Durant-Vesga Jennifer

Suzuki Nanoka

Ochi Haruki

Le Bouffant Ronan

Eschstruth Alexis

Ogino Hajime

Umbhauer Muriel

Riou Jean-François
email: jean-francois.riou@sorbonne-universite.fr

Jean-François Riou

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3

Keywords: Renal precursors, pax8 regulation, hox, kidney field, Xenopus pronephros

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Introduction

The nephron is the basic unit of vertebrate kidneys. Although renal function can be very different in animals living in fresh water and in terrestrial animals, the general organization of the nephron is remarkably conserved [START_REF] Desgrange | Nephron Patterning: Lessons from Xenopus, Zebrafish, and Mouse Studies[END_REF]. In fishes and amphibia, the pronephros is the functional kidney at larval stages. The Xenopus tadpole bears a pair of pronephroi located on each side of the body that are made of one single giant nephron [START_REF] Wessely | Xenopus pronephros development--past, present, and future[END_REF]. This nephron is filtering plasma at the level of the glomus, while filtrate collected in the nephrocoele is further transformed into definitive urine by reabsorption of ions and proteins in the tubule. Notably, the different cell types constituting the Xenopus pronephros are very similar to those of the metanephric nephron of mammals, as evidenced by the conserved expression of orthologs of genes of the solute-carrier and claudin gene families for tubular cell types [START_REF] Raciti | Organization of the pronephric kidney revealed by large-scale gene expression mapping[END_REF], and podocin or nephrin expressed in podocytes of the glomus [START_REF] White | Notch signaling, wt1 and foxc2 are key regulators of the podocyte gene regulatory network in Xenopus[END_REF]. Understanding the molecular mechanisms controlling specification of renal precursors of the Xenopus pronephros is therefore not only of interest for the study of vertebrate kidney development. It can also provide valuable information in the perspective of renal tissue engineering [START_REF] Lienkamp | Using Xenopus to study genetic kidney diseases[END_REF].

Renal precursors at the origin of the pronephros in Xenopus are grouped in a specific region of dorsolateral mesoderm arising at the onset of neurulation. This region called the kidney field is defined by the overlapping expression of pax8 and lhx1 genes [START_REF] Carroll | Synergism between Pax-8 and lim-1 in embryonic kidney development[END_REF]. Renal precursors of the kidney field also express osr1 and osr2 genes [START_REF] Tena | Odd-skipped genes encode repressors that control kidney development[END_REF]. These four different genes encode transcription factors (TF), all of which have been shown to be required for pronephric development [START_REF] Buisson | Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development[END_REF][START_REF] Cirio | Lhx1 is required for specification of the renal progenitor cell field[END_REF][START_REF] Tena | Odd-skipped genes encode repressors that control kidney development[END_REF]. We have previously shown that pax8 plays a critical role for the specification of tubular cell types. Pax8 depletion causes an inhibition of the formation of the pronephric tubule anlage at early tailbud stage, resulting in the total absence of the tubule in the tadpole. At neurula stages, expression of lhx1 and osr2 in the kidney field is not affected, but hnf1b expression is inhibited. Pax8 depletion is also causing cell proliferation defects in the kidney field in a canonical-wnt-dependent manner, potentially through the control of components of the wnt signaling pathway such as dvl1 or sfrp3 [START_REF] Buisson | Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development[END_REF].

Mechanisms controlling the emergence of the kidney field are only partially understood. Wnt11b can induce pronephric structures in unspecified lateral mesoderm explants, and could act as a potential inducer [START_REF] Tetelin | Xenopus Wnt11b Is Identified as a Potential Pronephric Inducer[END_REF]. However, retinoic acid (RA) signaling appears to be absolutely required. Its disruption causes a loss of pax8 and lhx1 expression in the kidney field, resulting in the inhibition of pronephric markers of tubule and glomus at tailbud stages, and eventually the absence of pronephros in the tadpole [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF]. Control of pax8 and lhx1 expression in the kidney field by RA is still poorly understood. Some clues indicate that control of pax8 and lhx1 involves different mechanisms. For example, up-regulation of lhx1 in response to exogenous RA is very fast both in embryos [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF] and animal caps treated with activin and RA [START_REF] Drews | The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps[END_REF], while pax8 response is delayed. Lhx1 response to RA treatment also occurs in the absence of protein synthesis, supporting the idea that lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF][START_REF] Drews | The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps[END_REF]. Pax8 expression in the kidney field involves TRPP2-dependent calcium signaling, which requires RA potentially through the control of TRPP2 trafficking to the plasma membrane [START_REF] Futel | TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus[END_REF]. However, this mechanism is likely to act in a permissive rather than in an instructive way, suggesting that more RA-dependent inputs control the expression of pax8 in the kidney field. Regulation of pax8 expression in the Xenopus embryo is taking place through a set of evolutionary conserved enhancers associated with a silencer located in the pax8 proximal promoter sequence. This silencer suppresses enhancer activity outside pax8 expression domain [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF]. How these regulatory elements are potentially used to control pax8 expression in the kidney field, and what are the inputs is largely elusive.

In order to better understand the transcriptional response to RA during pronephric precursor specification, we have performed expression profiling of RA target genes expressed in lateral mesoderm at the origin of the kidney field [START_REF] Ciau-Uitz | Genetic control of hematopoietic development in Xenopus and zebrafish[END_REF][START_REF] Dale | Fate map for the 32-cell stage of Xenopus laevis[END_REF][START_REF] Moody | Fates of the blastomeres of the 32-cell-stage Xenopus embryo[END_REF]. We have previously shown that lateral marginal zone (LMZ) explants taken at early gastrula stage can up-regulate pax8 and lhx1 genes when cultured until early neurula stage. This process is abolished when RA signaling is disrupted as the result of ectopic cyp26a1 expression (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF]. Although this explanted tissue does not experience the whole set of mesoderm patterning signals that normally occur during gastrulation [START_REF] Dosch | Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus[END_REF], it provides an excellent system to analyze how RA controls the initiation of the renal specification process in lateral mesoderm. Our previous results have shown that timed treatment with a RA receptor inhibitor interferes with pronephric development when applied during gastrula and early neurula stages, but has only limited effects on pronephric development when applied later [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF]. We have therefore analyzed the effect of RA disruption during this time window, in explanted LMZ cultured until early gastrula, mid-gastrula or early neurula stages. One prominent group of genes involved in the transcriptional response to RA corresponds to hox family members and the hox cofactor meis3. In order to evaluate the potential involvement of this set of genes in the context of a normal developing pronephros, we have further analyzed the effect of meis3 depletion in ventro-lateral mesoderm. This results in a severe inhibition of pax8 expression in the kidney field, suggesting that meis3 is part of the mechanisms acting upstream of pax8. We further provide results suggesting that meis3 is involved in the regulation of pax8 in combination with hox. First, we show that a combination of hoxb4 with pbx1 and meis3 cofactors is able to upregulate pax8 in isolated animal caps. We further show that the same combination is able to transactivate one conserved enhancer of the pax8 gene, Pax8-CNS1 [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF]. Finally, we have identified a 3.5kb region of the pax8 gene that is required for pax8 expression at neurula stage both in the kidney field and the otic placod. This region contains two conserved pax8 enhancers, Pax8-CNS1 and Pax8-CNS2. These results point to hox/meis3 are part of the complex regulation of pax8 during renal specification of the pronephros.

Materials and methods

Embryos, microinjection and microdissections

Xenopus embryos have been obtained as previously described by human chorionic gonadotropin stimulation of females and in vitro fertilization [START_REF] Futel | TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus[END_REF]. They were cultured in modified Barth's Solution (MBS) and staged according to [START_REF] Nieuwkoop | Normal table of Xenopus laevis (Daudin).: A systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis[END_REF]. All animal experiments were carried out according to approved guidelines validated by the Ethics Committee on Animal Experiments "Charles Darwin"(C2EA-05) with the "Autorisation de projet" number 02164.02.

Microinjection of morpholinos and mRNA was performed as described in 0.1x MBS containing 3% Ficoll [START_REF] Colas | Mix.1/2-dependent control of FGF availability during gastrulation is essential for pronephros development in Xenopus[END_REF]. Synthesis of capped mRNA was done as previously described [START_REF] Umbhauer | Signaling specificities of fibroblast growth factor receptors in early Xenopus embryo[END_REF]. Dissections of blastula animal caps and gastrula LMZ explants were carried out in 1xMBS in agar-coated dishes using platinum wires and loops. Culture of explants was carried out in 1xMBS. For LMZ dissection, care was taken to exclude blastocoel wall in order to avoid contamination by ectodermal tissue, as previously described (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF].

Constructs

X. laevis hoxb4.S and pbx1.L coding sequences were cloned in pCRII vector (InVitrogen) by RT-PCR amplification from NFst28 cDNA. X. laevis meis3.S coding sequence was PCR-amplified from a clone kindly provided by Dr AH Monsoro. Hoxb4.S, pbx1.L and meis3.S coding sequences were inserted into pCS2+ vectors for RNA synthesis or expression in HEK293 cells. PCR mutagenesis has been performed to introduce silent mutations downstream of the start codon in order to avoid binding to Momeis3 in rescue experiments (AUG GCA CAA AGG to AUG GCT CAG CGC). Use of p-ßSRN3-GFP [START_REF] Zernickagoetz | An indelible lineage marker for Xenopus using a mutated green fluorescent protein[END_REF] and pCS2XCyp26 [START_REF] Hollemann | Regionalized metabolic activity establishes boundaries of retinoic acid signalling[END_REF] plasmids has been previously described [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF]. For transactivation assays, X. tropicalis Pax8-CNS1 sequence was removed from the 1 pax8 -pax8(-2038)-GFP construct [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF] with bamH1 and inserted upstream of the basal promoter of pGL4.23[luc2]miniP (PROMEGA) to generate pax8CNS1-luc. Mutations of Pbx-Hox and Meis/TGIF motifs in were obtained with the QuikChange® II XL Site-Directed Mutagenesis Kit (Agilent Technologies) using primers listed in Supplementary Table 4. Momeis3 antisense morpholino meis3 5'-CCTTTGTGCCATTCCGAGTTGGGTC-3' has been previously characterized [START_REF] Der Rieden | XMeis3 is necessary for mesodermal Hox gene expression and function[END_REF].

Microarray analysis

Embryos were microinjected equatorially at the 4-cell stage into the four blastomeres with 10 nl each of a mixture of cyp26a1 mRNA (25pg/nl) and GFP mRNA (50pg/nl), or with GFP mRNA alone (50pg/nl). LMZ explants were dissected immediately after appearance of the dorsal blastoporal lip and were kept at 14°C during one hour for healing. At this stage siblings were at NFst10-10.25. Explants were further cultured for 3h at 20°C (siblings at NFst11) and then placed overnight at 14°C until siblings reached early neurula stage (NFst14). Batches of 15 explants were frozen at each stage in 300 µl RLT solution (Qiagen) for further RNA extraction with Qiagen RNeasy columns. Experiments were validated for microarray analysis after RT-qPCR control of equal proportions of endoderm (sox17ß) and mesoderm (eomes) in control and cyp26a1 gastrula stage samples, as well as minimal contamination of neural tissue (sox2). Proper RA disruption was controlled by scoring inhibition of pronephric markers pax8 and lhx1 in neurula stage samples as previously described (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF].

Expression profiling was performed on GeneChip™ Xenopus laevis Genome 2.0 Array (Affymetrix) at the Curie Institute Affymetrix platform. Probe Set analysis was conducted in R (R Core Team, 2019). Data from .CEL files (GEO accession number GSE205827) was read and normalized using the "affy" package [START_REF] Gautier | affy--analysis of Affymetrix GeneChip data at the probe level[END_REF] to create the expression set. Differential expression analysis was further performed using "limma" package [START_REF] Ritchie | limma powers differential expression analyses for RNA-sequencing and microarray studies[END_REF]. Batch effect between the three analyzed experiments was adjusted by setting eBayes function's argument robust to True [START_REF] Phipson | ROBUST HYPERPARAMETER ESTIMATION PROTECTS AGAINST HYPERVARIABLE GENES AND IMPROVES POWER TO DETECT DIFFERENTIAL EXPRESSION[END_REF]. Xenopus gene identification of Affymetrix probe Set IDs can be found at: http://ftp.xenbase.org/pub/GenePageReports/GenePageAffymetrix_laevis2.0_chd.txt [START_REF] Bowes | Xenbase: gene expression and improved integration[END_REF]. Gene annotation from Ensembl databases was retrieved using "biomaRt" R package [START_REF] Durinck | Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt[END_REF]. In several instances where gene annotation from Ensembl was missing, annotation was added manually from Xenbase gene pages (Xenbase (http://www.xenbase.org/, RRID:SCR_003280) [START_REF] Fortriede | Xenbase: deep integration of GEO & SRA RNA-seq and ChIP-seq data in a model organism database[END_REF].

Real time quantitative PCR and whole mounted in situ hybridization

Real time quantitative PCR (RT-qPCR) has been performed as previously reported (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF]. The Comparative Ct method was used to determine the relative quantities of mRNA, using ornithine decarboxylase (odc) or ß-actin mRNA as endogenous reporter. Every RNA sample was analyzed in duplicate. Data point represents the mean ± SEM of at least three independent experiments. Data were analyzed using R by paired Student's t-test. Primers used for RT-qPCR are given in supplementary table 4.

Whole mounted ISH has been carried out as previously described [START_REF] Colas | Mix.1/2-dependent control of FGF availability during gastrulation is essential for pronephros development in Xenopus[END_REF] with riboprobes for pax8, lhx1 [START_REF] Carroll | Synergism between Pax-8 and lim-1 in embryonic kidney development[END_REF], aldh1a2 [START_REF] Chen | Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos[END_REF], cyp26a1 [START_REF] Hollemann | Regionalized metabolic activity establishes boundaries of retinoic acid signalling[END_REF] and GFP [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF].

Transactivation experiments in HEK 293 cells

HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. They were plated into 12-well plates at a density of 2.5 x 10 5 cells/well. After 24 hours transfection, mixtures containing Opti-MEM® Reduced Serum Media (ThermoFisher), 500ng of each reporter vector (pax8CNS1-luc, pax8CNS1mut-lucMeis, pax8CNS1mut-lucHP1, pax8CNS1mut-lucHP2 or pax8CNS1mut-lucHP3, pax8CNS1mut-lucHP12, pax8CNS1mut-lucHP13, pax8CNS1mut-lucHP23, pax8CNS1mut-lucHP123), 5 ng of pCMV-renilla luciferase normalization vector and the transactivation vector mix (pCS2-hoxb4 and/or pCS2-meis3 and/or pCS2-pbx1 and pCS2+ empty vector-300ng total) were prepared. Subsequently, cells were transfected with these mixtures using 2.4μL of X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's protocol. Transfected cells were washed twice in PBS, followed by the addition of 200μl 1X passive lysis buffer (Promega, Madison, WI, USA). All values are shown as the mean ± SEM. All determinations were repeated at least three times. Statistical significance was using Dunn's multiple comparison test after Kruskal-Wallis test .

Transgenic reporter assay

Transgenic Xenopus embryos carrying the wild-type pax8-exon2-GFP construct (previously described as pax8-GFP fosmid) were generated as previously described [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF] by sperm nuclear transplantation method with oocyte extracts [START_REF] Hirsch | Xenopus tropicalis transgenic lines and their use in the study of embryonic induction[END_REF][START_REF] Kroll | Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation[END_REF]. A mutant construct lacking CNS1 (-CNS1) was generated from this wild-type construct by replacing the CNS1 with a kan gene cassette amplified from an EGFP-flpe-KAN plasmid using homologous recombination technique [START_REF] Delaurier | Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration[END_REF], followed by flipping out of the kan cassette by arabinose-induced Flpe recombinase [START_REF] Lee | A Highly Efficient Escherichia coli-Based Chromosome Engineering System Adapted for Recombinogenic Targeting and Subcloning of BAC DNA[END_REF]. A mutant construct lacking both CNS1 and CNS2 (-CNS1/2) was generated by replacing a region encompassing CNS1 and CNS2 with the kan gene cassette as with the -CNS1 construct. The kan cassette was not removed by the Flpe recombinase from this -CNS1/2 construct. The manipulated embryos were cultured until the late neurula stage (NFst18/19), and all normally developed embryos were subjected to in situ hybridization to examine their GFP expression with maximum sensitivity [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF].

Results

Expression profiling of RA targets in lateral marginal zone explants.

Active RA signaling is thought to be required for pronephric development during gastrulation and early neurulation, since treatment with a RAR inhibitor has only limited effects on pronephric development when it is applied after completion of gastrulation [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF]. During gastrulation, cells that will give rise to renal precursors of the kidney field are localized in the lateral marginal zone (LMZ). In order to identify RA targets potentially involved in renal precursors specification, especially those involved in its transcriptional control, we have performed expression profiling of genes modified by RA disruption in LMZ explants isolated at early gastrula stage (NFst10) (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF]. LMZ explants taken from embryos previously injected with a mixture of cyp26a1 and GFP mRNA, or from control sibling embryos injected with GFP mRNA alone were cultured for 1hr (NFst10-10.25), 3hrs (NFst11), or until early neurula stage (NFst14). Microarray analysis of three independent experiments was carried out on GeneChip™ Xenopus laevis Genome 2.0 Array (Affymetrix) (Fig. 1A). Data and metada have been deposited on Gene Expression Omnibus (GEO) repository under the accession number GSE205827.

Differential expression (DE) analysis between control and RA-disrupted samples was performed as described in Materials and Methods. It allowed to identify genes whose expression is affected by RA disruption as soon as NFst10 (Fig1B). At this stage, 426 probe sets were differentially expressed (268 up-regulated and 158 down-regulated as a result of RA disruption) with a false discovery rate (fdr) <5% (Fig. 1B). Likewise, 236 were differentially expressed at NFst11 (143 up-regulated and 93 downregulated), and 505 at NFst14 (224 up-regulated and 281 down-regulated) (Fig. 1B). Detailed results of this analysis are provided as supplementary material as "probe set analysis" tables for every stage. We further chose a log 2 fc > 0.5 (absolute value) as an arbitrary threshold for DE analysis. RA targets encoding transcription factors (TF) are listed in table 1 (positive RA targets) and supplemental table1 (negative RA targets), while a list of DE genes grouped by potential function of encoded proteins is displayed in supplemental table2. L and .S alloalleles are specified in these tables for every DE gene, but will not be further indicated in the text.

As expected, several components of the RA pathway appear as RA targets (cyp26a1, cyp26c1, dhrs3, rdh13, rara, rxra). Furthermore, dusp6 encoding MAP kinase phosphatase-3 (MKP3) that we previously identified as RA target antagonizing FGF signaling during gastrulation (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF] also appear among DE genes at Nfst10, 11 and 14, as well as fgf3 and fgf8 during gastrulation (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF]. DE at NFst14 of some identified genes was further confirmed by RT-qPCR (supplemental Fig. 1A).

Because we were principally looking at transcriptional response to RA signals, we focused on probe sets corresponding to genes encoding TF. Inspection of negative RA targets up-regulated upon RA inhibition did not provide any insight about a specific mechanism that would be acting upstream of renal master genes expression, for example inhibition of a putative specific repressor of these genes. We cannot rule out the possibility that such a mechanism may exist, but none of the identified TF expected to be repressed by RA signals may point to this. Among TF up-regulated as the result of RA disruption, and thus expected to be repressed by RA signals (supplemental table1) are genes involved in early mesendoderm formation such as vegt and eomes, as well as vegt targets sox7 and sox17b, and putative sox17 targets foxa1 and foxa2 [START_REF] Charney | A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs[END_REF]. This suggests that RA has an inhibitory effect on early transcriptional control of mesendoderm emergence, indicating that RA may control the timing of this process during gastrulation. Mesendoderm formation is certainly important for renal lineage, since it derives from mesendoderm, but we are rather considering genes responsible for renal precursor specification that takes place later. TF down-regulated as the result of RA disruption, and thus expected to be activated by RA signals (table1) are more likely involved in this process. At NFst14, they included TF known to play a central role in pronephros specification, like pax8 and lhx1 [START_REF] Carroll | Synergism between Pax-8 and lim-1 in embryonic kidney development[END_REF], which have been previously shown to be down-regulated in LMZ explants by RA disruption (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF], and osr2, irx3 and mecom whose depletion strongly affects pronephric development [START_REF] Alarcon | A dual requirement for Iroquois genes during Xenopus kidney development[END_REF][START_REF] Tena | Odd-skipped genes encode repressors that control kidney development[END_REF][START_REF] Van Campenhout | Evi1 is specifically expressed in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation[END_REF]. Several greb1,hand1,hnf1b,mespa,mespb,nr2f2,ripply3,tbx1,tcf21,znf703) have also been previously reported to be RA targets in various contexts [START_REF] Brophy | A Gene Implicated in Activation of Retinoic Acid Receptor Targets Is a Novel Renal Agenesis Gene in Humans[END_REF][START_REF] Deimling | Retinoic acid regulates anterior-posterior patterning within the lateral plate mesoderm of Xenopus[END_REF][START_REF] Gere-Becker | Retinoic acid-induced expression of Hnf1b and Fzd4 is required for pancreas development in Xenopus laevis[END_REF][START_REF] Janesick | Znf703 is a novel RA target in the neural plate border[END_REF][START_REF] Janesick | RIPPLY3 is a retinoic acid-inducible repressor required for setting the borders of the pre-placodal ectoderm[END_REF][START_REF] Moreno | Retinoic acid regulation of the Mesp-Ripply feedback loop during vertebrate segmental patterning[END_REF][START_REF] Tanibe | xCOUP-TF-B regulates xCyp26 transcription and modulates retinoic acid signaling for anterior neural patterning in Xenopus[END_REF][START_REF] Von Bubnoff | The Xenopus laevis homeobox gene Xgbx-2 is an early marker of anteroposterior patterning in the ectoderm[END_REF]. Another prominent group of DE TF corresponded to hox genes. Hox have also been reported to be RA targets in other processes [START_REF] Nolte | Hox genes: Downstream "effectors" of retinoic acid signaling in vertebrate embryogenesis[END_REF]. Hoxa1 and d1 were detected as soon as NFst10, and thus are among the earliest RA target in LMZ. At NFst11, hoxa1, a9, a10, b6, c5 and d1 appeared as DE. At NFst14 DE of hoxa2, a3, b3, b4, c5 and d1 was detected. Although not significant in DE analysis (fdr >5%) DE of hoxa1 at NFst14, was further confirmed by RT-qPCR, as well as that of hoxa3, b3, b4, c5 and d1 (supplemental Fig. 1B). Finally, the hox cofactor encoding-gene meis3 also appeared to be a prominent RA positive target of the LMZ detected at NFst11 and NFst14. This was confirmed by RT-qPCR (fig. 2A). In situ hybridization (ISH) further showed that meis3 mRNA can be detected in lateral mesoderm at neurula stage (NFst18-19), including the kidney field (Fig. 2B-D).

Depletion of the RA-target meis3 results in the inhibition of pax8 expression in the kidney field

The above data raise the question of the potential role of hox downstream of RA during pronephros precursors specification. Yet, directly challenging hox function in renal precursors to evaluate their function is expected to be difficult because of the functional redundancy between the different hox expressed in these cells. We rather attempted to inhibit the hox cofactor meis3, whose expression is also regulated by RA to address this question.

We have first analyzed the effects produced by meis3 depletion upon expression of pax8 in the kidney field. A previously characterized translation-blocking morpholino, Momeis3 (In der [START_REF] Der Rieden | XMeis3 is necessary for mesodermal Hox gene expression and function[END_REF] was microinjected into the left V2 blastomere at the 8-cell stage to target ventro-lateral mesoderm at the origin of the kidney field on the left side of the embryo (Fig. 3A). A 10ng dose was used that did not interfere with gastrulation (In der [START_REF] Der Rieden | XMeis3 is necessary for mesodermal Hox gene expression and function[END_REF]. Embryos were cultured until late neurula stage (NFst18-19), and pax8 expression was analyzed by ISH. Comparison of pax8 expression on left and right sides of injected embryos showed a severe inhibition of pax8 expression in the kidney field on the left side. Pax8 expression domain appeared much smaller, and was often fragmented into small patches (67% inhibition, n= 101, 5 independent experiments, supplemental table 3) (Fig. 3B). This effect was not observed in embryos similarly injected with a control morpholino (87% normal, n= 82, 5 independent experiments, supplemental table 3) (Fig. 3B). Furthermore, a mutated version of meis3 mRNA not targeted by Momeis3 was able to significantly rescue pax8 inhibition (Fisher's Exact Test for Count Data p-value = 5.892e-08), when co-injected with meis3 morpholino (Fig. 3C-D), demonstrating that pax8 inhibition is a specific effect resulting from meis3 depletion and further suggesting that meis3 plays an important role in the control of pax8 expression in the kidney field.

Since mesodermal expression of aldh1a2 that encodes RA-synthesizing enzyme retinaldehyde deshydrogenase has been previously shown to involve a direct regulation by Hoxa1-Pbx1/2-Meis2 in mouse embryos, and could be down-regulated by depletion of pbx1 and hoxa1 in Xenopus embryos [START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF], a possibility is that meis3 depletion actually only interferes with RA signaling, which in turn would result in pax8 inhibition. We have therefore analyzed to what extent meis3 depletion also affected aldh1a2. Meis3 depletion did not result in a strong inhibition of aldh1a2 expression in the mesoderm similar to that observed for pax8 (Fig. 4A). Aldh1a2 expression domain yet often appeared smaller on the injected side when compared to the control side (76% smaller, n=72, 4 independent experiments, supplemental table 3), while aldh1a2 expression domain was mostly unaffected by control morpholino (76% unaffected, n=75, 4 independent experiments, supplemental table 3) (Fig. 4A). Because lhx1 expression in the kidney field is very sensitive to RA signaling [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF][START_REF] Drews | The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps[END_REF], we may expect a similar effect of meis3 depletion upon lhx1. Meis3 depletion indeed only caused a limited inhibition of lhx1 expression. Lhx1 expression domain appeared narrower, when compared to control side (76% narrower, n=90, 5 independent experiments, supplemental table 3) (Fig. 4B), but was never affected in the same way as pax8. This effect was not observed in embryos injected with a control morpholino (82% unaffected, n=89, 5 independent experiments, supplemental table 3) (Fig. 4B).

Together, our observations show that meis3 depletion is resulting in a strong inhibition of pax8, suggesting that meis3 plays an important role upstream of pax8 expression in the kidney field. Meis3 might also act upstream of lhx1, but to a lower extent than for pax8. Meis3 might be involved in the mechanisms controlling RA synthesis in the kidney field by participating to the regulation of aldh1a2 expression, possibly in conjunction with hoxa1 and pbx1 [START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF]. Interference with RA synthesis resulting from meis3 depletion could explain the effect observed on lhx1. Yet, the strong effect observed on pax8 expression is unlikely to result only from this interference, thus raising the question of other inputs acting on pax8 that are also dependent on meis3.

Animal cap experiments suggest an involvement of hox/pbx1 and meis3 in the control of pax8 but not of lhx1 expression

Since we expect meis3 to act as a hox cofactor, we further investigated whether expression in isolated blastula animal caps of meis3 associated with one hox expressed in the kidney field can cause up-regulation of pax8 and lhx1. Hox are often acting as hox/pbx dimers [START_REF] Mann | Chapter 3 Hox Specificity[END_REF]. We therefore tested a combination of hoxb4 that is identified as RA target (see above) and is expressed in the kidney field (In der Rieden et al., 2010), pbx1 and meis3. Mouse orthologs of these three genes are also expressed in kidney progenitor cells from E15.5 embryos (supplemental Fig. 2). Messenger RNA mixtures were injected at the animal pole of every blastomere at the 4-cell stage and animal caps dissected when embryo reached the mid-blastula stage. Isolated caps were further cultured until sibling controls reached neurula stage NFst18-19, and were processed for RT-qPCR analysis (Fig. 5A).

The results show that hoxb4 combined with pbx1 and meis3 caused a significant up-regulation of pax8 expression. In contrast, expression of pbx1 and meis3 alone had little effect (Fig. 5B), showing that pax8 up-regulation requires hoxb4 expression and does not result from the induction of hox expression by meis3 (In der [START_REF] Der Rieden | XMeis3 is necessary for mesodermal Hox gene expression and function[END_REF]. Interestingly, hoxb4 combined with pbx1 and meis3 did not cause lhx1 up-regulation (Fig. 5C), indicating that pax8 activation does not result from a general induction of kidney field genes. We have further analyzed whether pax8 up-regulation resulting from hoxb4 co-expressed with meis3 and pbx1 co-factors is accompanied by an effect on RA metabolism. Neither expression of aldh1a2 (Fig. 5D) nor cyp26a1, which encodes RA catabolizing enzyme cyp26 was significantly affected by the expression of hoxb4, meis3 and pbx1 (Fig. 5D,E). These experiments show that combining a hox with pbx1 and meis3 cofactor can upregulate pax8 in a process that does not only rely on RA synthesis, raising the question of a direct input of hox and hox cofactors on pax8 expression.

Transactivation of Pax8-CNS1 by Xenopus hoxb4, meis3 and pbx1 suggests a direct regulation of pax8 by hox

Pax8 regulation in the late pronephric anlage at tailbud stage involves a set of conserved enhancers and a silencer located in the proximal promoter region of the gene that restricts pax8 expression to the pronephros. Four conserved enhancers (Pax8-CNS1-4) can drive reporter expression in the pronephric anlage in transgenics at tailbud stages when associated to a minimal promoter. One of these conserved enhancers, Pax8-CNS1 contains three conserved putative Pbx-Hox binding motifs and one putative Meis motif (Fig. 6A). When it is associated with the silencer region, expression is restricted to the pronephros in transgenesis assays [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF]. These data raise the question of a regulation of this enhancer by hox. We have therefore tested whether various combinations of Xenopus hoxb4, pbx1 and meis3 can transactivate Pax8-CNS1 in HEK293 cells.

HEK293 cells were transfected with a reporter construct containing Pax8-CNS1 sequence upstream of a minimal promoter followed by the luciferase coding sequence. HEK293 cells were either transfected with the reporter construct alone, or were co-transfected with the reporter construct and vectors allowing expression of the various combinations of transcription factors tested (see Materials and methods). Combination of hoxb4, pbx1 and meis3 elicited a reporter activation 10 fold higher than the basal activation observed in cells transfected with the reporter construct alone (Fig6B) (4 independent experiments, p<0.01, Dunn's multiple comparison test after Kruskal-Wallis test). This strong effect was never observed when cells were co-transfected with hoxb4, meis3 or pbx1 alone, or combinations of pbx1 and meis3, hoxb4 and meis3, or hoxb4 and pbx1 (Fig. 6B), suggesting that the three transcription factors are cooperating.

In order to identify which putative binding sites are involved during transactivation, reporter construct bearing different combinations of mutations of any of the three Pbx-Hox motifs, or of the Meis motif (Fig. 6A), were tested in transactivation assays. Neither single mutations of Pbx-Hox motifs nor mutation of the Meis motif appeared to affect reporter activation (Fig. 6C). The putative Meis motif present in Pax8-CNS1 is therefore not required for transactivation, since similar mutations of Meis binding sites have been previously shown to interfere with Meis2 interaction (Wassef et al., 2008). This suggests that either meis3 is interacting with another site of Pax8-CNS1, or that meis3 does not need to bind to DNA to exert its effect. The absence of effect of single mutations of any of the Pbx-Hox binding motives may rather suggest redundancy. We therefore tested combinations of PBX-Hox sites mutations. Combining mutations of Pbx-Hox motif 1 and 3 resulted in a strong inhibition of reporter activation (Fig. 6D) (4 independent experiments, p<0.05, Dunn's multiple comparison test after Kruskal-Wallis test), showing that these two sites are required for transactivation. In contrast, combining mutation of Pbx-Hox motif 2 with mutations of Pbx-Hox motives 1 or 3 does not have any significant effect on reporter activation, suggesting that this motif is not used. Together these results support the idea of a potential direct regulation of pax8 by hox.

A region encompassing Pax8-CNS1 and Pax8-CNS2 is critical for pax8 regulation at neurula stage

In order to identify regulatory sequences involved in pax8 regulation in the pronephric kidney field, we have performed reporter assays using a previously characterized transgene encompassing 36.5 kb of the X. tropicalis pax8 gene sequence with a GFP cassette inserted into the second exon. This transgene allows expression of a truncated pax8-GFP fusion protein. Transgenesis experiments in X. laevis embryos with this sequence have shown that expression of this transgene recapitulates endogenous pax8 expression in the developing pronephros and otic vesicle [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF]. The 36.5kb sequence includes three of the conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3 (Fig. 7A). We have first tested whether removing Pax8-CNS1 from the transgene affected its expression. Reporter expression was monitored by ISH at late neurula stage (NFst18/19). Transgenesis experiments performed with the wild-type transgene, or with the transgene lacking Pax8-CNS1 (-CNS1) showed that the absence of Pax8-CNS1 did not affect transgene expression; more than 61% of the embryos manipulated with the wild-type or the -CNS1 construct showed otic placode-and kidney field-specific GFP expression in both sides of their bodies (n = 109/177 and n = 91/149, respectively; Fig. 7A,B). It is possible that the presence of Pax8-CNS2 and/or Pax8-CNS3 may compensate for the absence of Pax8-CNS1. Previous transgenesis analyses performed with these different sequences associated to a minimal promoter have indeed shown that they all drive reporter expression in the pronephric anlage at tailbud stages [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF]. We have therefore tested whether removing Pax8-CNS1 and Pax8-CNS2 from the transgene would affect its expression. Transgenesis experiments were carried out with a transgene deleted of a 3.5kb sequence encompassing Pax8-CNS1 and Pax8-CNS2 (-CNS1/2, Fig. 7A). This resulted in a dramatic inhibition of reporter expression: none of the manipulated embryos showed GFP expression in both sides and less than 7% of them showed otic-and kidney field-specific GFP expression only in one side of their bodies (n = 9/130, Fig. 7A,B). These results point to this 3.5kb region as critical for pax8 regulation both in kidney field and the otic placode. It contains two conserved enhancers Pax8-CNS1 and Pax8-CNS2, and could be regulated by hox at the level of Pax8-CNS1.

Discussion

RA signaling is centrally involved in the specification of pronephric precursors of the kidney field by controlling the expression of master transcriptional regulators of renal development such as pax8 and lhx1 in a subset of lateral mesodermal cells [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF][START_REF] Drews | The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps[END_REF]. The present study provides evidence for a role of hox in the control of pax8 expression downstream of RA. Expression profiling of RA target genes in lateral mesendoderm during gastrulation and early neurulation allowed to identify several hox genes, including hoxa1, a3, b3, b4, c5 and d1 as RA targets, as well as meis3 that encodes a TALE homeodomain hox cofactor. We further show that targeted depletion of meis3 in the kidney field results in an inhibition of pax8 expression. Pax8 can also be induced in response to hoxb4 combined with cofactors meis3 and pbx1 in ectodermal animal caps. Supporting the view of a direct control of pax8 expression by hox, the same combination of TF is able to transactivate a previously identified pax8 enhancer, Pax8-CNS1 [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF],. Using transgenesis experiments with reporter fosmid constructs containing -24kb to +12kb of the Xenopus pax8 gene that recapitulate endogenous pax8 expression, we further show that deletion of a 3.5kb region encompassing Pax8-CNS1 and a second pax8 enhancer, Pax8-CNS2, totally abolishes reporter expression in the kidney field, showing that this region plays a critical role in pax8 regulation. Deletion of Pax8-CNS1 alone is yet not sufficient to affect reporter expression, indicating that hox may also interact with other regions of this 3.5kb sequence, and/or that other inputs are cooperating with hox during regulation of pax8 in renal precursors of the kidney field.

Hox are thought to play an important role in the control of RA synthesis in paraxial and lateral mesoderm through the control of aldh1a2 expression. Expression of aldh1a2 in paraxial and lateral mesoderm of Xenopus embryos has been indeed previously shown to be inhibited by depletion of hoxa1 and pbx1. Direct regulation of mouse Aldh1a2 was further shown to involve the interaction of hoxa1-pbx1/2-meis2 complex with a regulatory element that is required to maintain normal Aldh1a2 expression levels in mouse embryos, and a mechanism maintaining high levels of RA in mesoderm through a feed-forward loop has been proposed [START_REF] Vitobello | Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation[END_REF]. The present results show that meis3 depletion only marginally affects aldh1a2 expression, while it strongly affects pax8. This suggests that besides controlling RA synthesis, hox might also regulate pax8 in a more direct way. Although in an ectodermal context, our observations showing that a combination of hoxb4, pbx1 and meis3 is able to up-regulate pax8 in animal caps without significantly inducing aldh1a2 expression further support this view. Finally, transactivation of Pax8-CNS1 by hox and cofactors suggests a direct regulation in this process.

Although the present study points to hox in the control of pax8 downstream of RA, it is likely that pax8 expression in the kidney field does not depend only on hox, and hox may cooperate with other RA targets, as summarized in Fig. 8. Lhx1 can be a direct target of RA, since it is up-regulated in the embryo and in animal caps as an immediate-early response to exogenous RA [START_REF] Cartry | Retinoic acid signalling is required for specification of pronephric cell fate[END_REF][START_REF] Drews | The nephrogenic potential of the transcription factors osr1, osr2, hnf1b, lhx1 and pax8 assessed in Xenopus animal caps[END_REF]. Depletion of lhx1 results in a reduction of the pax8 expression domain of the kidney field, while expression of constitutively active forms of lhx1 expands this domain [START_REF] Cirio | Lhx1 is required for specification of the renal progenitor cell field[END_REF], suggesting that lhx1 may also act upstream of pax8 in renal precursors. A direct regulation of pax8 by lhx1 still remains elusive. RA can also influence pax8 expression in a more indirect way. We have previously shown that TRPP2-dependent intracellular calcium signaling is required for pax8 expression in the kidney field. Both intracellular calcium signaling and expression of the TRPP2 channel at the plasma membrane were inhibited upon RA disruption [START_REF] Futel | TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus[END_REF]. It is not known whether this increase in cytoplasmic calcium transients also results in an intranuclear calcium increase that could directly influence pax8 transcriptional control. Finally, another RA target dusp6 that encodes the enzyme mkp3 can also positively influence pax8 expression in the kidney field by inhibiting the FGF/erk pathway known to impede early pronephric development [START_REF] Colas | Mix.1/2-dependent control of FGF availability during gastrulation is essential for pronephros development in Xenopus[END_REF][START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF].

We have identified a 3.5kb regulatory region of the pax8 gene that is absolutely required for its expression at neurula stage. This region includes two previously identified enhancers, Pax8-CNS1 and Pax8-CNS2 [START_REF] Ochi | Evolution of a tissuespecific silencer underlies divergence in the expression of pax2 and pax8 paralogues[END_REF]. Reporter experiments show that a construct lacking Pax8-CNS1 is still able to drive reporter expression both in otic placode and kidney field expression domains of pax8. This suggests that additional inputs are interacting with other regions of the 3.5kb sequence, especially at the level of Pax8-CNS2. It is possible that this enhancer can be also regulated by hox. It indeed contains two conserved Hox-Pbx binding motives and one Meis motif, and bound by Hox and Meis proteins in a human acute lymphoblastic leukemia cell line (supplemental Fig. 2). As outlined above, it is also likely that TF(s) different from hox and their cofactors may be also interacting with this 3.5kb sequence.

In summary, we have shown that hox and their cofactor meis3 are transcriptional targets of RA in lateral mesoderm at the origin of the pronephric kidney field. Our results further show that meis3 is playing a central role in the control of pax8 expression in the developing renal precursors during neurulation and can directly regulate pax8 expression in association with hox/pbx. Future work will identify additional inputs cooperating with meis3 and hox/pbx to finely tune pax8 expression in developing renal precursors.

Legends to figures

Figure 1: Transcriptomic profiling of RA target genes in lateral mesoderm. A. Experimental workflow. LMZ explants are dissected at early gastrula stage from embryos previously injected with cyp26a1 and GFP mRNA (disruption of RA signaling), or GFP mRNA alone (control). Profiling is performed with batches of explants cultured until early gastrula, mid-gastrula or early neurula stages. B. Overview of DE analysis. Mean-difference plots of log-intensity ratios (differences) versus log-intensity averages (means) of Affymetrix probe sets at early gastrula, mid-gastrula or early neurula stages. Black dots correspond to probe sets with fdr > 0.05, and thus excluded from DE analysis. Dots highlighted with blue correspond to probe sets whose expression is down-regulated upon RA disruption (RA-positive targets). Dots highlighted with red are probe sets whose expression is up-regulated upon RA disruption (RA-negative targets). (hg38,chr2: 113,341,342,169),mouse (mm10,chr2: 24,539,539,292),and frog (Xenopus tropicalis;xenTro10,chr3: 125,792,792,572) with three highlighted conserved putative Pbx-Hox binding motives (blue), and one Meis (orange). Nucleotides identical in two or three sequences are shaded in grey. Mutations tested for every site are shown in red. Note that the putative binding motives mapped here show close similarity to the consensus binding sequences of Pbx-Hox (5'-ATGATTNATNN-3') or Meis (5'-TGACAGST-3') [START_REF] Chang | Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins[END_REF][START_REF] Chang | Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus[END_REF]. B. Reporter expression measured for HEK293 cells transfected with pax8CNS1-luc, and various combinations of plasmids allowing expression of Xenopus hoxb4, meis3 or pbx1 indicated in the bottom of the figure. Values are expressed as fold changes relative to HEK293 cells transfected with pax8CNS1-luc alone. A significant increase of reporter expression is only observed with cells expressing a combination of hoxb4, meis3 and pbx1. Other combinations tested do not elicit any significant increase of reporter expression. ** p<0.01Dunn's multiple comparison test after Kruskal-Wallis test). C. Analysis of mutations of single Pbx-Hox and Meis motifs in transactivation assays. HEK293 cells were transfected with the three plasmids allowing expression of hoxb4, meis3 and pbx1 combined with wild-type pax8CNS1-luc, pax8CNS1mut-lucMeis, pax8CNS1mut-lucHP1, pax8CNS1mut-lucHP2 or pax8CNS1mut-lucHP3. Values are expressed as fold changes relative to HEK293 cells transfected with wild-type pax8CNS1luc. None of the single mutation tested significantly affected transactivation. D. Analysis of multiple mutations of Pbx-Hox motifs in transactivation assays. HEK293 cells were transfected with the three plasmids allowing expression of hoxb4, meis3 and pbx1 combined with wild-type pax8CNS1-luc, pax8CNS1mut-lucHP1+2, pax8CNS1mut-lucHP1+3, pax8CNS1mut-lucHP2+3 or pax8CNS1mut-lucHP1+2+3. Values are expressed as fold changes relative to HEK293 cells transfected with wild-type pax8CNS1-luc. Combining mutation of Pbx-Hox 1 and 3 motifs results in a strong decrease of reporter expression, pointing to these two sites as active hox-Pbx binding sites. * p<0.05 (Dunn's multiple comparison test after Kruskal-Wallis test). [START_REF] Cirio | Lhx1 is required for specification of the renal progenitor cell field[END_REF] (3), and a yet unidentified mechanism dependent on RA-dependent intracellular calcium signaling [START_REF] Futel | TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus[END_REF] (2). The FGF/erk pathway was previously shown to inhibit pax8 expression [START_REF] Colas | Mix.1/2-dependent control of FGF availability during gastrulation is essential for pronephros development in Xenopus[END_REF] (5) and can be negatively regulated by MKP3 that is encoded by dups6, another RA target (Le [START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF])(this study) (4). ER: endoplasmic reticulum.

Aknowledgements

We thank S. Authier, A. Bois and E. Manzoni for excellent technical assistance in the maintenance of the Xenopus animal facility, and I. Buisson for help with in situ hybridization. We are also extremely grateful to D. Gentien at Genomics platform at Curie Institute for Affymetrix microarray analysis, as well as to C. Antoniewski at the ArtBio bioinformatics platform of IBPS for transcriptomics data interpretation. We also thank Dr AH Monsoro for the X. laevis meis3 clone.

Figure 2 :

 2 Figure 2: Targeted meis3 depletion results in the inhibition of pax8 expression in the kidney field. A. RT-qPCR analysis of meis3 expression in LMZ explants in response to RA depletion. Expression of meis3 is shown as relative expression to odc. Meis3 expression appears to be dependent on RA from midgastrula stage onward (NFst11) and at early neurula stage (NFst14). * p<0.05, ** p<0.01 (paired Student's t-test). Error bars correspond to SEM. B-D. ISH with meis3 antisense probe. Late neurula stage (NFst18). Lateral (B) and dorsal (C) views. D. Embryo bisected according to the dotted line shown in C. Meis3 is expressed in dorsal and lateral mesoderm (arrowheads), including the kidney field (KF). s: somite.

Figure 3 :

 3 Figure 3: Targeted meis3 depletion results in the inhibition of pax8 expression in the kidney field. A. Morpholinos are injected in the V2 blastomere at the 8-cell stage to target the kidney field on the left side of the embryo. Injected embryos are cultured until late neurula stage for ISH analysis of pax8 expression. B. Representative injected embryos. Views of dorsal region, left and right sides of the same embryos are shown. White arrowheads indicate kidney field expression and black arrowhead expression in the otic placode. Pax8 expression is strongly inhibited in the kidney field on the left side of embryos injected with Momeis3 morpholino. It is almost absent or fragmented into small patches. A control morpholino (Cmo) has no effect. C, D. Rescue experiments with a mRNA encoding a version of meis3 transcript not targeted by Momeis3. C. Representative examples of meis3-depleted and meis3 mRNA rescued embryos. D. Cumulative results from 3 independent experiments.

Figure 4 :

 4 Figure 4: Targeted meis3 depletion has a moderate effect upon aldh1a2 and lhx1 expression in the kidney field. A, B. Representative injected embryos showing aldh1a2 expression (A) or lhx1 expression (B).A. Adh2a1 expression is very dynamic and can vary from one embyo to the other. Yet comparing Momeis3 injected left side with control right side shows that expression domain in lateral mesoderm (arrowheads) is smaller on the injected side. This mild effect is not observed on embryos injected with a control morpholino (Cmo), although Cmo can slightly affect expression. B. Lhx1 expression is moderately inhibited as a result of Momeis3 injection. Lhx1 expression domain appears narrower. This is not observed after injection of Cmo, although Cmo can also slightly affect lhx1 expression.

Figure 5 :Figure 6 :

 56 Figure 5: Combined expression of hoxb4, meis3 and pbx1 results in pax8 up-regulation in animal cap assays, but not of lhx1. A. Mixtures of mRNA encoding Xenopus hoxb4, meis3 and pbx1 are injected into the four blastomeres of 4-cell stage embryos close to the animal pole. Animal caps are dissected at mid-blastula stage and cultured until siblings reached late neurula stage, and are processed for RT-qPCR analysis. B-E. RT-qPCR analyses. Expression of the analyzed genes is shown as relative expression to odc. Error bars correspond to s.e.m. B. Expression of pax8 is up-regulated in caps expressing hoxb4 combined to meis3 and pbx1. Expression of meis3 and pbx1 alone has little effect on pax8 expression. The same combination of hoxb4, meis3 and pbx1 neither significantly affect lhx1 expression (C), nor RA pathway components aldh1a2 (D) and cyp26a1 (E). * p<0.05 (paired Student's t-test) Figure 6: Transactivation of Pax8-CNS1 by Xenopus hoxb4 combined with meis3 and pbx1. A. Alignment of pax8-CNS1 sequences from the human (hg38, chr2: 113,341,342,169), mouse (mm10, chr2: 24,539,539,292), and frog (Xenopus tropicalis; xenTro10, chr3: 125,792,792,572) with three highlighted conserved putative Pbx-Hox binding motives (blue), and one Meis (orange). Nucleotides identical in two or three sequences are shaded in grey. Mutations tested for every site are shown in red. Note that the putative binding motives mapped here show close similarity to the consensus binding sequences of Pbx-Hox (5'-ATGATTNATNN-3') or Meis (5'-TGACAGST-3')[START_REF] Chang | Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins[END_REF][START_REF] Chang | Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus[END_REF]. B. Reporter expression measured for HEK293 cells transfected with pax8CNS1-luc, and various combinations of plasmids allowing expression of Xenopus hoxb4, meis3 or pbx1 indicated in the bottom of the figure. Values are expressed as fold

Figure 7 :

 7 Figure7: A genomic region encompassing the CNS1 and CNS2 is required for otic placode-and pronephric kidney field-specific expression of pax8. A. GFP reporter constructs used for transgenesis experiments in X. laevis embryos (left) and bar graphs summarizing GFP expression patterns in the resulting embryos (right). The bar graphs show ratio of embryos showing distinct otic placode-and kidney field-specific expression in both sides (scored as the Category 1), no or faint expression in one side (Category 2), and no expression in both sides (Category 3). B. Representative embryos showing GFP expression scored as the Category 1, Category 2, and Category 3; dorsal view with anterior side to the top. Black and white triangles indicate expression in the otic placode and kidney field, respectively.

Figure 8 :

 8 Figure 8: Working model based on present data and previously published results (Cartry et al., 2006; Cirio et al., 2011; Colas et al., 2008; Futel et al., 2015; Le Bouffant et al., 2012) illustrating the mechanisms that control pax8 expression in the kidney field. The present data (1) show that meis3 and hox are RA targets, and are involved in the direct regulation of pax8. They are cooperating with other RA-dependent inputs such as lhx1[START_REF] Cirio | Lhx1 is required for specification of the renal progenitor cell field[END_REF] (3), and a yet unidentified mechanism dependent on RA-dependent intracellular calcium signaling[START_REF] Futel | TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus[END_REF] (2). The FGF/erk pathway was previously shown to inhibit pax8 expression[START_REF] Colas | Mix.1/2-dependent control of FGF availability during gastrulation is essential for pronephros development in Xenopus[END_REF] (5) and can be negatively regulated by MKP3 that is encoded by dups6, another RA target (Le[START_REF] Bouffant | Retinoic aciddependent control of MAP kinase phosphatase-3 is necessary for early kidney development in Xenopus[END_REF])(this study) (4). ER: endoplasmic reticulum.

Funding: This work was supported by grants from CNRS and Sorbonne Université. JDV was financed by a 2017-2019 "Contrat Doctoral" from the doctoral school "Complexité du Vivant". This work was also supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Grant No. 19K06689 to H. Ogino). Xenopus tropicalis was provided by Hiroshima University Amphibian Research Center through National BioResource Project (NBRP) of MEXT.

Legend to supplemental figure 2:

Co-expression of Pax8, Hoxb4, Meis3, and Pbx1 in mouse kidney progenitor cells. Single cell RNA-seq data of E15.5 mouse embryonic kidney [START_REF] Lawlor | Nephron progenitor commitment is a stochastic process influenced by cell migration[END_REF](GEO accession: GSM3330879) were visualized by UMAP analysis at PanglaoDB [START_REF] Franzén | PanglaoDB: a web server for exploration of mouse and human single-cell RNA sequencing data[END_REF]( https://panglaodb.se/index.html). Cells expressing Pax8, Hoxb4, Meis3, and Pbx1 are colored brown, respectively. Putative kidney progenitor cells expressing Pax8 were circled in blue.

Legend to supplemental figure 3: pax8-CNS2 may be regulated by Hox and Meis proteins. A. Alignment of pax8-CNS2 sequences from the human (hg38, chr2: 113,324,980-113,325,232), mouse (mm10, chr2: 24,521,500-24,521,747), and frog (Xenopus tropicalis;xenTro10,chr3: 125,795,795,890) with two highlighted conserved putative Pbx-Hox binding motives (blue), and one Meis (orange). These putative binding motives show close similarity to the consensus binding sequences of Pbx-Hox (5'-ATGATTNATNN-3') or Meis (5'-TGACAGST-3'). Nucleotides identical in two or three sequences are shaded in grey. B. ChIP-seq analysis indicates that MEIS1 and HOXA9 bind to human CNS2 in an acute lymphoblastic leukemia cell line (SEM). The ChIP-seq mapping data collected in ReMap2020 database [START_REF] Hammal | ReMap 2022: a database of Human, Mouse, Drosophila and Arabidopsis regulatory regions from an integrative analysis of DNAbinding sequencing experiments[END_REF] was shown with schematic Multiz alignments of the human, mouse and X. tropicalis CNS2 sequences on UCSC Genome Browser [START_REF] Kent | The Human Genome Browser at UCSC[END_REF].


Jianwei W Qiao

Shuai Zhang

Xinquan Q Zhou

Prof. Z Weibin B G Chen

Romain Gautier

Zhiguo Xia

Prof. Z G Xia

Prof R Gautier

Near-infrared light-emitting diode utilizing europium-activated calcium oxide phosphor with external quantum efficiency of up to 54.7%

Keywords: near-infrared luminescence, phosphor, light-emitting diodes

Near-infrared (NIR) luminescence materials with broadband emissions are necessary for the development of light-emitting diodes (LEDs) based light sources. However, most known NIR emitting materials are limited by their low external quantum efficiency. Here, we report how the photoluminescence quantum efficiency of europium-activated calcium oxide (CaO:Eu) NIR phosphor can be significantly improved and stabilized at operating temperatures of LEDs. A carbon paper wrapping technology was innovatively developed and used during the solid-state sintering to promote the reduction of Eu 3+ into Eu 2+ . In parallel, the oxygen vacancies in the CaO lattice were repaired utilizing GeO 2 decomposition. Through this process, a record high external quantum efficiency of 54.7% at 740 nm is obtained with a thermal stability greatly improved from 57% to 90% at 125 °C. The as-fabricated NIR-LEDs reach a photoelectric efficiency of 100 mA@23.4% and an output power of 100 mA@319.5 mW, which are also the record values reported so far. This discovery of high-performance phosphors will open new research avenues for broadband NIR LEDs light sources in a variety of photonics applications.

Introduction

Near-infrared (NIR) light sources play important roles in various crucial fields, such as biomedical imaging, remote sensing, night vision and food monitoring, etc.. [1] While organic fluorophores, quantum dots, polymers and metal halides have shown benefits as NIR emitters, they so far failed to produce efficient NIR light sources because of their stability issues, low photoluminescence quantum yield (QY) and/or narrowband emissions. [2] In the light of high stability and low fabrication cost, high QY and tunable emission, inorganic luminescent materials show a great potential in the fabrication of highly efficient NIR phosphor-converted light-emitting diodes (LEDs) with broadband emissions. [3] Recently, Cr 3+ activated inorganic solid-state phosphors have attracted much attentions due to the broadband NIR emission. [3][4] However, the low absorption strengths of the parity forbidden d-d transitions of Cr 3+ activators lead to low external quantum efficiency (EQE).

For example, Ca 3 Sc 2 Si 3 O 12 :Cr 3+ and Na 3 ScF 6 :Cr 3+ possess a very high internal quantum efficiency (IQE) of 92.3% and 91.5% respectively, but low EQE values of 25.5% and 40.8% fail to meet the requirement of practical applications. [5] Higher EQE values have been also obtained in some NIR phosphors, such as LaMgGa 11 O 19 :Cr 3+ (42.5%) and SrGa 12 O 19 :Cr 3+ (45%), but EQE still remain lower than 50%. [6] In contrary, Eu 2+ activated solid-state phosphors with the parity-allowed d-f emission are normally endowed with strong absorption and high EQE, and have been commercialized for solid-state lighting. [7] Unfortunately, seldom Eu 2+ -doped NIR emitters have been discovered, and the existed emitters exhibit much lower emission efficiency (EQE < 15%) and weaker thermal stabilities. [1d, 8] Therefore, developing high efficiency and high thermal stability NIR phosphors has become a crucial challenge for the ongoing developments of broadband NIR-LED light sources.

To reach such requirements of high efficiency and high stability for NIR Eu-doped phosphors, two important parameters must be considered: (i) the formation of luminescence "killer" Eu 3+ should be prevented, [9] and (ii) the structural rigidity as well as the construction of defects should be controlled. [10] Europium-activated calcium oxide phosphor exhibiting a rigid structure, a binary oxide composition and a NIR emission appeared to be of interest.

However, the coexistence of the Eu 2+ and Eu 3+ centers in this material is known to be responsible of its low quantum efficiency. [11] Here, we succeed to improve simultaneously the efficiency and thermal stability of CaO:Eu NIR phosphor to reach the commercial application standards. By introducing carbon paper or using a graphite crucible to further optimize the reduction process under N 2 +H 2 gas, more Eu 3+ luminescent killers can be reduced into beneficial Eu 2+ emission centers. Furthermore, GeO 2 addition during the synthesis effectively eliminates the oxygen vacancies in CaO:Eu due to the insertion of O 2-ions from the GeO 2 decomposition. These conditions finally lead to an unprecedent improvement of the EQE by three times from 17.9% to 54.7%, and a giant enhancement of the thermal stability of about 33% for the treated material at 125 °C compared with the untreated materials. We further proved that the defect elimination mechanism also accounted for the improvement of the thermal stability. Such results suggest the general possibility to design new doped phosphors for emerging high-power applications. [7a, 10c] The design strategies proposed here can be extended to develop other europium-activated inorganic phosphors for efficient and highpower LED light source applications.

Results and discussion

As shown in Figure 1a, the as-prepared CaO:Eu phosphor via the optimized technique exhibits a broad emission band centered at 740 nm under 470 nm excitation, attributed to the characteristic 4f 6 5d 1 →4f 7 transition of Eu 2+ . The corresponding photoluminescence excitation (PLE) spectrum monitored at 740 nm contains two bands peaking at 245 and 470 nm, which are ascribed to the 4f 7 ( 8 S 7/2 )→4f 6 5d(T 2g ) and 4f 7 ( 8 S 7/2 )→4f 6 5d(E g ) transitions respectively. [11] The PL intensity of sample wrapped with carbon paper or placed in graphite crucible (marked as CaO:Eu@C) shows a significant enhancement compared with the PL intensity of the initial CaO:Eu phosphor. Furthermore, the integrated PL intensity of GeO 2 treated sample (marked as CaO:Eu@C+GeO 2 ) is more than 2.7 times higher than that of the untreated sample. In addition, the bulk color of CaO:Eu turns from light purple to dark purple under natural light illumination. These results suggest that enhancing the reduction via carbon paper wrapping technology and adding GeO 2 can be acted as an effective approach to increase the luminescence intensities of CaO:Eu. As shown in the Figure S1a (Supporting Information), the structure remains invariable, as all the diffraction peaks are well consistent with the calculated patterns of CaO. Rietveld methods used on powder XRD data also demonstrate that the GeO 2 addition does not modify the phase, even at a high concentration of 8% (Figure S1b).

Figure 1b shows the IQE, EQE and absorption efficiency (Abs) of CaO:Eu and CaO:Eu@C+xGeO 2 (x = 0-6%) phosphor, respectively. As one can see, all the samples possess a relatively high absorption efficiency at around 70%, which coincides with the values measured from the diffuse reflectance spectra (Figure S2a) and helps to obtain high EQE values. Obviously, the IQE and EQE values increase for the samples treated by carbon and GeO 2 , and reach a maximum at x = 4%. The EQE value (54.7%) of CaO:Eu@C+4%GeO 2 is about 3-fold higher than that of CaO:Eu (17.9%) counterpart. In particular, CaO:Eu@C+4%GeO 2 exhibits distinct advantages in both Abs and EQE values compared with the currently reported most efficient NIR phosphors, such as Ca 3 Sc 2 Si 3 O 12 :Cr 3+ , Na 3 ScF 6 :Cr 3+ and LaMgGa 11 O 19 :Cr 3+ , as shown in Figure 1c and Table S1 (Supporting Information). [5][6] For the purpose of getting deep insights into the EQE enhancement mechanism, the valence state of Eu was analyzed in detail. From the Eu-L3 edge X-ray absorption near-edge structure (XANES) spectra in Figure S3a, all the samples contain two peaks centered at 6974 and 6983 eV, ascribing to the 2p 3/2 → 5d transition of Eu 2+ and Eu 3+ , respectively. [9] The relative intensity of Eu 2+ absorption band increased obviously after carbon treatment and is further enhanced with GeO 2 addition. Accordingly, the high resolution XPS spectra are recorded in the energy region of the Eu-3d transition and presented in Figure S3b. This range consists of four peaks, which are derived from the 3d state spin-orbit splitting of both Eu 3+ and Eu 2+ ions into 3d 5/2 and 3d 3/2 . The peak at the binding energy of 1134, 1164 eV and 1124, 1154 eV correspond to the Eu 3+ and Eu 2+ ions, respectively. Similar change trend is observed for the XPS intensity of Eu 2+ and Eu 3+ , which once again confirms the effective reduction of Eu 3+ to Eu 2+ . Based on above analyses, the increase of Eu 2+ /Eu 3+ ratio in the CaO:Eu@C+4%GeO 2 phosphor is the major reason for the improved EQE, and such a schematic model is partially demonstrated in Figure 1d.

Since the decay time is directly proportional to the luminescence efficiency, the decay curves monitored at 740 nm are measured and presented in Figure S4 under pulse laser excitation at 450 nm. [12] All the decay curves can be well-fitted by the bi-exponential functions: [13] (1)

where A 1 , A 2 is the corresponding fitting constant; I is luminescence intensity; are lifetimes for the fast and slow components; t is time. The details of fitting results are listed in Table S2. The decay time reveals an increasing tendency for CaO:Eu (0.8 μs), CaO:Eu@C (0.99 μs) and CaO:Eu@C+4%GeO 2 (1.47 μs) phosphors, in agreement with the trend of emission intensity in Figure 1a. It is worth noting that only one type of Eu 2+ luminescent centers existed in CaO:Eu lattice, while the decay curves were well fitted by bi-exponential functions. This mismatch between the numbers of luminescent centers and fitting index is due to the additional nonradiative transitions originated from lattice thermal vibration participating in the luminescence process. Especially, the sharp increase in lifetime for CaO:Eu@C+4%GeO 2 sample is attributed to the restraint of nonradiative transition process, which will be discussed in detail in the thermal quenching section. [12] Another key assessment index for an excellent phosphor is the thermal quenching (TQ)

behavior. Figure 2a shows the temperature dependent integrated emission intensity for the CaO:Eu, CaO:Eu@C and CaO:Eu@C+4%GeO 2 phosphors measured at temperatures ranging from 25 to 125 °C. The PL intensity of all the samples decreases with increasing temperature due to the TQ effect. It should be noted that both CaO:Eu and CaO:Eu@C exhibit a similar poor thermal stability. This result indicates that strengthening the reducing atmosphere with carbon paper or graphite crucible alone can hardly improve the thermal stability of CaO:Eu.

By contrast, the thermal stability of CaO:Eu@C+xGeO 2 (x = 1%-6%) samples are greatly improved, as shown in Figure 2b and Figure S5. In particular, the integrated intensity of CaO:Eu@C+4%GeO 2 remains 90% at 125 °C of the initial intensity at room temperature.

Such superior thermal stability is conducive to its application in high-power NIR light source.

According to the thermal ionization model proposed by Dorenbos, the thermal stability is decided, in great extent, by the energy barrier ΔE between the conduction band and 5d level of Eu 2+ . [14] Hence, the variation of bandgap is of first importance. Figure 2c shows the plots of (αhν) 2 versus photo energy hν based on the diffuse reflectance spectra of the materials (Figure S2b). The bandgaps of all the samples determined by the transformed Kubelka-Munk function remain almost unchanged with the value of about 3.86 eV. [15] This result is expected because it can be inferred from the fact that Ge 4+ ions do not enter the CaO lattice. In addition, the same emission peaks at 740 nm (Figure 1a) indicates that the 5d energy level of each sample does not change. Therefore, another quenching mechanism must be considered. Recent studies revealed that energy transfer from the thermally activated defect levels to the Eu 2+ 5dband can enhance thermal stability. [7a, 10c] This model involves two basic factors: defect level and thermoluminescence. As such, DFT calculations were performed to determine the defect types in CaO:Eu phosphor, as illustrated in Figure 2d and Table S3. Such calculations show that oxygen vacancies are easily formed due to a negative formation energy (-3.27 eV) of V O , while the defect V Ca can be hardly generated with a formation energy of 5.69 eV.

Moreover, the strong electron paramagnetic resonance (EPR) signals observed around g = 1.996 further approve the existence of V O (Figure 2e). [8b, 16] Nevertheless, no spectral signal was detected from both the 2-and 3-dimensional thermoluminescence (TL) measurement for the selected samples, as shown in Figure S6. These results suggest that the defect levels do not assist the thermal stability for the CaO:Eu phosphor.

As exhibited in Figure 2e, the intensity of EPR signal for CaO:Eu@C+4%GeO 2 , which shows the best thermal stability, is significantly weaker than the ones of CaO:Eu and CaO:Eu@C (Figure 2a). Compared with the EPR spectra of CaO:Eu@N 2 prepared in N 2 atmosphere, one can deduce that the strong reducibility of carbon and H 2 /N 2 flowing atmosphere at high temperatures leads to the formation of V O . The formation of similar oxygen defects induced by treatments under reducing atmosphere were also observed for other phosphors such as BaMgSiO 4 :Eu. [17] Paradoxically, the reducing atmosphere not only contributes to Eu 3+ →Eu 2+ conversion but also introduces oxygen defects simultaneously.

With our material, this contradiction is skillfully solved by GeO 2 addition. O 2-ions can be introduced during GeO 2 decomposition by the following reaction: 2GeO 2 (s) → 2GeO (s) + O 2 (g), and GeO (g) + H 2 (g) → Ge (s) + H 2 O (g). [18] The O 2-ions from GeO 2 decomposition effectively compensate for the loss of oxygen ions, leading to the decrease of V O (Figure 2e and Figure 1d). Moreover, the GeO 2 decomposition occurring at high temperatures will also lead to the formation of Ge metal (Figure S7a), suggesting that Ge 4+ ions do not enter CaO host lattice. Thus, the cell volumes and bond lengths of [CaO 6 ] are maintained nearly unchanged with the increasing of GeO 2 amount (Figure S7b). This view is further confirmed by the Ge3d XPS spectrum (Figure S8a), elemental mapping images (Figure S8b) and DFT calculations (Table S3). The details of the Rietveld refinements for CaO:Eu@C+xGeO 2 (x = 0-8%) samples are exhibited in Figure S9, S10 and Table S4-7.

Based on the above results, a simplified schematic (Figure 2f) was drawn to explain the mechanism of TQ. The bandgap of CaO was predicted to be 3.97 eV by DFT calculation, close to the measured value (3.86 eV). The removal of O atom creates a defect energy level (V O ∼2.36 eV) within the bandgap (Figure 2d). Under 470 nm excitation (2.63 eV), the electrons are promoted to 5d band, and then emits in the NIR region after a Stokes shift.

When the temperature increases, electrons partly transfer to conduction band (CB) and cause TQ [process (1)]. Some electrons will be captured by the oxygen defect levels and then thermally ionized to CB under electron-phonon interaction [process (2)]. As for CaO:Eu@C+4%GeO 2 sample, the TQ process (2) was effectively suppressed due to the decrease of defect concentration, thus leading to the excellent thermal stability and long decay time. Not only GeO 2 discussed here but also SnO 2 have a similar positive effect on the improvement of emission intensity and thermal stability of CaO:Eu. As depicted in the Figure S11, all the XRD patterns of SnO 2 treated samples are well indexed to the CaO phase, and the emission intensity of CaO:Eu@C+1.5%SnO 2 was enhanced about 1.6 times comparing to the PL intensity of CaO:Eu@C. Moreover, about 84% of the NIR emission at room temperature can be maintained at 125 °C. Therefore, such a method utilizing the characteristics of GeO 2 and SnO 2 decomposition during a high temperature reduction atmosphere can be generally adopted and it helps to optimize the thermal stability and the EQE of Eu 2+ -activated phosphors.

The practical feasibility of high-performance CaO:Eu@C+4%GeO 2 phosphor for photon conversion in broadband NIR light sources has been tested and realized based on a commercial blue LED (455 nm) chip. Figure 3a shows the emission spectra of the fabricated NIR-LED under different drive currents in the range of 20-300 mA. The emission peaks are stable and the intensity grows continuously with the increase of the drive currents. To evaluate the performance of the CaO:Eu@C+4%GeO 2 phosphor in the NIR LED light source, the electricity-to-NIR power conversion, the electricity-to-blue power conversion and NIR output power were achieved and shown in Figure 3b. When the drive current increases from 20 to 300 mA, the NIR output power raises from 70 to 766 mW, while the electricity-to-NIR efficiency gradually decreases due to the "efficiency droop" of the InGaN blue chip. In particular, the NIR output power and photoelectric efficiency under current of 100 mA (319.5 mW@23.4%) and 300 mA (766.1 mW@17.1%) are the highest values reported so far, as exhibited in the comparison in Table S1. To highlight the application prospect of highefficiency and thermally stable CaO:Eu@C+4%GeO 2 phosphor in the field of night vision, sapphire, UV adhesives, beam homogenizer, condenser and four high-power blue LEDs (455 nm, 10 W) were used to fabricate a high-power NIR light device. A 5.2 W NIR light source was obtained (Figure S12 and Figure 3c). Landscape photography taken with ordinary cameras in the daytime are clear (Figure 3d), but neither visible light cameras nor NIR cameras can capture clear photos in a dark night (Figure 3e). In contrast, with the aid of the as-prepared NIR light source, a clear black-and-white photo of the pavilion at a distance of 65 meters was successfully taken by using the same near-infrared camera (Figure 3f). These results demonstrate that CaO:Eu@C+4%GeO 2 phosphor with high luminous efficiency and thermal stability can be successfully used for high-power NIR light sources.

Conclusion

In summary, the external quantum efficiency and thermal resistance of europium activated calcium oxide phosphor have been greatly improved and reach record values. The carbon paper wrapping strategy contributed to optimize the formation of Eu 2+ centers leading to high quantum efficiency. In parallel, the O 2-ions formed in-situ from GeO 2 decomposition further decrease the concentration of oxygen vacancies, and result in the improvement of thermal stability from 57% to 90% at 125 °C. High EQE (54.7%) and photoelectric efficiency (100 mA@23.4%) make it possible to be used in high-power NIR devices. The general effect observed in SnO 2 treated samples certified the universality and effectiveness of this strategy.

Our work will motivate future research on optimizing the luminescent properties of doped solid-state phosphors, showcasing the potential of this strategy in high-power broadband NIR devices.

Experimental Section/Methods

Materials and Preparation: CaO:Eu and CaO:Eu@C+xGeO 2 phosphors were synthesized by the traditional high temperature solid state reaction method. CaCO 3 (99.99%), Eu 2 O 3 (99.99%) and GeO 2 (99.99%) were used as starting materials and weighed, grounded in stoichiometric proportions. Then the mixture was divided into two parts, and one part transferred into an alumina crucible directly, another part wrapped with carbon paper or place in graphite crucible. Subsequently, the powders were sintered at 1400 °C for 6 h in a 80%N 2 /20%H 2 atmosphere. After cooling down naturally to room temperature, the products were reground into fine powders for further investigations.

Characterization: X-ray powder diffraction (XRD) patterns were measured in the range of 5 o -120 o on a D8 Advance X-ray diffractometer (XRD, Bruker Corporation, Germany). The structure refinement was performed by TOPAS 4.2 software. [19] The photoluminescence (PL), photoluminescence excitation (PLE) spectra and quantum efficiency (QE) were measured by a FL1000 fluorescence spectrophotometer (Edinburgh Instruments, UK) with visible and NIR PMT detectors. The temperature-dependent spectra and decay curves were also collected by FL1000 equipped with a temperature control instrument (OXFORD) and a 450 nm pulse laser diode, respectively. The diffuse reflectance spectra were recorded on a Hitachi UH4150 UVvis-NIR spectrophotometer. The morphology and microstructure of phosphors were investigated using a field-emission scanning electron microscope (SEM, JEOL JSM-6510A).

Thermoluminescence (TL) spectra were measured using a TOSL-3DS instrument (Ai-di-ruisheng Company, China) equipped with a Hg lamp as the UV radiation source. Electron paramagnetic resonance (EPR) spectra were measured at room temperature using an EMXplus X-band spectrometer (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) spectra were recorded by a VG ESCALABMK II electron spectrometer with a Mg Kα (1200 eV) line as an excitation source.

WLED Fabrication: The low-power NIR pc-LEDs were fabricated by encapsulating the mixture of CaO:Eu@C+4%GeO 2 phosphor and transparent epoxy resin on blue (455 nm, 3

W) LED Chips. The emission spectra and photoelectric properties of the fabricated pc-LEDs were collected by a NOVA Laboratory Class Spectrometer equipped with Morpho 3.2 software. For high-power NIR LED application, the mixture of CaO:Eu@C+GeO 2 phosphor and transparent epoxy resin was firstly coated on the surface of sapphire, then the sapphire were fixed onto four high-power blue LED (455 nm, 10 W). The photoelectric properties of the fabricated high-power NIR LED were measured on an integrating sphere spectroradiometer system (ATA-1000, Ever fine).

Computational method: In order to study the structure well, we used the Vienna ab initio simulation package (VASP) codes to calculate the lattice structure and electronic structure of CaO based on the density functional theory (DFT). [20] The generalized gradient approximation with the Perdew-Burke-Ernzerhof functional was employed as the exchange-correlation potential. To evaluate the Ge, V Ca and V O distribution in GaO, a 2 × 2 × 2 CaO supercell was adopted. The cut-off energy E cut of 400 eV was used throughout all the calculations. A gamma-centered 4 × 4 × 4 k-mesh grid in the Brillouin zone was applied to determine the self-consistent charge density using the Monkhorst-Packscheme. The crystal lattice was fully relaxed until the atomic force was less than 0.02 eV Å -1 . The energy convergence criterion for self-consistent electronic calculation was set to 1.0 × 10 -5 eV/atom. Here we report how the photoluminescence quantum efficiency of CaO:Eu NIR phosphor was significantly improved and stabilized. By utilizing GeO 2 decomposition, the oxygen vacancies in the CaO lattice were effectively repaired. A record high EQE of 54.7% at 740 nm is obtained with a thermal stability greatly improved from 57% to 90% at 125 °C. ; ;

Keywords

where ε is the number of photons emitted by the sample, and α is the number of photons absorbed by the sample. L S is the luminescence emission spectrum of the sample; E S is the spectrum of the light used for exciting the sample; E R is the spectrum of the excitation light without the sample in the sphere; Abs is absorption efficiency. Obviously, EQE is proportional to IQE and Abs. Note: There are no XPS spectra peaks observed in the region from 26 to 35 eV, suggesting that no Ge 4+, Ge 3+, Ge 2+, Ge 1+ and Ge 0+ existed in CaO:Eu, 4%GeO 2 phosphor. Comparing with the scanning electron microscopy (SEM) images of the CaO:Eu@C (i), the surface and size of CaO:Eu@C+4%GeO 2 microcrystals become more smoother and uniform after doping GeO 2 . This optimization in morphology is related to the decomposition of GeO 2 and the newly introduced O 2-ions. The elemental mapping images reveal that Ca, O, Eu element homogeneously dispersed within phosphor particles, while nearly no Ge element was observed. These results combined with the DFT calculations and XRD analysis suggest that Ge element did not enter the host lattice. Note: (1) The Eu dopant concentration is about 0.2 % for all samples mentioned in the article.

With the increase of the Eu 2 O 3 dopant concentration, the emission intensity increases and reaches a maximum at x = 0.2%, and then decreases due to the concentration quenching, as shown in the following figure. Thus, the optimal dopant concentration of Eu is about 0.2 %.

FiguresFigure 1

 1 Figures

Figure 2

 2 Figure 2 Thermal quenching property of CaO:Eu@C+GeO 2 phosphor. a) Temperature dependence of the relative integrated photoluminescence intensity for CaO:Eu, CaO:Eu@C and CaO:Eu@C+GeO 2 phosphor. b) Temperature dependence of the spectral emission. c) The calculated energy gap by Tauc equation for each samples. d) Electronic band structure of CaO and the calculated defect levels of V O . e) EPR spectra of CaO:Eu, CaO:Eu@C and CaO:Eu@C+GeO 2 compounds. f) Schematic illustration of the mechanism of the thermal quenching process of the CaO:Eu and CaO:Eu@C+GeO 2 phosphor.

Figure 3

 3 Figure 3 NIR performance and application of CaO:Eu@C+GeO 2 phosphor based LEDs device. a) Photoluminescence spectra and photographs of as-fabricated NIR-LED by combining a commercial blue LED and CaO:Eu@C+GeO 2 phosphor. b) NIR output power, electricity-to-blue light and electricity-to-NIR light power conversion efficiency under various drive current. c) Photographs and schematic illustration of high-power NIR-LED device by combining the blue LEDs and phosphorsapphire composite. d-e) Photographs of pavilion captured by visible camera at day and (e) night. f) Photograph of pavilion captured by NIR camera under the illumination of the fabricated high-power NIR-LED device.

  : near-infrared luminescence, phosphor, light-emitting diodes Jianwei Qiao, Shuai Zhang, Xinquan Zhou, Weibin Chen, Romain Gautier, Zhiguo Xia* Near-infrared light-emitting diode utilizing europium-activated calcium oxide phosphor with external quantum efficiency of up to 54.7%

Figure S1

 S1 Figure S1 Structural characterization of CaO:Eu phosphor. a) X-ray powder diffraction patterns of CaO:Eu, CaO:Eu@C, CaO:Eu@C+GeO 2 and the calculated pattern based on Rietveld refinement data (gray line). b) XRD patterns of CaO:Eu, xGeO 2 (x = 0-8%) samples.

Figure

  Figure S2 a) UV-vis diffuse reflectance spectra of CaO, CaO:Eu, CaO:Eu@C and CaO:Eu@C+4%GeO 2 . powder. b) UV-vis diffuse reflectance spectra of CaO, CaO@C and CaO@C+GeO 2 host.

Figure

  Figure S3 a) XANES spectra of the Eu L3 edge for CaO:Eu, CaO:Eu@C and CaO:Eu@C+GeO 2 samples. b) XPS spectra of Eu3d region recorded for CaO:Eu, CaO:Eu@C and CaO:Eu@C+GeO 2 samples.

Figure S4

 S4 Figure S4 Photoluminescence decay curves of the different phosphors under 450 nm pulse laser diode excitation. The statistical photons are 10000. All the datas were fitted by the FAST software attached with FL1000.

Figure

  Figure S5 a-e) Temperature-dependent emission spectra of CaO:Eu@C+xGeO 2 (x = 0 -6%) phosphor under 470 nm excitation in the temperature range 25-150 o C with a temperature interval of 25 o C. f) Temperature dependence of the relative integrated photoluminescence intensity for CaO:Eu@C+xGeO 2 .

Figure

  Figure S6 a) Thermoluminescence curves of CaO:Eu, CaO:Eu@C and CaO:Eu@C+4%GeO 2 in the temperature range 25-300 o C. b-d) 3D-thermoluminescence spectra of CaO:Eu, CaO:Eu@C and CaO:Eu@C+4%GeO 2 after 254, 395 and 450 nm irradiation for 100 s at room temperature.

Figure

  Figure S7 a) XRD diffraction patterns of compounds volatilized and adhered to the surface of graphite crucible during sintering process of CaO:Eu@C+8%GeO 2 sample. b) Cell volume V(x) and average bond length d(Ca-O) dependence of the GeO 2 doping concentration (x = 0-0.08); the inset shows crystal structure of CaO:Eu, where gray and red spheres represent calcium and oxygen ions respectively. Note: The GeO 2 decompose into Ge metal under high temperature and reducing atmosphere, and deposited on the crucible wall and lid, suggesting that Ge 4+ ions do not enter CaO host lattice.

Figure

  Figure S8a) XPS spectra of CaO:Eu, 4%GeO 2 . b) SEM images of CaO:Eu@C (i) and CaO:Eu@C+4%GeO 2 (ii) particles; and (iii) the element mapping images of Ca, O, Eu, and Ge for the selected particle. Note: There are no XPS spectra peaks observed in the region from 26 to 35 eV, suggesting that no Ge 4+, Ge 3+, Ge 2+, Ge 1+ and Ge 0+ existed in CaO:Eu, 4%GeO 2 phosphor. Comparing with the scanning electron microscopy (SEM) images of the CaO:Eu@C (i), the surface and size of CaO:Eu@C+4%GeO 2 microcrystals become more smoother and uniform after doping GeO 2 . This optimization in morphology is related to the decomposition of GeO 2 and the newly introduced O 2-ions. The elemental mapping images reveal that Ca, O, Eu element homogeneously dispersed within phosphor particles, while nearly no Ge element was observed. These results combined with the DFT calculations and XRD analysis suggest that Ge element did not enter the host lattice.

Figure

  Figure S9 a-e) Rietveld refinements of CaO:Eu, xGeO 2 samples: x = 0-8% based on XRD datas.

Figure S10 a- b )

 b Figure S10 a-b) Rietveld refinement of the synchrotron XRD profile for CaO:Eu and CaO:Eu, 8%GeO 2 .

Figure

  Figure S11 a) XRD patterns of CaO:Eu@C+xSnO 2 (x = 0-1.5%) phosphor. b) PL spectra of CaO:Eu@C+ xSnO 2 under 470 nm excitation, and the photographs of crucible lid and CaO:Eu@C+SnO 2 phosphor. c) Temperature-dependent emission spectra of CaO:Eu@C+ xSnO 2 phosphor in the temperature range 25-150 o C. Note: Similar Sn particles observed on the crucible lid suggests that Sn do not enter CaO host lattice.

Figure S12

 S12 Figure S12 PL spectra of the fabricated high-power NIR-LED based on four InGaN chips (460 nm, 10 W) and CaO:Eu@C+4%GeO 2 phosphor.

Figure S13 .

 S13 Figure S13. PL spectra of CaO:xEu (x = 0.04%-0.4%) under 470 nm excitation.

Figure S14 .

 S14 Figure S14. a) The PL spectra of the pristine CaO:Eu and samples treated in degradation conditions at 85% relative humidity (RH), 85 o C for different time, respectively. b) The normalized integrate PL intensities dependent on times and photographs of these samples under natural light.

Table S6

 S6 Bond lengths (Å) of CaO:0.15%Eu, xGeO 2 .

	Table S5 Fractional atomic coordinates and isotropic displacement parameters (Å 2 ) of
	CaO:0.15%Eu, xGeO 2 .
	Atom	x	y	z	B iso	Occ.
	x = 0
	Ca	0.0000	0.0000	0.0000	0.005(23)	1
	O	0.5000	0.5000	0.5000	0.223( 52)	1
	x = 0.02
	Ca	0.0000	0.0000	0.0000	0.315( 17)	1
	O	0.5000	0.5000	0.5000	0.516( 41)	1
	x = 0.04
	Ca	0.0000	0.0000	0.0000	0.204( 18)	1
	O	0.5000	0.5000	0.5000	0.291( 42)	1
	x = 0.06
	Ca	0.0000	0.0000	0.0000	0.552(15)	1
	O	0.5000	0.5000	0.5000	0.321(23)	1
	x = 0.08
	Ca	0.0000	0.0000	0.0000	0.593(15)	1
	O	0.5000	0.5000	0.5000	0.452(32)	1
		x=0	x = 0.02	x = 0.04	x = 0.06	x = 0.08
	Ca-O	2.4064(0)	2.4072(0)	2.4058(0)	2.4065(0)	2.4056(0)
				TOC

Table S7

 S7

	Main parameters of refinement for the CaO:0.15%Eu and CaO:0.15%Eu, 0.08GeO 2
	samples based on the synchrotron XRD profiles.
	x(GeO 2 )	Space Group	Cell parameters (Å ), Cell Volume (Å 3 )	R wp , R p , R exp
			a = 4.8122(0)
	0	Fm-3m	β = 90.0000	2.39, 5.34, 4.65
			V = 111.44(0)
			a = 4.8119(0)
	0.08	Fm-3m	β = 90.0000	2.10, 4.78, 1.27
			V = 111.42(0)
	Note: In our work, the internal quantum efficiencies (IQE) and external quantum efficiencies
	(EQE) test were performed at room temperature by using a barium sulfate coated integrating
	sphere (150 mm in diameter) that attached to FL1000. The IQE and EQE were calculated by
	using the following equations:

Acknowledgements

This research was supported by the National Key Research and Development Program of China (2021YFB3500400 and 2021YFE0105700), National Natural Science Foundations of China (Grant No. 52102169, 51972118 and 51961145101), Guangzhou Science & Technology Project (202007020005), China Postdoctoral Science Foundation (2021M691053), the State Key Laboratory of Luminescent Materials and Devices (Skllmd-2021-09). This work is also funded by Young Elite Scientists Sponsorship Program by China Association for Science and Technology No. YESS20200053).

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Conflict of Interest

The authors declare no conflict of interest.

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Supporting Information

Near-infrared light-emitting diode utilizing europium-activated calcium oxide phosphor with external quantum efficiency of up to 54.7% Jianwei Qiao, Shuai Zhang, Xinquan Zhou, Weibin Chen, Romain Gautier, Zhiguo Xia* K 2 NaScF 6 :Cr 3+ 74%/-100 193.9@14.02 % [6] LiScP 2 O 7 :Cr 3+ , Yb 3+ 74%/-100 36@12% [7] YAl 3 (BO 3 ) 4 :Cr 3+ 86.7%/-100 50.6@17.4% [8] Ga 2-x Sc x O 3 :Cr 3+ 99%/-350 66.1@6.57% [9] BaMgAl 10 O 17 :Cr 3+ 94%/-100 3.4@3.4% [10] ScF 3 :Cr 3+ 45%/-100 8.5@3% [11] LiGaP 2 O 7 :Cr 3+ 47.8%/28.3 % 120 28.1@7.8% [12] LaMgGa 11 O 19 :Cr 3+ 82.6%/42. Average decay times are calculated by the following question:

Where A 1 and A 2 are the corresponding fitting constants; , are lifetimes for the fast and slow components.

Table S3 The formation energy calculated by DFT. The formation energies for each model were calculated using the following formula: [15] where is the total energy derived from a supercell calculation containing the defect X, and is the total energy for the perfect crystal using an equivalent supercell. The integer indicates the number of atoms of type i (host atoms or impurity atoms) that have been added to ( > 0) or removed from ( < 0) the supercell to form the defect, and the are the corresponding chemical potentials of these species. Note: DFT calculations revealed that Ge 4+ can hardly to substitute Ca sites, because of the formation energies is about 8.74 eV higher than that of the initial model. Moreover, oxygen vacancy is easy to form with a negative formation energy -3.27 eV, while the defect V Ca can be hardly generated with a formation energy of 5.69 eV.