal (green) and nervous systems (yellow). Pink arrows point to changes related to metabolic pathways. f. RT-qPCR analyses of Lyl-1 expression in A1 to A3 MΦ subsets isolated from Cx3cr1 WT/GFP brain at E10.

Lyl-1 is expressed by the 3 subsets, with levels decreasing with differentiation. Expression levels were normalized to the mean value obtained for Cx3cr1 WT/GFP YS A1 progenitors (n=3).

g. Defective differentiation of brain MΦ progenitor in Lyl-1 mutant embryos. Distribution of A1-A2 and A3 MΦ subsets in E10 brain from Cx3cr1 WT/GFP :Lyl-1 WT/WT , Cx3cr1 WT/GFP :Lyl-1 WT/LacZ and Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ embryos. The size of the whole MΦ population was similar in the three genotypes (Top panel), but Lyl-1 deficiency modified the distribution of the MΦ subsets (middle and lower panel) with an increased size of the A1 subset and a reduced A3 pool (5-12 independent analyses, each sample cumulating brains from 6-8 embryos. Plots show mean ± s.e.m.; Unpaired, two-tailed t Test).

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The copyright holder for this this version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570 doi: bioRxiv preprint 36 d. In Lyl-1 LacZ/LacZ new-borns, the cellularity of the brain was consistently lower than in WT (left), and so was the estimated microglia number (right). Plots show mean ± s.e.m.; Two tailed, unpaired t-test.

e. Kinetic evolution of Lyl-1 expression levels in WT microglia from embryonic stages to adulthood. An increased expression of Lyl-1 from embryonic stages to adulthood was also inferred from timeline RNAseq. data (Matcovitch-Natan et al., 2016) (GEO accession number GSE79812).

f. Quantitative RT-PCR analyses also point to E12 and P0 as key development stages regulated by Lyl-1.

CD11b + F4/80 + CD45 low microglia were isolated at sequential development stages. Bar graphs show the kinetic of expression of genes modified in Lyl-1 LacZ/LacZ microglia (arrowheads), normalized to the mean expression value in WT E12 microglia (n=3). Error bars indicate s.e.m. Two tailed, unpaired t-test.

g. Cx3Cr1 and Lyl-1 expression in mutant microglia. The expression level of Cx3CR1, analysed as in f, was decreased in Lyl-1 mutant at E12 (left), while Lyl-1 expression level was unmodified in CX3CR1 GFP/GFP microglia at E12 and in new-borns (right).

h. Mafb expression in mutant microglia. Mafb expression level, analysed as in f, was reduced in the microglia of Lyl-1 LacZ/LacZ new-borns. i. The expression of genes enriched in microglia and/or essential for their function are deregulated in Lyl-1 LacZ/LacZ MΦ progenitors at E9. Relative expression levels (read counts) in WT and Lyl-1 lacZ/lacZ MΦ progenitors from E9 YS (P values were determined by unpaired, two-tailed t-Test).

Figure 1 :

 1 Figure 1: Lyl-1 expression discriminates MΦ Prim from MΦ T-Def progenitors in the early YS a. Lyl-1 deficiency leads to an increased production of MΦ progenitors in the early YS: Left: Clonogenic potential of E8 OrgD1-YS cells: The production of MΦ progenitors (CFU-M) was increased in Lyl-1 LacZ/LacZ

  panel: Volcano plot of E9 WT vs E10 WT MΦ progenitors. Red and green dots indicate genes with statistically significant changes in expression level. (p-value <0.05, absolute fold change≥2.0) (NDE: not deregulated genes; DE-Up: up-regulated genes; DE-Down: down-regulated genes). g. Upper panel: Venn diagram comparing DEGs in E9 WT versus E10 WT MΦ progenitors to the EMP (Left) or MΦ signatures defined by (Mass et al., 2016) (GEO accession number GSE81774). The number and percentage of DEGs common to the EMP or MΦ signatures is shown. Lower panel: Expression profiles of the overlapping genes identified by the Venn diagram (Heatmap displays transformed log2-expression values; Unpaired t-Test, two-tailed). Note the higher expression at E10 of genes involved in erythroid (Haemoglobins: Pink arrow; Transcription factors: green arrow), as well as megakaryocytic and granulocytic-related genes (blue arrow). h. Relative expression levels of Gata1 and Spi1/PU.1, as indicated by their relative Transcripts per million kilo-bases (TPM). i. Enriched Pathways in E9 and E10 WT MF progenitors with absolute z-score ≥2, from QIAGEN's Ingenuity® Pathway Analysis (IPA). Bars: minus log of the p-value of each canonical pathway; Orange line: threshold p-value of 0.05. Ratio: genes detected/genes per pathway. j. Expression profiles of DEGs related to IFNγ and IFNβ response, identified by g:Profiler. (Heatmap displays transformed log2-expression values; unpaired t-Test, two-tailed). k. Expression profiles of DEGs related to MHC-II complex (Heatmap displays transformed log2-expression values; unpaired t-Test, two-tailed). l. Expression profiles of DEGs related to cytokine signalling (Heatmap displays transformed log2expression values; unpaired t-Test, two-tailed).

  Figure 2: a. Lyl-1 regulates the production of E8 MΦ Prim progenitors.

Figure 3 :

 3 Figure 3: The differentiation MΦ progenitors is defective in Lyl-1 LacZ/LacZ YS. a. Distribution of A1-A2 and A3 MΦ subsets in E10-YS from Cx3cr1 WT/GFP :Lyl-1 WT/WT , Cx3cr1 WT/GFP :Lyl-1 WT/LacZ and Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ embryos. While the size of the whole MΦ population is similar in the three genotypes (Top panel), Lyl-1 deficiency leads to a modified distribution of the MΦ subsets (middle and lower panel) with an increased size of the A1 subset and a reduced A3 pool (5-12 independent analyses, each sample cumulating 6-8 YS. Plots show mean ± s.e.m.; Unpaired, two-tailed t-Test). b. GSEA pathway indicates a deficit in Jak1-Stat signalling in Lyl-1 LacZ/LacZ MΦ progenitors compared to WT at E9 (NES: normalised enrichment score; FDR: false discovery rate). c. Relative expression levels (read counts) of haematopoietic markers in WT and Lyl-1 LacZ/LacZ MΦ progenitors at E9 (unpaired t-Test, two-tailed). d. Top 1 GSEA pathway indicates that the IFN signalling pathway (left) which characterize E9 MΦ Prim progenitors, and particularly Irf8 (right), is defective in Lyl-1 LacZ/LacZ MΦ progenitors (NES: normalised enrichment score; FDR: false discovery rate). e. From the 53 canonical pathways identified by IPA in the DEGs, 9 were enriched with an absolute Z score ≥ 1. Bars: minus log of the p-value of each canonical pathway; Orange line: threshold p-value of 0.05. Ratio: genes detected/genes per pathway. f. Upper panel: Venn diagram comparing the DEGs in E9 Lyl-1 LacZ/LacZ vs E9 WT to those in E10 Lyl-1 LacZ/LacZ vs E10 WT MΦ progenitors. Lower panel: Expression profiles of the DEGs common to both stages identified by the Venn comparison (Heatmap displays transformed log2-expression values; unpaired t-Test, two-tailed).

  Figure 4: a. Left panel: All MΦ progenitors from E10-Brain (Left) expressed Lyl-1, contrary to the corresponding YS (Fig. 1b right) which harbour both FDG + /Lyl-1 + and FDG -/Lyl-1 -subsets. MΦ progenitors from E10 Lyl-1 LacZ/LacZ FL (right) harboured both FDG + /Lyl-1 + and FDG -/Lyl-1 MΦ progenitors subsets. Right panel: in E10 brain, mature MΦ (CD11b + F4/80 + gate) were all FDG + /Lyl-1 + The contour plots in WT samples indicate the level of non-specific background β-Gal activity/FDG labelling in WT samples. Representative profiles of 3 independent samples, each consisting of 3-4 Brain or 8-12 E10-FL. b. Quantification of cKit + CD45 + CD11b + MΦ progenitors in E10-FL (plots show mean ± s.e.m.; Unpaired, two-tailed t-Test). c. Lyl-1 marks the entire F4/80+ microglia/BAM population from the onset of brain colonisation. The rare CD11b+ F4/80 low-neg cells present in the brain at E9 are FDG/Lyl-positive (Top Panel). Grey histograms indicate non-specific background β-Gal activity/FDG levels in WT samples. d. MΦ progenitors from E10 brain expressed cMyb levels similar to E9-YS MΦ Prim progenitors. RT-qPCR quantification of cMyb expression levels in cKit + CD45 + CD11b + MΦ progenitors sorted from WT E9-YS and from WT and Lyl-1 WT/LacZ brain at E10. Lin -Sca + cKit + (LSK) progenitors from WT E12 FL were used as positive control. cMyb expression levels, shown on a Log 2 scale, were normalized to the mean expression value obtained for WT E10-YS, considered as 1 (Unpaired, two-tailed t-Test). e. Heatmap showing the expression profiles of the DEGs in E9 WT vs E10 WT MΦ Prim progenitors that mark the development of tissue resident MΦ (Heatmap displays transformed log2-expression values; Unpaired, two-tailed t-Test).

Figure 5 :

 5 Figure 5: Lyl-1 deficiency leads to transient reductions of the microglia pool at E12 and P0-P3. a. Quantification of the microglia population in E12 and E14 brain showing the decreased size of the microglia pool at E12 and its recovery to a normal pool size at E14. Plots show mean ± s.e.m.; Two tailed, unpaired t-test. b. A reduced microglia proliferation may account for the reduced microglia number at E12, as shown by the two folds decrease (right) of BrdU-labelled cells in Lyl-1 LacZ/LacZ (middle) compared to WT (left) brains. Plots show mean±s.e.m.; Two tailed, unpaired t-test. c. At E12, Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ microglia displayed a reduced number and extent of ramifications compared to their Cx3cr1 WT/GFP counterpart. Bottom: Microglia morphology was classified into subtypes depending on the number of main ramifications (A: none, B: 2, C: 3 and D:>3). Top: Microglia deprived of ramifications predominated in Lyl-1-deficient microglia. 65 and 61 cells were respectively acquired from the midbrain of E12 Cx3cr1 WT/GFP and Cx3cr1 WT/GFP :Lyl-1 LacZ/LacZ embryos (for each genotype, brains from 12 embryos were acquired in 3 independent experiments). Microglia were identified by Cx3cr1-driven GFP expression and F4/80-APC immuno-staining. Bar=10mm. Plots show mean±s.e.m.; Two tailed, unpaired ttest.

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2020.09.28.316570	this version posted September 28, 2020.	The copyright holder for this
						,	Morpheus
	(https://software.broadinstitute.org/morpheus/)	and	Venny
	(https://bioinfogp.cnb.csic.es/tools/venny/) softwares.
	Statistical analysis. Statistical tests were performed using Prism 7 (GraphPad) Software.
	Statistical significance is indicated by the exact p-value and/or as *p < 0.05, **p < 0.01, ***p <
	0.001 and ****p < 0.0001.

preprint

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	bioRxiv preprint	doi:	; https://doi.org/10.1101/2020.09.28.316570	this version posted September 28, 2020.	The copyright holder for this
				35

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) to D. Ren, Grant Agency of the Czech Republic (GACR n°19-23154S) 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 Scolarship Council fellowships to S. Wang and D. Ren.

Author Information.

The authors declare no competing financial interests.

Correspondence should be addressed to I.G. (Isabelle.Godin@gustaveroussy.fr).

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.