Katarzyna J Radomska

Fanny Coulpier

Aurelie Gresset

Alain Schmitt

Amal Debbiche

Sophie Lemoine

Pierre Wolkenstein

Jean-Michel Vallat

Patrick Charnay
email: patrick.charnay@ens.fr

Piotr Topilko
email: topilko@biologie.ens.fr

Mechanisms of Cutaneous Neurofibromas Revealed by Mice with Nf1 Knockout in Boundary Cap Cells

Keywords: NF1, Prss56, boundary caps, Schwann cells, neurofibroma

come

SIGNIFICANCE

Patients affected by NF1 develop numerous cutaneous neurofibromas (cNFs). We present a mouse model that faithfully recapitulates cNFs, identify a candidate cell type at their origin, analyze the steps involved in their formation and show that their development is dramatically accelerated by skin injury. These findings have important clinical/therapeutic implications.

INTRODUCTION

Neurofibromatosis type 1 (NF1) is a common human genetic disorder caused by heterozygous loss-of-function of the tumour suppressor gene NF1, encoding neurofibromin 1.

It predisposes patients to develop benign nerve sheath tumours, termed neurofibromas (NFs).

While about 20% of NF1 patients suffer from plexiform NFs (pNFs) along peripheral nerves tracts, with significantly higher incidence at nerve roots, all of them develop cutaneous NFs (cNFs) localised at nerve terminals in the skin [START_REF] Ratner | A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor[END_REF]. cNFs typically appear at puberty and tend to increase in number throughout life, so that they may reach thousands. Although of benign character, these tumours can be disfiguring and painful, thus significantly affecting quality of life. All NFs are composed of a mixture of Schwann cells (SCs) with other nerve fibre elements, such as axons, fibroblasts, mastocytes, macrophages and endothelial cells. Despite this marked cellular heterogeneity, NFs are known to arise from cells belonging to the SC lineage in which the remaining functional copy of NF1 is mutated, resulting in complete NF1 loss-of-function and permanent hyperactivation of the Ras proto-oncogene and its downstream effectors [START_REF] Ratner | A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor[END_REF]. However, the specific cell type within the SC lineage in which the second-hit mutation occurs is still controversial. Although non-myelinating SCs (nmSC) were initially considered the primary pathogenic target, several recent studies suggest that at least pNFs arise as a result of Nf1 mutation at earlier stages in the SC lineage, possibly as early as neural crest stem cells [START_REF] Parrinello | Neurofibroma development in NF1--insights into tumour initiation[END_REF].

Recently, significant efforts have been made to develop genetically engineered mouse (GEM) models to study Nf1-associated NFs [START_REF] Parrinello | Neurofibroma development in NF1--insights into tumour initiation[END_REF]. These genetic tools have allowed major advances in the understanding of the mechanisms implicated in pNF formation. However, as none of these models recapitulate the development of cNFs, their origin and pathogenic mechanisms remain largely unknown. There is therefore an urgent need to develop new animal models that recapitulate cNFs and allow their experimental analysis.

As possible candidates for the cells at the origin of paraspinal pNFs (ppNFs), neural crest-derived boundary cap (BC) cells, which are transiently located at the embryonic nerve root entry/exit zone, have attracted particular attention. Using the Krox20/Egr2 gene as a marker for BC cells, we have demonstrated that Krox20-expressing BC cells give rise to the entire SC component of the nerve roots, precisely where ppNFs develop [START_REF] Maro | Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS[END_REF]. Furthermore, conditional inactivation of Nf1 in Krox20-expressing cells gives rise to ppNFs, but not cNFs [START_REF] Zhu | Neurofibromas in NF1: Schwann cell origin and role of tumor environment[END_REF]. This suggests that Nf1 inactivation in the Krox20-positive BC cell lineage leads to pNFs and that cNFs do not originate from these cells. Recently, we have identified another BC cell marker, Prss56, and developed a mutant allele, Prss56 Cre , in which the Cre recombinase coding sequence is inserted into the locus [START_REF] Coulpier | Novel features of boundary cap cells revealed by the analysis of newly identified molecular markers[END_REF]. In vivo fate mapping analyses with this allele indicated that BC derivatives migrate into the nerve roots and, along peripheral nerves, into nerve terminals in the skin, precisely the two locations where NFs are observed [START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF]. These observations led us to hypothesize that the lineage of Prss56-expressing BC cells might be the target for Nf1 mutation in both plexiform and cutaneous forms of NFs.

In the present study, we have generated and characterised mice carrying bi-allelic Nf1 deletions in Prss56-expressing BC cells and their progeny, by introducing Prss56 Cre and the Cre-activable reporter R26 tdTom alleles into Nf1-floxed backgrounds (Nf1 fl/fl or Nf1 fl/-), susceptible to loss-of-function upon Cre recombination. This approach allows conditional deletion of Nf1 in Prss56-expressing BC cells and in vivo tracing of mutant cells due to permanent expression of the fluorescent reporter. We found that both types of mutants develop numerous cNFs and pNFs. As inactivation of Nf1 in the peripheral nervous system (PNS) is restricted to Prss56-expressing BC cells and their derivatives, this population is clearly directly involved in the development of NFs. Our model allows a detailed analysis of cNF development, leading to critical observations on the aetiology, progression and properties of the disease that are of prime importance for clinical and therapeutic applications.

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RESULTS

Prss56-expressing BC cells give rise to glial and non-glial derivatives in nerve roots and skin nerve terminals

We have previously shown that derivatives of Prss56-expressing BC cells migrate into nerve roots and nerve terminals in the skin [START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF]. Here, using Prss56 Cre ,R26 tdTom mice, we present a detailed characterisation of tdTomato (TOM)-traced cells in these two structures. As expression of Prss56 was never detected in nerve roots nor in the skin, we consider that all traced cells in these tissues correspond to BC derivatives ((6), and Fig. S1A). Consistent with our previous report [START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF], numerous traced cells were present along the adult dorsal and ventral nerve roots and in the dorsal root ganglia (DRG), but there were only a few in the proximal segment of the spinal nerves (Fig. 1A). In the trunk of the sciatic nerve, TOM staining was absent from SCs (S100-positive; S100+) and remained restricted to a small subset of axons (TUJ1+) (Fig. S1B). We performed immunohistochemical (IHC) analyses in the nerve roots with SC marker S100 and neural crest lineage marker SOX10 (Fig. 1B-D). All traced cells were derived from the neural crest, whereas only a subset of the SOX10+ population was traced (Fig. 1C). Quantification in cervical dorsal nerve roots indicated that 76.1 ± 10.2% (mean ± SD) of the SOX10+ cells originated from Prss56-expressing BC cells. Most traced cells were S100+ and correspond to myelinating (mSC) and non-myelinating (nmSC) SCs, according to their morphology and organisation with respect to axons. The level of S100 expression was much weaker in nmSCs than mSCs (Fig. 1B,D).

In addition to SCs, we observed a small subset of TOM+ endoneurial cells that lacked S100-immunoreactivity (Fig. 1B), possibly corresponding to neural crest-derived endoneurial fibroblasts (eFBs) [START_REF] Joseph | Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells[END_REF]. To explore this possibility, we immunolabelled adult nerve roots with TOM, a generic fibroblast marker PDGFRα and CD34, recently identified as a putative eFB marker in human and rat peripheral nerves [START_REF] Richard | Endoneurial fibroblast-like cells[END_REF]. Whereas numerous cells expressing these markers were observed in the endoneurial space, no overlap with TOM labelling was observed. (Fig. S1C,D). Further studies will therefore be required to characterise the identity of this TOM+/S100-population. Finally, we have never observed TOM+ perineurial fibroblasts (Fig. 1B and Fig. S1E).

In the adult skin, traced cells were found in the dermis (Fig. 1E) and hypodermis (subcutis) (Fig. 1F) but were absent from the epidermis (Fig. 1G). In the dermis, they showed a highly heterogeneous distribution, with patches aligned with some, but not all nerves and nerve terminals (Fig. 1E,G). IHC analysis with S100 revealed that most of these cells correspond to nmSCs (Fig. 1G). Nevertheless, we also observed a few traced mSCs (S100+/MBP+) enwrapping rare large-diameter fibres, mainly located in the lower dermis or innervating hair follicles (Fig. S1F). Dermal traced nmSCs exhibited broad heterogeneity and could be classified into four categories: (i) typical nerve-associated nmSCs unsheathing small-calibre axons (Remak SCs; Fig. 1G), (ii) hair follicle-associated glia unsheathing lanceolate nerve endings (Fig. 1G,H) (iii) glia associated with the arrector pili muscle (Fig. 1G,I), and (iv) terminal glia associated with nociceptive free nerve endings (Fig. 1G, J and Fig. S1G). This later population, residing directly beneath the epidermis (thus further referred to as subepidermal SCs), is characterised by a complex multipolar morphology and remains largely uncharacterised, although it has been identified in the human hairy skin (9) [START_REF] Reinisch | The dimensions and characteristics of the subepidermal nerve plexus in human skin -Terminal Schwann cells constitute a substantial cell population within the superficial dermis[END_REF].

Analysis of large skin fragments also revealed the presence of pigmented TOM+ and KIT+ melanocytes, in a subset of hair follicle bulbs (Fig. 1K, K' and Fig. S1H,H'). Finally, while BC derivatives are absent from the trunk of peripheral nerves, they populate subcutaneous nerve branches, where they correspond to a subset of mSCs, nmSCs and eFBs (Fig. 1F and ( 6)).

Conditional Nf1 knockout in BC cells leads to skin lesions and progressive limb paralysis

Since BC derivatives populate nerve roots and nerve terminals in the skin, two highly favoured sites for NF development, we explored whether Nf1 knockout in Prss56-expressing BC cells was sufficient to promote formation of NFs in the mouse. We transferred the Prss56 Cre ,R26 tdTom alleles into Nf1 flox/-or Nf1 floxflox backgrounds to generate Prss56 Cre ,R26 tdTom ,Nf1 flox/-and Prss56 Cre ,R26 tdTom ,Nf1 flox/flox progeny, collectively referred to as Nf1-KO mutants. These mutant animals should carry bi-allelic Nf1 knockout in BC cells and their derivatives, in a Nf1 heterozygous or wild-type background, respectively. As Prss56 expression is restricted to BC cells between embryonic day (E) E11 and E13 and is absent from any other component of the PNS, recombination at the Nf1 locus should occur during this period [START_REF] Coulpier | Novel features of boundary cap cells revealed by the analysis of newly identified molecular markers[END_REF]. Moreover, as TOM is activated in parallel to Nf1 inactivation, this should allow identification of the cells carrying the Nf1 knockout. The efficiency of Cre-mediated recombination at the Nf1 locus was evaluated using semi-quantitative RT-PCR. No Nf1 expression was observed in Nf1 mutant cells, demonstrating efficient recombination (Fig. S1A).

The use of the two genetic backgrounds (Nf1 flox/-or Nf1 flox/flox ) for the analysis of the phenotypes associated with the targeted mutation is important, because in some of the existing Nf1 GEM models, tumourigenicity requires inactivation of both Nf1 alleles in the cells that become neoplastic, along with inactivation of one allele in the tumour environment [START_REF] Zhu | Neurofibromas in NF1: Schwann cell origin and role of tumor environment[END_REF]. As controls (CTRL), we used Prss56 Cre ,R26 tdTom ,Nf1 flox/+ littermates, which carry only a heterozygous Nf1 inactivation in the BC cell lineage in a wild-type background. Control animals did not develop any abnormalities and were phenotypically indistinguishable from wild-type animals. Both types of Nf1-KO mutants were born at the expected Mendelian frequency and all developed identical symptoms, at 10.0 ± 4.5 months and 13.1 ± 3.9 months (mean ± SD) of age for Nf1 flox/-and Nf1 flox/flox backgrounds, respectively (Table S1). The symptoms included lethargy and numerous skin lesions, mainly localised on the neck, and were often accompanied by progressive limb paralysis. Skin lesions were typically accompanied by pruritus that required the sacrifice of the animals. Gross anatomicalpathological analysis of Nf1-KO mice revealed enlarged nerve roots/DRGs, often accompanied by tissue masses compressing the spinal cord at the cervico-thoracic level. In subsequent analyses, as the phenotypes in Nf1 flox/-and Nf1 flox/flox backgrounds appeared to be similar apart from the mean age of appearance, animals from both backgrounds were used interchangeably, except when explicitly indicated.

BC-derived non-myelinating Schwann cells constitute the major pathogenic cell type in plexiform neurofibroma

Gross dissection of Nf1-KO mutant spinal cord with the attached nerve roots revealed the presence of tumours that are largely TOM+, localised at the thoracic and/or cervical nerve roots (Fig. 2A-C). All remaining nerve roots showed signs of marked hyperplasia (Fig. 2A-C). The number of tumours varied from 1 to 4 and often affected contralateral sides. Severe spinal cord compression was frequent and likely accounted for the observed paralysis (Fig. 2C,D). Paraspinal lesions contained abnormal accumulations of disorganised, slender TOM+ cells that expressed low levels or no S100, likely corresponding nmSCs (Fig. 2E-G''). In the mutants, unlike controls, most TOM+ cells coexpressed SOX2, a marker of precursor/immature SCs (Fig. S2A-B'). In contrast, TOM+ mSCs, enwrapping large-calibre axons, expressed high levels of S100 and showed no obvious abnormalities (Fig. 2E-G''). We also observed increased infusion of KIT+ mastocytes and IBA1+ macrophages (both nontraced), which are always detected in human pNFs (Fig. S2C-F'). Ultrastructure analysis of paraspinal tumours revealed a substantial expansion of an interstitial compartment filled with collagen-rich extracellular matrix, and accumulation of abnormal Remak bundles (Fig. 2H-K). Mutant nmSCs (identified by continuous basal lamina) showed profound morphological abnormalities, including partial or complete dissociation from axons, and extension of numerous cytoplasmic protrusions surrounding collagen fibrils or myelinated fibres (Fig. 2I-K).

In addition, subcutaneous nerves containing traced BC derivatives were markedly enlarged, and localised enlargements (ballooning) were often observed beneath the skin lesions (Fig. 2L). Subcutaneous lesions were very similar to paraspinal tumours in their histology and ultrastructure (Fig. 2M-P), and contained large numbers of traced, S100+ nmSCs with disrupted morphologies and axonal interactions, intermixed with numerous nontraced, PDGFRα+ fibroblasts. Fibroblasts present in the peripheral part of a tumour had typical characteristics of perineurial fibroblasts, such as patchy basal lamina and the presence of pinocytic vesicles (Fig. S2G). However, unlike the control nerves, where perineurial cells form a multilayered, tightly compacted protective sheath, the perineurial compartment of the Nf1-KO nerves appeared much thicker and contained a high number of loosely organised fibroblasts intermingled with numerous SC processes and large amounts of collagen (Fig. 2O,P). Despite these abnormalities, nerve integrity seemed to be preserved. Finally, as in paraspinal lesions, there was an increased influx of mastocytes and an even more pronounced infiltration of macrophages in the tumour masses (Fig. S2H,H').

Together, our observations support the previously proposed role of BC-derived nmSCs in the formation of ppNFs and a similar involvement in the development of subcutaneous tumours.

Nf1 inactivation in Prss56-positive cells leads to the development of diffuse cutaneous neurofibromas

In Nf1-KO mice, the first macroscopically detectable cutaneous symptoms were areas of hair thinning on the neck, which rapidly evolved into pruritus (Fig. 3A,B). After shaving the hair, we frequently observed additional smaller lesions (with or without scabs) containing wellcircumscribed, dense aggregates of traced cells (Fig. 3B). Such dense clusters of TOM+ cells were never observed in control skin. Although affected skin regions were significantly thicker than non-affected skin from the same individual, their basic architecture, including integrity of hair follicles, remained preserved. IHC analyses of lesioned areas indicate that (i) the diffuse accumulation of traced cells is restricted to the dermis; (ii) traced cells correspond to S100+ nmSCs. However, the level of S100 was significantly lower than in rare mSCs. These nmSCs appear highly disorganised and extend numerous slender cytoplasmic processes (Fig. 3C-D'). Their SC identity is further supported by expression of other known nmSC/immature SC markers, including p75NTR, NCAM and L1, as well as SOX2 (Fig. S3A-D); (iii) mutant nmSCs express high levels of phosphorylated Mitogen-Activated Protein Kinase (ERK1/2), a downstream effector of Ras (Fig. S3E); (iv) numerous non-traced KIT+ mastocytes, IBA1+ macrophages and PECAM+ blood vessels infiltrate the lesion (Fig. 3E-F' and Fig. S3F); (v) the dermal fibroblast population (non-traced and defined by PDGFRα expression) is drastically enlarged (Fig. 3G,G') and accompanied by heavy deposition of collagen (Fig. 3H); (vi) both traced and non-traced pigmented cells, presumably melanocytes, are often concentrated in extrafollicular dermis within the lesion (Fig. 3I).

Ultrastructural analysis of the Nf1-KO skin lesions confirmed that numerous nmSCs with severely affected morphologies are present in the dermis. These SCs form a dense network of cytoplasmic extensions that often enwrap isolated axons and/or are devoid of axonal contact, and enclose clusters of collagen fibrils, forming apparent collagen pockets (Fig. 3J,K). Immuno-electron microscopy confirmed that most of these cells express TOM and correspond to mutant nmSCs (Fig. 3K). Finally, numerous fibroblasts (identified by morphology and the absence of basal lamina) and collagen fibres were observed at the level of the lesions (Fig. S3G). Since endoneurial and perineurial fibroblasts are rare or absent in the dermis, we assume that this accumulation of fibroblasts mainly corresponds to recruitment and/or proliferation of mesodermally-derived dermal population (non-traced). Lack of basal lamina and pinocytic vesicles, typical characteristics of perineurial fibroblasts, further supports their dermal identity. While in control skin, dermal fibroblasts are rare, of round shape, with few ribosomes and poorly developed endoplasmic reticulum (ER), in the Nf1-KO mutant dermis, they often form long cytoplasmic extensions with or without collagen pockets and contain numerous ribosomes and highly developed ER (Fig. S3G-I), supporting their engagement in the active synthesis of collagen. Finally, in addition to infiltration by mastocytes and macrophages, ultrastructural analysis revealed accumulations of polymorphonuclear neutrophils, which were only sporadically observed in control skin (Fig. S3J).

In conclusion, all morphological, cellular and molecular characteristics associated with the cutaneous lesions are highly similar to those of diffuse human cNFs [START_REF] Peltonen | Molecular and Cellular Basis of Human Cutaneous Neurofibromas and Their Development[END_REF]. These data indicate that the loss of Nf1 in dermal BC derivatives is sufficient to promote development of cNFs. This is correlated with activation of the ERK signalling pathway in mutant SCs.

Development of cNFs involves progressive alterations of the dermis

Although NF1 patients develop cNFs during adulthood after a long asymptomatic period, it is unclear whether these tumours appear suddenly in the adult skin or whether they slowly evolve from micro-tumoural loci already present at younger ages. To investigate this issue, we compared the skin of Nf1-KO mice and control littermates from embryonic day E13.5, when the first traced BC derivatives settle in the skin, until 6 months after birth, when most mutants do not show obvious cutaneous manifestations. For this study, we used only Prss56 Cre ,R26 tdTom ,Nf1 flox/-mutants which, similar to NF1 patients, harbour heterozygous germline Nf1 mutation along with homozygous Nf1 knockout in the SC lineage. The inactivation of Nf1 in BC cells around E11 did not affect the migration of BC-derived progenitors along the nerves to the skin nor the number of traced SC precursors or immature SCs in the embryonic skin (Fig. S4A-F). This suggests that the early specification and migration of the SC lineage is not affected in the absence of Nf1. At birth, however, the number of traced SCs in Nf1-KO was nearly double that in control skin (Fig. 4A-C), although all mutant SCs remained in contact with axons and did not show any morphological abnormalities. Intraperitoneal 5-ethynyl-2'-deoxyuridine (EdU) injection into pregnant females at E18.5, followed by analysis of newborn skin, revealed a small but significant (p = 0.036) increase in the proportion of proliferating SCs (EdU+/TOM+) in Nf1-KO (22.6 ± 1.37%) compared to control animals (18.19 ± 1.89%, mean ± SEM). Slow SC proliferation is later maintained in cNFs, as immunolabelling of cNFs from 10-month-old Nf1-KO mutants against the mitotic marker phospho-histone-H3 (PHH3) indicates that a small proportion of traced cells undergo mitosis (Fig. S3K). This is in line with clinical observations demonstrating rare mitoses in human NFs [START_REF] Stemmer-Rachamimov | Comparative pathology of nerve sheath tumors in mouse models and humans[END_REF]. Overall, our data suggests that inactivation of Nf1 provides a small proliferative advantage to neonatal mutant SCs.

As BC derivatives include a subset of skin melanocytes, we compared the number of cells co-expressing TOM and the melanocyte lineage marker TRP2 between Nf1-KO and control, newborn animals. While we observed no difference in the number of traced melanoblasts within the hair follicles, the extrafollicular (dermal) fraction was substantially increased in Nf1-KO skin (Fig. 4D-F). Interestingly, there was already enhanced melanocytogenesis in embryonic mutant skin at E15.5 (Fig. S4G-L). At P9, when melanoblast migration into developing hair follicles is complete in control skin, numerous double-positive (TOM+/TRP2+) cells were still observed in the mutant dermis. Conversely, there was no difference in the numbers of traced follicular melanocytes (Fig. S4M), suggesting a selective expansion of the extrafollicular melanocyte fraction in the mutant.

We next investigated whether the elevated SC content in mutant newborn skin is accompanied by modifications of the other cell types. Although Nf1-KO skin had a slight (1.1-fold) elevation in the number of KIT+ mastocytes compared to controls (Fig. 4G), the densities of PDGFRα+ fibroblasts (Fig. 4H,I) and of IBA1+ macrophages (Fig. 4J) were comparable between the two groups.

Analysis of the postnatal back skin between P0 and 3 months of age revealed a further progressive increase in the numbers of TOM+ SCs in Nf1-KO mutants compared to controls (Fig. 4K-M and Fig. S4N). Although at three months, most traced SCs were still well-aligned with axons, many appeared hypertrophic and extended numerous cytoplasmic protrusions that detached from axons (Fig. 4N-O''). In some cases, we observed an entire SC soma "escaping" from the nerve bundle (Fig. 4O''). These abnormal morphologies preferentially occurred in the upper dermis (Fig. S5A-D). In addition, we observed a dramatic increase in the density of dermal fibroblasts, as revealed by labelling for PDGFRα and increased density of nuclear staining (Fig. 4P,Q). However, as in the newborn skin, the number of mastocytes was only slightly (1.4-fold) higher in Nf1-KO dermis than in controls (Fig. 4R), whereas the density of macrophages was unaffected (Fig. S4O,P).

It is surprising that despite their increased number, mutant SCs mostly remain in contact with axons for up to 3 months. This observation raises the possibility of an increase in the density of cutaneous innervation to cope with elevated SC numbers. To examine innervation density, we performed whole-mount immunostaining of the 3-month-old Nf1-KO and control skin with antibodies against TOM and the pan-axonal marker ß-III-tubulin (TUJ1). The density of innervation appeared higher in the regions of Nf1-KO skin corresponding to patches of traced SCs as compared to controls or regions of mutant skin poor in traced cells (Fig. S5E-G). This suggests that enhanced local innervation in the mutant skin is linked to SC hyperplasia. To investigate if enhanced innervation might result from increased sensory DRG neurogenesis, we compared the numbers of traced sensory neurons in Nf1-KO mutant and control newborn DRGs from the same A-P level (Fig. S5H). There was no significant difference in the number of traced sensory neurons, suggesting that enhanced innervation is rather due to branching of nerve terminals in the mutant dermis.

We next analysed the skin of 6-month-old Nf1-KO mice, which still did not show any visually detectable abnormalities. However, numerous microscopic lumps were detected upon tactile examination. Histological analysis of the skin revealed SC hyperplasia along with the presence (over the entire back skin) of multiple small (up to 1 mm) aggregates of disorganised nmSCs, positive for TOM and S100 (Fig. 5A-C), which were intermixed with numerous nontraced fibroblasts, mastocytes, macrophages and polymorphonuclear neutrophils, all present in typical cNFs. Quantification of distinct cell populations was performed by electron microscopy (Fig. 5D). Interestingly, the presence of inflammation-related cells, such as neutrophils, mastocytes and macrophages in these micro-lesions suggests these cell types are involved in the development and/or progression of tumours rather than being a simple consequence of pruritus-related inflammation. Ultrastructural characteristics of nmSCs in the micro-lesions were also typical of classical cNFs and included abnormal extension of cytoplasmic processes that enclosed isolated axons and/or groups of collagen fibrils, or were devoid of axonal contact (Fig. 5E,F). Whole-mount immunostaining of the mutant dermis confirmed accumulation of traced SCs with profound morphological aberrations (Fig. 5G-H').

SC hypertrophy in the upper dermis was far more severe than in 3-month-old mutants, and some cells seemed to lose contact with axons. In the lower dermis, most mutant SCs had detached from axons and extended multiple filipodia-like structures. Together these data indicate that the micro-lesions, apart from their size, share essential characteristics with bona fide cNFs and consequently, we refer to them as micro-cNFs.

Ultrastructural examination of dermal innervation also revealed that intact Remak bundles, containing small groups of tightly packed axons, were extremely rare in the mutant dermis. Instead, we commonly observed numerous isolated axons that were separated by large quantities of collagen and individually ensheathed by small fragments of SC cytoplasm (Fig. 5F). This suggests decompaction of nerve bundles. Moreover, we observed a 2-fold increase in axon numbers between control and Nf1-KO dermis (Fig. 5D). Immunolabelling with CGRP, a marker of peptidergic nociceptive neurons [START_REF] Basbaum | Cellular and molecular mechanisms of pain[END_REF] further supported an increased density of dermal innervation in micro-cNFs compared to control dermis (Fig. S5I-L).

Overall, these data point toward a marked decompaction of nerve bundles combined with increased branching of axon terminals in the mutant dermis.

If micro-cNFs constitute a precursor stage of full-blown tumours, it would be expected that the number of tumours arising in older individuals should be much larger than what was actually observed (typically up to a few well-demarcated lesions per mutant at the cervical or cervico-thoracic level). As mutant mice had to be sacrificed as soon as pruritic lesions appeared, it is likely that most of the developing tumours did not have enough time to reach a macroscopically detectable stage. Indeed, this idea is supported by the observation of a unique Nf1-KO individual that survived until 20 months of age without developing pruritus. In this case, dozens of raised skin bumps containing dense clusters of traced cells were observed all over the back skin. Analysis of their cellular composition with the set of markers described above confirmed their cNF identity (Fig. S3L-N). This strongly supports the hypothesis that many of the micro-cNFs observed in the 6-month-old skin could evolve into full-blown diffuse cNFs in older animals.

Skin injury accelerates the development of cNFs

In NF1 patients, surgical and laser ablation of cutaneous tumours are routinely used for tumour resection. However, several case reports suggest that skin injury (trauma) might promote development of additional cNFs [START_REF] Riccardi | Cutaneous manifestation of neurofibromatosis: cellular interaction, pigmentation, and mast cells[END_REF]. To test whether an injury favors the appearance of tumours, small skin incisions were made at the thoracic level on 3-month-old Nf1-KO mutants (n=6) and control individuals (n=4). These animals were sacrificed at 10 months of age and their skin was analysed and compared with that of uninjured age-matched Nf1-KO mutants (n=6). All injured Nf1-KO mutants developed large, mostly non-pruritic lesions at the site of injury (Fig. 6A-a). Furthermore, all injured mutants also developed numerous, smaller (≤ 5 mm), well-demarcated lesions all over the back skin (Fig. 6A-d). Both types of lesions, at the injury site or distant from it, contained dense accumulations of TOM+ cells (Fig. d',f,g). Thoracic and lumbar lesions were histologically identical (except for their size) and corresponded to typical diffuse cNFs (Fig. 6A-f, g, i-k). No abnormalities in the distribution or densities of traced cells were ever observed in injured control animals (Fig. 6A-c). Moreover, we did not observe any morphologically visible skin lesions at the thoracic or lumbar level in 10-month-old uninjured Nf1-KO mutants (Fig. 6A-e). The accumulation of traced cells in this region was limited and corresponded to micro-cNFs (Fig. 6A-e', h). Furthermore, in our original Nf1-KO cohort (Table S1), morphologically visible lesions were rare at the thoracic level (4 animals out of 26) and only found in aging individuals (17.5 ± 3.6 months). Finally, while the thickness of the skin in the uninjured mutants in the lumbar region was typical of early stage micro-NFs (Fig. 6A-h), it was increased in the same region in injured mutants (Fig. 6A-g), consistent with cNF stage. Skin thickness reached an unprecedented level in the thoracic region of the injured mutants, with a dramatic expansion of the number of traced cells (Fig. 6A-f).

Wound healing following skin injury is a highly dynamic process that involves soluble mediators, extracellular matrix components, resident cells, and infiltrating leukocytes, which differentially participate in three sequential phases: inflammation, proliferation, and remodeling [START_REF] Singer | Cutaneous wound healing[END_REF]. Initially, neutrophils accumulate in wound sites, followed by a large influx of macrophages that augment inflammatory response. Recent studies highlighted the role of nerves and SCs as pivotal elements promoting skin repair [START_REF] Parfejevs | Injury-activated glial cells promote wound healing of the adult skin in mice[END_REF]. As soon as 2 days after injury, SCs detach from sectioned nerves, proliferate and begin to invade the wound bed, where they support extracellular matrix remodeling via paracrine signaling. This delamination is transient as 2 weeks later many SCs at the site of injury are associated with nerves. To investigate the impact of skin injury on BC-derived Nf1-KO SCs, we analyzed TOM+ cells at 2, 4, 7 and 14 day post-injury (dpi) in 3-month-old control (Prss56 Cre , R26 tdTom ) and Nf1-KO animals (Fig. 6B). During the peak of the inflammation phase (2-4 dpi), traced Nf1-KO and control SCs from the damaged nerves were similarly distributed into the wound bed (Fig. 6B-a-d).

However, by day 7, the number of Nf1-KO SCs drastically increased as compared to control SCs, and this phenomenon was even more pronounced at 14 dpi (Fig. 6B-e-h). Rapid expansion of the mutant SC lineage was also supported by the observation that the majority of Nf1-KO SCs expressed the proliferation marker PH3 at 7dpi (inset in Fig. 6B-f). In addition, detailed analysis of 14 dpi mutant wounds revealed the presence of morphologically abnormal Nf1-KO SCs, similar to those observed in cNFs (Fig. 6B-h'). At this stage, numerous Nf1-KO SCs were still dissociated from the axons, in contrast to control SCs (Fig. 6B-g', h').

Furthermore, macrophage and fibroblast, but not mastocyte, densities were increased in Nf1-KO as compared to control wounds, suggesting enhanced inflammatory response in the mutant.

To identify candidate factors involved in the abnormal behavior of Nf1-KO SCs and their microenvironment upon injury, and possibly in the acceleration of cNF development, we analyzed the distribution of mRNAs by high throughput sequencing in wounded (14 dpi) and intact skin from Nf1-KO and control mice (n=3). Prss56 mRNA was undetected across all compared samples, excluding the possibility of de novo activation of this gene in the skin upon injury. Among the 9627 genes found to be differentially transcribed (p<0.05; fold change, FC>4x) between injured and uninjured control skin, 13.4 % were upregulated and 86.6% downregulated. Among the 4112 genes differentially transcribed between injured and uninjured Nf1-KO skin, 34% were upregulated and 66% downregulated (Fig. 6C-a). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses performed on the upregulated transcripts identified "Cytokine-cytokine receptor interaction", "ECMreceptor interaction", "Complement and coagulation cascades", "Protein digestion and absorption" and "Focal adhesion" among the top ranked pathways in injured vs uninjured skin comparisons, in both Nf1-KO and control (Fig. 6C-b, c and Table S2). As expected, many genes from these lists have previously been implicated in wound healing and tissue repair [START_REF] Eming | Wound repair and regeneration: Mechanisms, signaling, and translation[END_REF]. Remarkably, similar to the wounded Nf1-KO skin, "Cytokine-cytokine receptor interaction" has also been reported as the most enriched pathway in mouse neurofibromaderived SCs and macrophages [START_REF] Choi | An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system[END_REF], suggesting the involvement of similar paracrine/autocrine signaling pathways. More specifically, among the cytokines identified as significantly upregulated in injured vs uninjured Nf1-KO skin, Ccl2 (FC=10.5x), Ccl5 (FC=17.4x), Ccl12 (FC=22.6x), Cxcl5 (FC=37.2x), Cxcl12 (FC=11.3x), Cxcl13 (FC=7.2x), Il1b (FC=9.2x) and Pf4 (FC=13.4x) have previously been reported as overexpressed in human and/or mouse neurofibromas compared to healthy nerves [START_REF] Choi | An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system[END_REF][START_REF] Karaosmanoglu | Heightened CXCR4 and CXCL12 expression in NF1-associated neurofibromas[END_REF]. Furthermore, 39% of the upregulated genes in this KEGG category were specifically activated in Nf1-KO and not in control animals, suggesting the existence of differences in injury-induced inflammatory response and tissue repair between mutant and control mice (Table S2). This observation is in line with the increased density of mutant SCs and macrophages observed in Nf1-KO wound beds. Of particular interest, Ccl5, which encodes a potent chemoattractant for immune effector cells, including macrophages, and has been reported as upregulated in both neurofibroma Nf1 -/-SCs and macrophages [START_REF] Choi | An inflammatory gene signature distinguishes neurofibroma Schwann cells and macrophages from cells in the normal peripheral nervous system[END_REF], was among the genes specifically activated in Nf1-KO wounds. Its receptors, Ccr5 and Ccr1, were upregulated in both Nf1-KO and control wounds, although with a higher FC in mutant (8.8x and 12.6x) than in control (4.4x and 6.7x) skin. This makes Ccl5 an interesting candidate for being involved in early development of cNFs. Cx3cr1 was also found specifically upregulated in Nf1-KO wounds (FC=5.8x). It encodes a macrophage and fibroblast chemokine receptor known to promote macrophage and fibroblast accumulation in the wound site, as well as collagen deposition [START_REF] Ishida | Chemokine Receptor CX3CR1 Mediates Skin Wound Healing by Promoting Macrophage and Fibroblast Accumulation and Function[END_REF]. Furthermore, Cx3cr1mediated signaling has recently been implicated in NF1-related optic glioma formation [START_REF] Pong | Reduced microglial CX3CR1 expression delays neurofibromatosis-1 glioma formation[END_REF].

Finally, the mRNA levels of Ccl2, its receptor Ccr2 and Ccl3, which are also implicated in macrophage recruitment, were more strongly increased in Nf1-KO (10.5x, 13.2x and 23.8x) than in control (6x, 4.8x and 4.8x) skin comparisons.

Together our findings demonstrate that skin injury promotes cNF development. The injury has a rapid impact on Nf1-KO SC proliferation, morphology and interactions with axons.

RNA-seq analysis of the skin supports the implication of inflammation-related signals in the acquisition of an invasive, pro-tumorigenic SC phenotype and provides several gene candidates susceptible to play key roles in this process.

Subepidermal glia are the likely cell type at the origin of cutaneous neurofibroma

It is striking that mice subjected to targeted Nf1 inactivation in Krox20+ BC cells or Dhh+ SC precursors develop pNFs, but never diffuse cNFs [START_REF] Wu | Plexiform and dermal neurofibromas and pigmentation are caused by Nf1 loss in desert hedgehogexpressing cells[END_REF]. A likely explanation is that the BC derivatives that migrate into the skin and participate in development of cNFs do not activate these two genes. To test this hypothesis, in vivo fate mapping and characterisation of derivatives of either Krox20or Dhh-expressing progenitors in the adult skin was achieved by morphological and molecular analysis of TOM+ cells from Krox20 Cre ,R26 tdTom and Dhh Cre ,R26 tdTom adult animals. In Krox20 Cre ,R26 tdTom mice, the TOM+ cells in the skin correspond to the rare S100+/MBP+ mSCs, NG2+ pericytes and previously described keratinocytes and hair follicle cells [START_REF] Topilko | Krox-20 controls myelination in the peripheral nervous system[END_REF][START_REF] Gambardella | Pattern of expression of the transcription factor Krox-20 in mouse hair follicle[END_REF] (Fig. 7A-B''). Notably, traced nmSCs were never observed. Expression of TOM in mSCs is likely to reflect Krox20 locus activation in mSC precursors around E15.5 [START_REF] Topilko | Krox-20 controls myelination in the peripheral nervous system[END_REF], rather than a BC origin of these cells.

Analysis of Dhh Cre ,R26 tdTom adult skin revealed that a majority of SCs were traced in the hypodermis (Fig. 7C). In the dermis, TOM+ cells corresponded to various glial and nonglial derivatives (Fig. 7D-G'). However, subepidermal glia, unsheathing nociceptive fibres at the dermis/epidermis boundary and melanocytes were not traced, in stark contrast to the situation in Prss56 Cre ,R26 tdTom skin (Fig. 7D,H,I).

Together these observations indicate that Prss56-, Krox20-and Dhh-traced populations give rise to overlapping and distinct types of glial populations in the skin (summarised in Fig. 7J). Among them, the only cell type uniquely derived from Prss56+ cells is the subepidermal glia. As diffuse cNFs do not develop upon conditional Nf1 loss in Krox20-and Dhh-traced lineages, subepidermal SCs are likely to be the cell type at the origin of cutaneous neurofibromas.

DISCUSSION

Several mouse models of NF1 have been previously developed with two major objectives: to recapitulate the various aspects of the human disease and to provide cues on the cellular origins of the different types of neurofibromas. These objectives have been so far only partially fulfilled, as diffuse cNFs are not recapitulated in these models [START_REF] Parrinello | Neurofibroma development in NF1--insights into tumour initiation[END_REF]. In the present study, we performed targeted inactivation of Nf1 in Prss56-positive BC cells and observed the development of bona fide plexiform and cutaneous NFs. Comparison with previous models, together with an intrinsic tracing system, allow us to suggest a precise hypothesis on the cell type at the origin of cNFs. Furthermore, follow up of mutant animals from birth until the appearance of cNFs has allowed a detailed description of disease progression and revealed decisive steps in tumour development. Understanding these steps is likely to impact the search for therapeutic molecules against cNFs. Finally, the discovery that the skin trauma might stimulate the systemic progression of micro-cNFs into full-blown cNFs is also of clinical relevance.

The data derived from the alternative NF1 GEM models suggest that the cells at the origin of cNFs are not targeted for Nf1 inactivation in these cases. More specifically, comparison of fate tracing performed with the Cre drivers Prss56 Cre and Dhh Cre , combined with the fact that in our model, development of cNFs appears to be initiated in the upper dermis, points to subepidermal SCs as the likely candidate for the tumour-initiating cell.

Identification of specific markers for this type of glia will pave the way for direct analysis of their role in the development of cNFs in both mice and human patients. In humans, the second-hit mutation responsible for cNFs could therefore happen in subepidermal glia and/or their precursors. As our targeting system is restricted to BC cells (within PNS) and we observed the different types of NF1-associated tumours in our mutants, this work demonstrates that BC cells give rise to all cell types at the origin of NFs. More specifically, our tracing analyses indicate that Prss56-positive BC cells generate nerve root glia, hypodermal and dermal glia, the latter including subepidermal SCs. These three BC-derived cell types are likely to be at the origin of ppNFs, spNFs and cNFs, respectively. Finally, this work confirms that the BC cell population is heterogeneous, as previously suggested by differences in gene expression [START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF]. Krox20-positive BC cells mostly give rise to nerve root glia ((3) and this study), and accordingly Nf1 loss-of-function in Krox20-positive BC cells only give rise to ppNFs [START_REF] Zhu | Neurofibromas in NF1: Schwann cell origin and role of tumor environment[END_REF]. The development of cNFs upon Nf1 inactivation in Prss56positive BC cells indicates that the heterogeneity of BC cells has profound physiopathological relevance.

It is striking that, despite Nf1 inactivation in BC cells around E11, most Nf1-KO animals develop cNFs only after 10 months of age. Our time-course analysis reveals that this late onset is due to a very slow but steady progression of the cutaneous disease. We have defined four stages in the development of the disease, shown in a schematic in Fig. S6. There is no indication of disease until E15.5, apart from an increased number of traced melanoblasts. At birth (Fig. S6A, stage 1), the number of traced SCs in the dermis has increased to almost the double the control number (mild hyperplasia), however with no significant morphological modifications of the cells. At three months (Fig. S6B, stage 2), the fold increase in traced SCs in Nf1-KO over control animals is even greater (severe hyperplasia) and their morphology is modified (hypertrophy), although most are still in contact with axons, which have undergone abnormal defasciculation and branching. This stage also involves an accumulation of fibroblasts, due to accretion and/or proliferation, and of collagen fibres presumably synthesized by these fibroblasts. At six months (Fig. S6C,stage 3), numerous micro-cNFs are present all over the back skin, which share all the characteristics of full-blown cNFs, apart from their size. They contain dense aggregates of mutant SCs that show altered morphologies and frequent loss of contact with the axons. These aggregates are massively infiltrated by fibroblasts, mastocytes, macrophages and neutrophils. Inflammation is therefore a major hallmark of this stage. From 12 months of age (Fig. S6D, stage 4), morphologically visible, diffuse cNFs are often associated with pruritus. On the basis of these observations, we believe that tumour progression in cNFs involves three decisive transitions: i) an increase in the level of proliferation of SCs during the late embryonic period; ii) initiation of morphological changes of SCs and the accumulation of fibroblasts between stage 1 and 2, establishing a pre-tumourous state; iii) loss of SC axonal contact and the establishment of an inflammatory process that corresponds the progression to a tumourous state, between stage 2 and 3. The transition between stage 3 and 4 may simply be a slow increase in the tumour size.

Interestingly, before stage 2, the elevation of the SCs number appears to be compensated by increased branching of nerve bundles, allowing the supernumerary SCs to maintain appropriate contacts with axons. This observation also raises the possibility that the defasciculation/branching phenomenon might actually be induced by the excess of SCs. It is possible that this putative compensatory mechanism has limitations and, when overridden, SCs can no longer interact with axons, possibly favouring a pro-tumourigenic phenotype. The branching capacity of nerve terminals in the upper dermis might be related to the absence of perineurium in this layer. Increased skin innervation might explain the skin hypersensitivity and neuropathic pain reported in approximately 10% of NF1 patients [START_REF] Drouet | Neurofibromatosis 1-associated neuropathies: a reappraisal[END_REF]. If confirmed, this link could be used to predict sites of cNF development. Intriguingly, skin trauma is also accompanied by active proliferation of SCs, their dissociation from nerves and local increase of density of innervation, further supporting the impact of peripheral glia and/or inflammation on axonal branching [START_REF] Parfejevs | Injury-activated glial cells promote wound healing of the adult skin in mice[END_REF].

Presence of highly innervated microscopic neurofibromas, often associated with hair follicle apparatus was reported in clinically asymptomatic skin of NF1 patients ((26) and F.

Rice, personal communication)). In our model, despite an identical cellular composition of diffuse cNFs, their association with skin adnexa cannot be firmly established due to a higher density of hair follicles present in the mouse skin. More generally, the fact that micro-cNFs in the asymptomatic patient skin might prefigure cNFs may have profound consequences in terms of therapy and raises exciting possibilities for the development of preventive treatments. For instance, reducing proliferation of SCs, preventing their hypertrophy and/or dissociation from axons, or inhibiting the recruitment and/or activity of dermal fibroblasts and immune effector cells might constitute key entry points for developing effective anti-tumour agents. Such approaches would strongly benefit from the development of non-invasive methodologies for the early detection and monitoring of micro-cNFs. The systematic development of cNFs in our mouse model makes it a powerful tool for the screening of candidate therapeutic molecules. Furthermore, the availability of the Cre-inducible fluorescent reporter that allows identification of Nf1 mutant cells allows a continuous survey of the impact of the drug.

The similarity between patients and mice at the "pathological" level is surprising in view of their differences at the genetic level. Previous studies suggested that NF1 heterozygosity in the cellular microenvironment (particularly in mastocytes) was necessary for initiation and development of cNFs [START_REF] Yang | Nf1-Dependent Tumors Require a Microenvironment Containing Nf1+/--and c-kit-Dependent Bone Marrow[END_REF]. However, more recent studies performed in mice and humans suggest that heterozygosity in mastocytes is dispensable for tumour development [START_REF] Liao | Contributions of inflammation and tumor microenvironment to neurofibroma tumorigenesis[END_REF]. Our observations are consistent with these latter findings as NFs develop in both Nf1 +/- and wild type backgrounds. Another difference is that all BC cell derivatives have undergone a homozygous loss of Nf1 in our model, whereas this condition is sporadic in human. A key factor might be the difference in lifetime of both species. Only a minority of the possible tumours might develop during the short lifetime of the mouse, an interpretation supported by the dramatic density of tumours observed in a 20-month-old mutant animal. In addition to NFs, all NF1 patients develop a number of pigmentary lesions, including café au lait macules that correspond to accumulation of epidermal melanocytes. In our model, BC-derived melanocytes carry biallelic Nf1 loss whereas all other melanocytes are either Nf1 +/-or wild type (TOM-). Although café au lait macules do not develop in mice, due to the absence of melanocytes in the interfollicular epidermis, we observed accumulation of Nf1-KO melanocytes in dermal compartment starting from late embryogenesis. Moreover, we noticed marked accumulation of Nf1-KO and wild type pigmented melanocytes in cNFs. This is in line with the presence of pigmented cNFs in NF1 patients. In conclusion, NF1-associated dysregulation of melanocytogenesis and/or melanin synthesis is a common feature in NF1 patients and in our model.

MATERIALS AND METHODS

Animals

Mice used in this study were housed in a temperature-and humidity-controlled vivarium on a 12 h dark-light cycle with free access to food and water. The following mouse lines were used and genotyped as described in the original publications: Nf1 flox/flox and Nf1 flox/-(4), Prss56 Cre (6), R26 tdTom [START_REF] Madisen | A robust and high-throughput Cre reporting and characterization system for the whole mouse brain[END_REF], Dhh Cre (30), Krox20 Cre [START_REF] Voiculescu | Expression pattern of aKrox-20/Cre knock-in allele in the developing hindbrain, bones, and peripheral nervous system[END_REF]. We bred Prss56 Cre ,R26 tdTom mice with the Nf1 flox/flox mice to obtain the F1 generation (Prss56 Cre/+ ,R26 tdTom/+ ,Nf1 flox/+ ); we bred F1 mice with Nf1 flox/flox and Nf1 +/-mice to obtain Prss56 Cre ,R26 tdTom ,Nf1 flox/flox and Prss56 Cre ,R26 tdTom ,NF1 flox/-mutants, respectively. Prss56 Cre ,R26 tdTom ,Nf1 flox/+ animals were used as controls. For embryo analysis, we established timed pregnancies with the morning of a vaginal plug defined as E0.5. All animal manipulations were approved by a French Ethical Committee (APAFIS#1882-2015092215426712) and were performed according to French and European Union regulations.

Skin incisions and tissue processing

3-month-old control and Nf1-KO mice were anesthetized with Isoflurane. Dorsal skin at the lateral thoracic level was shaved and disinfected with 70% ethanol. Preoperative analgesia was applied using subcutaneous injection of Buprenorphine (0.1 mg/kg). 5 mm-long fullthickness incisions were performed unilaterally with surgical micro-scissors and sutured.

Animals were perfused with 4% paraformaldehyde (PFA) on day 2, 4, 7 and 14 after surgery, and 7 months after surgery. The back skin was dissected and processed for immunohistochemical analysis. The 14 dpi wounds and unwounded skin of three Nf1-KO and three control animals were excised post-portem, cleaned of subcutaneous fat and homogenized in RNA lysis buffer for subsequent RNA isolation (RNesay Micro Kit, Qiagen).

Electron microscopy

For standard ultrastructure analysis, mice were perfused with 4% PFA/1.6% glutaraldehyde (Polysciences) in 0.1 M phosphate buffer. Dorsal skin, nerve roots and subcutaneous nerves were dissected and post-fixed in the same solution overnight at 4°C, followed by embedding in Epon as previously described [START_REF] Gresset | Boundary Caps Give Rise to Neurogenic Stem Cells and Terminal Glia in the Skin[END_REF]. Semi-thin sections (1 µm) were stained with toluidine blue and ultra-thin sections were stained with Uranyl Acetate and analysed using a Jeol microscope. For immuno-electron microscopy, mice were perfused with 4% PFA/0.5% glutaraldehyde and samples were embedded in LR white (Sigma Aldrich) as described [START_REF] Peltonen | Molecular and Cellular Basis of Human Cutaneous Neurofibromas and Their Development[END_REF].

TOM-expressing cells were detected using rabbit anti-RFP antibody (1:20, Rockland #600-401-379).

Statistical analysis

Statistical analyses were carried out using two-tailed Student t-tests or non-parametric Mann-Whitney tests (for quantification of the melanocyte lineage and electron microscopy-based counting), and p-values considered significant are indicated by asterisks as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical analyses and scatter plots were generated using the GraphPad Prism 6.0 package. The data are represented as mean values ± standard deviation (SD) or standard error of the mean (SEM).

Analysis of cell proliferation

Pregnant mice were intraperitoneally injected with EdU at the dose of 20 mg per kg of body mass at embryonic age E18.5 and newborn pups were collected 24h later. Three Nf1-KO and two control littermates were decapitated, and their skin was dissected and fixed overnight in 4% PFA. EdU detection was performed on 20 µm-thick cryosections using the Click-IT Plus EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) according to the manufacturer's instructions, followed by immunolabelling for TOM and TRP2. Numbers of proliferating Schwann cells (TOM+/TRP2-/EdU+) were normalised against the total number of traced (TOM+/TRP2-) SCs. Quantifications were performed on 4 distant sections from each individual (~24 fields per sample) using 25X magnification under a TCS SP5 confocal microscope. The two-tailed Mann-Whitney test was used for statistical analysis. P values < 0.05 were considered statistically significant.

Library preparation, RNA sequencing and data analysis cDNA libraries, RNA sequencing library preparation and Illumina sequencing were performed at the Ecole normale supérieure genomic core facility (Paris, France). 10 ng of total RNA was amplified and converted to cDNA using SMART-Seq v4 Ultra Low Input RNA kit (Clontech). An average of 200 pg of amplified cDNA was used for library preparation using Nextera XT DNA kit (Illumina). Libraries were multiplexed by 38 on 3 high output flow cells. 75 bp sequencing reads were performed on a NextSeq 500 device (Illumina). The analyses were performed using the Eoulsan pipeline [START_REF] Jourdren | Eoulsan: a cloud computing-based framework facilitating high throughput sequencing analyses[END_REF], including read filtering, mapping, alignment filtering, read quantification, normalization and differential analysis. Prior to mapping, poly-N read tails were trimmed, reads ≤40 bases and with mean quality ≤30 were discarded. Reads were then aligned against the Mus Musculus genome from Ensembl version 91 using STAR (version 2.5.2) [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF]. Alignments from reads matching more than once on the reference genome were removed using Java version of SamTools [START_REF] Li | The Sequence Alignment/Map format and SAMtools[END_REF]

Finally, we found

  that local mechanical skin injury promotes the development of cNFs, not only at the site of the injury, but also at distant sites. Wounding Nf1-KO skin triggers massive local proliferation of denervated Nf1 -/-SCs within the first week postsurgery, suggesting the involvement of inflammation-related signals. Consistently, transcriptome profiling of wounded vs intact control and Nf1-KO skin led to the identification of several candidate molecules linked to inflammation that are specifically overexpressed in Nf1-KO skin upon injury and may play a role in this activation of SCs. Some of them have been previously reported as highly expressed in human and/or mouse neurofibromas. Soluble factors attract particular attention as they might promote development of cNFs both locally and remotely. Modulation of the activity of such factors and/or of their receptors might constitute an interesting option to impede cNF growth.

Figure 1 :

 1 Figure 1: Tracing of Prss56-positive BC cell derivatives in the adult PNS (A-D) Analysis of BC cell derivatives (TOM+) in the adult nerve roots of Prss56 Cre , R26 tdTom

Figure 2 :

 2 Figure 2: Targeted Nf1 inactivation in the BC cell lineage gives rise to paraspinal and

Figure 4 :

 4 Figure 4: Nf1 loss-of-function in BC cells promotes proliferation of dermal Schwann cell and

Figure 5 :

 5 Figure 5: 6-month-old Nf1-KO mutant skin contains numerous micro-cNFs

Figure 6 :

 6 Figure 6: Skin injury promotes development of cNFs in Nf1-KO mice

  Downloaded from http://aacrjournals.org/cancerdiscovery/article-pdf/doi/10.1158/2159-8290.CD-18-0156/2929956/2159-8290_cd-18-0156v1.pdf by guest on 20 October 2022 Together, our observations indicate that Prss56-expressing BC cells participate in the development of nerve roots and cutaneous/subcutaneous innervation as a source of glial and non-glial derivatives.

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  .05 and FC>4x were used as cutoffs criteria to select differentially expressed genes (DEGs). The RNA-seq gene expression data and raw Fastq files are available on the GEO repository (www.ncbi.nlm.nih.gov/geo/) under accession number GSE117781.

	Pathway over-representation analysis was performed using the WebGestalt web server
	. To (http://www.webgestalt.org). DEG sets were queried against the KEGG database using FDR q<0.05 cutoff to select significantly enriched pathways.	Downloaded from http://aacrjournals.org/cancerdiscovery/article-pdf/doi/10.1158/2159-8290.CD-18-0156/2929956/2159-8290_cd-18-0156v1.pdf by guest on 20 October 2022
	compute	gene	expression, Mus	musculus GFF3	genome	annotation
	version 91 from Ensembl database was used. All overlapping regions between alignments and

referenced exons were counted using HTSeq-count 0.5.3

[START_REF] Anders | HTSeq--a Python framework to work with highthroughput sequencing data[END_REF]

. The sample counts were normalized, then statistically analyzed using DESeq 1.8.3. P-value <0

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DECLARATION OF INTERESTS

The authors declare no competing interests.


Lydia Yefsah
email: lydia.yefsah@lepmi.grenoble-inp.fr

Giuseppe Sdanghi

Giuseppe Sassone

Jean-Marc Bassat

Maxime Hubert

Elisabeth Djurado

Jérôme Laurencin

Reaction mechanisms of mixed ionic and electronic conductors used as oxygen

A deep understanding of the reaction mechanisms taking place in a SOC oxygen electrode is a prerequisite before microstructural optimization. In this work, we propose a coupled experimental and modeling approach aimed to investigate the reaction mechanisms for both under-and over-stoichiometric oxygen electrode materials. Two main reaction pathways have been considered, the oxygen incorporation/excorporation at the gas/electrode interface for the bulk path and the direct charge transfer at the Triple Phase Boundary lengths (TPBls) for the surface path. Polarization curves and impedance diagrams under various operating conditions (650-750°C, 0.15 -1 atm, and ± 100 mA cm -2 ) have been recorded by using a three-electrode setup. These data were used to calibrate the models based on elementary reactions. In addition, all the microstructural parameters required for the simulation have been computed on a 3D electrode reconstruction obtained by FIB-SEM tomography. A change from the bulk towards the surface path was observed for the LSCF electrode under low anodic polarization, which was explained by the progressive depletion of oxygen vacancies when increasing the anodic polarization. Conversely, a limitation of the bulk path was observed under cathodic polarization for the LNO electrode, which was due to the significant reduction of the oxygen over stoichiometry under these conditions. The impact of the temperature and the pO2 on the reaction pathways has been also investigated for both materials and is discussed in this article.

Introduction

SOCs are high-temperature electrochemical energy-conversion devices that produce efficiently clean electricity (solid oxide fuel cell, SOFC mode) or hydrogen (solid oxide electrolysis cell, SOEC mode). These technologies are composed of an assembly of two porous electrodes separated by a thin dense electrolyte in Yttria Stabilized Zirconia (YSZ). The porous hydrogen electrode is usually a Nickel and YSZ cermet while mixed ionic and electronic conductors (MIECs) are mostly developed as oxygen electrode materials. This mixed property allows extending the oxygen reaction on the whole gas/electrode interface. The classical MIECs material is the lanthanum doped strontium cobalt ferrite La1-xSrxCoy-1FeyO3-δ (LSCF) [1] [2]. This oxygen-deficient perovskite presents a good ionic and electronic conductivity as well as a high oxygen surface exchange rate. However, LSCF suffers from large degradation rates due to chemical decomposition at high operating and reactivity with electrolyte [START_REF] Pan | Effect of Sr Surface Segregation of La0.6Sr0.4Co0.2Fe0.8O3 -δ Electrode on Its Electrochemical Performance in SOC -IOPscience[END_REF] [4] [START_REF] Uhlenbruck | Element interdiffusion at electrolyte-cathode interfaces in ceramic high-temperature fuel cells[END_REF]. These degradations induce significant loss in SOCs performances, especially when they are operated in SOEC mode. In this context, rare earth nickelates Ln2NiO4+δ (Ln = La, Pr, Nd) are considered nowadays as alternative oxygen electrode materials. The crystal structure of this over-stoichiometric material consists of LnNiO3 perovskite block inter-grown between LnO rock-salt layers. The excess oxygen occupies interstitial sites within the LnO layers. Among the different components, La2NiO4+δ (LNO) has been chosen in this study thanks to its chemical stability in a wide range of temperature [START_REF] Vibhu | Influence of La/Pr ratio on the ageing properties of La2-xPrxNiO4+ δ as cathodes in IT-SOFCs[END_REF]. This work aims to analyze, by an experimental and a modeling approach, the effect of the temperature, polarization, and oxygen partial pressure on the reaction pathways for LSCF and LNO electrodes.

Scientific Approach

Experiments

The two electrode materials, LSCF and LNO, were deposited by screen-printing on each side of a circular and dense 8YSZ electrolyte (thickness = 260 µm, diameter = 25 mm). To prevent the reactivity with YSZ that leads to the formation of insulating phases [START_REF] Anderson | Reactivity of lanthanide ferrite SOFC cathodes with YSZ electrolyte[END_REF] [8], a Ce0.8Gd0.2O2-δ (CGO) barrier layer of 2 µm of thickness was added in between the electrodes, and the electrolyte. The symmetrical cells were then tested in a 3-electrodes setup. A platinum wire was positioned at the periphery of the electrolyte membrane and used as a reference electrode (RE). Two platinum grids with a mesh of 3600 meshes.cm -2 and a surface equal to the electrodes were used as current collectors. It can be noticed that a thick disk in porous gold was also added between the ceramic housing and the platinum grids so that each electrode surface can be considered equipotential. A load of 0.7 kg/cm² was applied to ensure the electric contact between the metallic grids and the electrodes. At the beginning of the test, the cells were heated up to 800°C at a rate of 1°C/min. This temperature has been maintained during several hours until the stabilization of the electrochemical impedance spectra (EIS) measured at open circuit potential (OCP).

Electrochemical characterizations were then carried out at a temperature of 750, 700 and 650°C under air condition, with a gas flow rate of ≈ 2NL.min -1 . For each temperature, polarization curves have been recorded for both cells whereas EIS diagrams were acquired at OCP, in a frequency range of 10 6 to 10 -2 Hz, with an amplitude of ± 10 mA/cm -2 . For the temperature of 700°C, the electrochemical characterizations were complemented by EIS B1601 / Page 3-10 diagrams measured under anodic and cathodic current (with idc = ± 50 mA/cm² for LSCF electrode and idc = ± 100 mA.cm² for LNO electrode) and over a large range of oxygen partial pressure from 0.1 to 1 atm.

Model Description

An elementary model framework for MIEC electrodes has been developed and used to investigate the LSCF and LNO reaction mechanisms occurring under anodic and cathodic polarization. In this approach, surface and bulk species are taken into account as well as their transport in the electrodes phases. A temporal term is also added to each mass/charge balance for the EIS computations. The mass transfer in the LNO and LSCF is calculated through the chemical diffusivity of the oxygen interstitials or vacancies, respectively, while the electronic current density is modeled using a classical Ohm's law. Both molecular and Knudsen diffusions are taken into account for the mass transport in the gas phase. Finally, the diffusion of oxygen adatoms 𝑂𝑂 𝑎𝑎𝑎𝑎𝑎𝑎 , oxygen ions 𝑂𝑂 𝑎𝑎𝑎𝑎𝑎𝑎 -and molecular oxygen 𝑂𝑂 2 𝑎𝑎𝑎𝑎𝑎𝑎 at the surface of both materials is computed using a classical Fick's law. The proposed model considers two possible reaction pathways for oxygen oxidation/reduction divided into a sequence of elementary steps as represented in Figure 1 in the electrolysis mode. The "bulk path" is characterized by an ionic transfer at the electrode/electrolyte interface (R1). After diffusion of the oxygen interstitials (for LNO) or oxygen vacancies (for LSCF) in the bulk of the electrode material, the oxygen is excorporated to produce oxygen adatoms on the electrode surface (R2). In parallel, the "surface path" considers direct oxidation at the TPBls (R3) leading to the formation of oxygen ions on the electrode surface. Then, in both cases, the second reaction of charge transfer (R4) takes place on the surface to produce oxygen adatoms that can be associated (R5) and desorbed (R6) in the porosity of the electrodes. The kinetic constants have been estimated by calibrating the model on the LSCF and LNO polarization curves. Due to the influence of the microstructure on the electrochemical response, all the microstructural properties used as inputs in the model have been extracted from 3D reconstructions obtained for both electrodes. Thereafter, the model has been validated using the electrochemical impedance diagrams recorded at OCP and under polarization for different oxygen partial pressures. The elementary kinetic model has been already published for the LSCF electrode [START_REF] Effori | An Elementary Kinetic Model for the LSCF and LSCF-CGO Electrodes of Solid Oxide Cells: Impact of Operating Conditions and Degradation on the Electrode Response[END_REF] while the one for the LNO is an adaptation of a simplified version available in [START_REF] Sdanghi | Reaction Mechanisms of La 2 NiO 4+δ Oxygen Electrodes Operated in Electrolysis and Fuel Cell Mode[END_REF].

Experimental Results

The experimental electrode polarization curves η-i at 650°C, 700°C, and 750°C are represented in Figure 2 for the LSCF and LNO oxygen electrodes. As expected, it can be noticed that the electrode performances are thermally activated [START_REF] Tong | Performance and stability of Ruddlesden-Popper La2NiO4+δ oxygen electrodes under solid oxide electrolysis cell operation conditions[END_REF] [12]. To the OCP, a non-symmetrical shape of the η-i curves is observed at 650°C and 700°C for both electrodes. However, it is worth mentioning that the electrode performances are better under anodic polarization (SOEC mode) for the LNO electrode, whereas the LSCF electrode exhibits lower overpotentials under cathodic current (SOFC mode). Within the narrow investigated overpotential range, a linear behavior of the η-i curves is observed at 750°C. The dissymmetry of the polarization curves at a lower temperature can be explained either by a change in the reaction mechanism or the rate-determining steps. Conversely, a unique dominant mechanism with the same co-limitations could explain the linearity of η-i curves at 750°C in the studied conditions. The EIS diagrams acquired at OCP under air at 650 °C, 700 °C, and 750 °C are shown in Figure 3 for the LNO and LSCF materials. It is worth noting that the absolute values of the polarization resistances cannot be directly compared between the two electrodes since they present rather different microstructures due to the fabrication. Nevertheless, the shape of the impedance spectra can give key information about the reaction mechanisms. Indeed, for the LSCF electrode, the impedance spectra show a "Gerischer-type" element, whatever the temperature. A small contribution can be noticed at low frequencies on the EIS diagram at 750°C which can be attributed to a limitation in the gas conversion reaction [START_REF] Nielsen | Impedance of porous IT-SOFC LSCF:CGO composite cathodes[END_REF]. On the contrary, the EIS diagrams at OCP of the LNO electrode show a different evolution depending on the temperature. A "Gerisher-type" element is observed at 650°C whereas the diagrams present a more depressed semi-circle at 700°C and 750°C. This evolution suggests an influence of the temperature on the reaction mechanism at OCP for the LNO electrode. On the other hand, the non-evolution of the shape of the EIS diagrams for the LSCF electrode may suggest the presence of a unique dominant reaction mechanism in the range of the studied temperature.

B1601 / Page 5-10 For both cases, the EIS diagrams recorded under dc current density at 700°C have not revealed a change in the shape of the impedance spectra [START_REF] Effori | An Elementary Kinetic Model for the LSCF and LSCF-CGO Electrodes of Solid Oxide Cells: Impact of Operating Conditions and Degradation on the Electrode Response[END_REF] [10] compared to the OCP. These results suggest that, for both electrodes, there is no change in the reaction mechanism at 700°C under polarization for the limited investigated range of dc current. The evolution of the impedance spectroscopy with the oxygen partial pressure is shown in Figure 4. As expected, the polarization resistance of both electrodes decreases with increasing pO2.

Modeling Results and Discussion

Model calibration and validation

The missing parameters for the models (i.e. the forward kinetic constants for all the reactions, the surface diffusion coefficients, and surface coverages at equilibrium for pO2=0.21 atm [START_REF] Effori | An Elementary Kinetic Model for the LSCF and LSCF-CGO Electrodes of Solid Oxide Cells: Impact of Operating Conditions and Degradation on the Electrode Response[END_REF] [10]) have been obtained by adjusting the simulations on the experimental polarization curves. The results of this fitting procedure are given in Fig. 2 for the two B1601 / Page 6-10

electrodes. Once the model was calibrated, the validation was carried out by simulating the impedance spectra without additional fitting. As shown in Fig. 3, the model for LSCF or LNO electrode can simulate accurately the impedance diagrams at OCP for the three investigated temperatures. Moreover, the reaction order n was deduced from the experimental and simulated impedance spectra (Fig. 4) according to : 𝑅𝑅 𝑝𝑝𝑝𝑝𝑝𝑝 ∝ (𝑝𝑝𝑂𝑂 2 ) -𝑛𝑛 (1) These simulated reaction orders are in good agreement with the experimental ones. Indeed, the errors between the computed and experimental coefficients do not exceed 12% for LSCF and only 9% for LNO. Therefore, the model for the two electrodes can be considered validated.

Effect of polarization, temperature, and pO2 on the reaction pathways

Thanks to the model, the electrode reaction mechanism can be studied as a function of the temperature, polarization, and oxygen partial pressure. For this purpose, the parameter ξ is introduced to quantify the relative contribution of the surface or bulk path in the electrode operation. It is expressed as the kinetic rate ratio between (i) the oxygen ad-ions produced at the TPBls for the surface path and (ii) the oxygen ad-ions produced by excorporation all along with the electrode thickness for the bulk path:

𝜉𝜉 = 𝑣𝑣 3 � 𝑣𝑣 2 𝑑𝑑𝑑𝑑 𝑧𝑧=ℓ 𝑧𝑧=0 � (1)
where 𝑣𝑣 3 and 𝑣𝑣 2 are kinetic rates for the charge transfer reactions R3 and R2, respectively. The term ℓ represents the electrode thickness. According to this definition, the reaction mechanism is dominated by the "bulk path" when the ratio is lower than one (ξ < 1). On the opposite, a value higher than one (ξ > 1) indicates a mechanism controlled by the "surface path".

For the LSCF electrode, Figure 5a shows an increase of the ratio ξ when increasing the anodic polarization. A transition from the bulk path (ξ < 1) to the surface path (ξ > 1) is thus observed at low anodic current density for the three temperatures. This behavior can be explained by the evolution of the kinetics rates plotted in Figure 5b as a function of the current density. Indeed, when the anodic polarization increases, the oxygen excorporation rate becomes bounded while the direct oxidation at TPBs remains activated. This behavior is related to the strong decrease of the oxygen vacancies concentration in the LSCF when increasing the anodic polarization [14] [15]. In other words, the under-stoichiometry δ falls to zero under anodic polarization in such a way that the material can no longer accept oxygen [START_REF] Bishop | Surface and Bulk Defect Equilibria in Strontium-Doped Lanthanum Cobalt Iron Oxide[END_REF]. In parallel, the surface concentrations of the adsorbed species involved in the surface path are increased and remain unlimited (since the sum of surface coverages of all the adsorbates remains lower than the coverage of the available sites). On the other hand, the opposite trend is observed under cathodic polarization. In this case, the incorporation reaction is activated because the concentration of vacancies is increased when increasing the cathodic polarization without limitation (indeed, δ remains lower than δ max ). Conversely, the charge transfer at TPBs becomes limited since the surface coverages decreases down to zero.

The same behavior is obtained for the three investigated temperatures (Figure 5a). Nevertheless, the influence of temperature on the reaction mechanism is not negligible. Indeed, the contribution of the surface path becomes rapidly more important at 650°C (Figure 5a). At intermediate temperatures (700°C and 750°C), the slopes of the curves are B1601 / Page 7-10 less steep. These results explain the strong dissymmetry of the experimental polarization curves at 650°C while a more linear behavior is observed at 750°C. To summarize, for the LSCF oxygen electrode, the bulk path appears as the dominant pathway under fuel cell conditions regardless of the temperature. For electrolysis mode, a transition from the bulk path to the surface path appears when the anodic polarization is increased. This transition occurs when the oxygen vacancy concentration tends to zero. The same analysis has been conducted on the over-stoichiometric LNO which exhibits an opposite behavior compared to the LSCF. Indeed, as shown in Figure 6a, the surface-tobulk path ratio decreases with increasing the overpotentials from cathodic to anodic polarization. This behavior can also be explained by the kinetics rates of the charge transfer at TPBls and the oxygen excorporation/incorporation reaction (integrated along with the electrode thickness) plotted as a function of the electrode overpotential (Figure 6b). Indeed, under cathodic polarization, the charge transfer at TPBls remains strongly activated while the oxygen incorporation reaction becomes bounded. This behavior, which is responsible for the limitation of the bulk path, is directly linked to the concentration of oxygen interstitial in the LNO. Indeed, the simulations have revealed that the concentration of oxygen interstitial becomes very low under cathodic polarization. According to Fig. 6b, the surface path is thus predominant under the investigated range of the cathodic currents. Nevertheless, the charge transfer at TPBls should also tend towards a limitation when the surface coverage at the LNO surface falls to zero. However, it seems that this limitation is not reached even for the higher cathodic polarization achieved in this work.

Under anodic polarization, both surface and bulk paths remain activated (Fig. 6b). Indeed, the concentration of oxygen interstitial is increased with increasing the polarization without reaching the maximum value of oxygen over-stoichiometry (i.e. δ remains much lower than 𝛿𝛿 𝑚𝑚𝑎𝑎𝑚𝑚 = 0.15 for LNO [START_REF] Nakamura | The determining factor for interstitial oxygen formation in Ruddlesden-Popper type La 2 NiO 4 -based oxides[END_REF]). On the other hand, the direct oxidation at TPBs is still promoted since the adsorption sites are not saturated under the applied anodic polarization. These observations allow explain the results obtained on the experimental electrode polarization curves η-i (Figure 2b). Indeed, the higher performances of the LNO electrode under anodic polarization are explained by the contribution of both bulk and surface paths to the reaction mechanism while only the surface path remains activated under cathodic polarization.

B1601 / Page 8-10

The impact of the temperature on the ratio is shown in Figure 6a. At OCP, the bulk path at 650°C controls the reaction mechanism (ξ < 1) while the surface path becomes predominant at higher temperature (ξ > 1). Thus, it can be stated that the temperature promotes the contribution of the charge transfer at the TPBls for the LNO electrode. Simulations have also been carried out by changing the oxygen partial pressure from 0.10 atm to 1 atm (Figure 7). Whatever the current density, it has been found that the ratio increases with increasing pO2 for the LSCF electrode. This statement is explained because the under-stoichiometry is reduced at high oxygen partial pressure [START_REF] Bishop | Surface and Bulk Defect Equilibria in Strontium-Doped Lanthanum Cobalt Iron Oxide[END_REF]. The opposite behavior has been obtained for the LNO electrode. Indeed, the concentration of interstitials increases with the oxygen partial pressure. From this analysis, it appears that the LNO over-stoichiometric material is especially well adapted for an operation in the electrolysis mode. Indeed, it has been shown that both surface and bulk paths are promoted under anodic polarization. This result is consistent with the higher performances obtained for the material when operated in SOEC mode. Regarding the oxygen-deficient LSCF material, it has been found that the bulk path is predominant for cathodic polarization whereas the surface path becomes favored under anodic current.

B1601 / Page 9-10

These statements mean that the material can be operated in both modes. However, its performances are slightly higher in fuel cell mode. To enhance the surface path and thus increase the electrode efficiency, it has been shown that a composite electrode made of LSCF/CGO is a relevant solution [START_REF] Effori | Reaction Mechanism and Impact of Microstructure on Performances for the LSCF-CGO Composite Electrode in Solid Oxide Cells[END_REF]. The precise role of CGO in an LNO/CGO composite electrode on its operation and performances still needs to be studied. Moreover, the stability of these electrodes for long-term operations remains also a subject of investigation for the two materials.

Conclusion

Elementary models have been developed and used to predict the electrochemical response of the classical oxygen-deficient oxide La1-xSrxCoy-1FeyO3-δ (LSCF) and the alternative oxygen over-stoichiometric material La2NiO4+δ (LNO). To validate the models, dedicated experiments have been performed on symmetrical cells using a three-electrode configuration. For the two materials, the determination of the unknown parameters of the models has been done by fitting the experimental electrode polarization curves at three temperatures (650°C, 700°C, and 750°C). For the validation, the impedance spectra at OCP and under polarization for different oxygen partial pressures have been computed without additional fitting. A good agreement between the experimental and the simulations has been obtained.

Once validated, the models have been used to analyze the electrochemical response of the LSCF and LNO materials by investigating the reaction mechanisms as a function of the polarization. For the LSCF, the 'bulk' path related to the oxygen exchange with the material appears as the dominant pathway under cathodic current regardless of the temperature. However, the 'surface' path associated with the charge transfer at TPBls is activated under anodic polarization. This behavior has been ascribed to the strong decrease of the oxygen vacancies concentration in the LSCF when increasing the anodic polarization.

For the LNO electrode, it has been found that both the 'bulk' and 'surface' path are favored under anodic current while only the 'surface' path remains active under cathodic polarization. In this case, the limitation of the 'bulk' path is due to the decrease of the oxygen interstitial concentration in the LNO lattice. These results have allowed explain the better performances obtained for this material in SOEC mode.

Figure 1 -

 1 Figure 1 -Schematic description of the reaction pathway considered in the elementary model (in electrolysis mode) represented for the LSCF electrode.

Figure 2 -

 2 Figure 2 -Experimental (marks) and simulated (solid lines) polarization curves obtained at 650, 700, and 750 °C under air for a) LSCF and b) LNO electrodes.

Figure 3 -

 3 Figure 3 -Experimental (marks) and simulated (solid lines) EIS diagrams under air at 650 °C, 700 °C, and 750 °C for LSCF (a, b, c) and LNO (c, d, f) electrodes

Figure 4 -

 4 Figure 4 -Impedance diagrams (Nyquist plots) recorded at 700°C for the investigated range of oxygen partial pressure for a) LSCF and b) LNO electrodes.

Figure 5 -

 5 Figure 5 -(a) Ratio of the surface to the bulk path as a function of the current density plotted for the LSCF electrode at 650°C, 700°C, 750°C, and 800°C. (b) Kinetics rates for the charge transfer at TPBs and the oxygen excorporation/incorporation reaction (integrated along with the electrode thickness) as a function of the current density at 700°C, for the LSCF electrode.

Figure 6 -

 6 Figure 6 -(a) Ratio of the surface to the bulk path as a function of the current density plotted for the LNO electrode at 650°C, 700°C, and 750°C. (b) Kinetics rates for the charge transfer at TPBs and the oxygen excorporation/incorporation reaction (integrated along with the electrode thickness) as a function of the current density at 700°C, for the LNO electrode. (c) Oxygen over stoichiometry in the electrode thickness, under air, at 700°C, at different polarization.

Figure 7 -

 7 Figure 7 -Ratio of the surface to the bulk path as a function of the electrode overpotential at OCP and 700°C for different oxygen partial pressure for (a) LSCF and (b) LNO.


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The association between macrovascular complications and intensive care admission, invasive mechanical ventilation, and mortality in people with diabetes hospitalized for coronavirus disease-2019 (COVID-19)

Gemma Llauradó, Bogdan Vlacho, Matthieu Wargny, Yue Ruan, Josep Franch-Nadal, Pere Domingo, Pierre Gourdy, Pierre-Jean Saulnier, Samy Hadjadj, Sarah Wild, et al.

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Gemma Llauradó, Bogdan Vlacho, Matthieu Wargny, Yue Ruan, Josep Franch-Nadal, et al.. The association between macrovascular complications and intensive care admission, invasive mechanical ventilation, and mortality in people with diabetes hospitalized for coronavirus disease-2019 (COVID-19). Cardiovascular Diabetology, 2022, 21 (1), pp.216. 10.1186/s12933-022-01657-8. hal-03822575