Mao Kuriki

Glenda Comai

Shahragim Tajbakhsh

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle progenitors

Keywords: Extraocular muscles, Pitx2, Myf5, Pax7, muscle stem cells, morphogenesis

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression and showed that this gene becomes progressively dispensable for specification and maintenance of the extraocular muscle (EOM) stem cell pool, yet it is the major EOM upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally showed a greater loss of muscle stem cells in the EOM compared to the limb, pointing to a relay between Pitx2, Myf5 and Pax7 for maintenance of the EOM stem cells. Further, we demonstrate that EOM stem cells adopt a quiescent state earlier that those in limb muscles and do not spontaneously re-enter in proliferation in the adult as previously suggested, yet EOMs have a significantly higher content of Pax7+ muscle stem cells per area pre-and post-natally. This unique feature could result from different dynamics of lineage progression in vivo, given the lower fraction of committed and differentiating EOM myoblasts. Finally, significantly less MuSCs are present in EOM compared to the limb in the mdx mouse model for Duchenne muscular dystrophy. Overall, our study provides a comprehensive in vivo characterization of muscle stem cell heterogeneity along the body axis and brings further insights into the unusual sparing of EOM during muscular dystrophy.

INTRODUCTION

Skeletal muscle satellite (stem) cells (MuSCs) arise during late foetal development, and they contribute to postnatal growth of muscle fibers. They are the main players for reconstituting muscle fibres during regeneration. Interestingly, this population of stem cells emerges in an asynchronous manner in different anatomical locations and it exhibits an unusual diversity in developmental origins and genetic regulation depending on the muscle group in which it resides (Comai et al., 2014;[START_REF] Grimaldi | Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse[END_REF][START_REF] Grimaldi | Diversity in cranial muscles: Origins and developmental programs[END_REF]. Understanding the physiological mechanisms controlling the development of different muscle groups, and how they are selectively deregulated or spared during disease and ageing, is crucial for devising therapeutic strategies particularly for a wide range of myopathies [START_REF] Emery | The muscular dystrophies[END_REF][START_REF] Porter | Crush injury and crush syndrome: a consensus statement[END_REF]. Extraocular muscles (EOMs) are known to be spared during ageing and in Duchenne, Becker, and some limb girdle muscular dystrophies [START_REF] Emery | The muscular dystrophies[END_REF][START_REF] Kallestad | Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis[END_REF][START_REF] Porter | Crush injury and crush syndrome: a consensus statement[END_REF]. While the underlying causes for their sparing are unclear, intrinsic differences in EOM MuSCs have been proposed as contributing factors (Di Girolamo et al., 2023;[START_REF] Evano | Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation[END_REF][START_REF] Randolph | A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups[END_REF]. Yet, the precise mechanisms governing the emergence and maintenance of these and other cranial MuSCs remain obscure.

Skeletal muscle cell commitment and differentiation throughout the body is regulated by basic helixloop-helix myogenic regulatory factors (MRFs) comprising Myf5,Mrf4,Myod and Myogenin (Myog).

Mouse knockout models have established a genetic hierarchy where Myf5, Mrf4 and Myod control commitment and proliferation of myogenic progenitors, and Myod, Mrf4 and Myog are involved in terminal differentiation (Comai and Tajbakhsh, 2014;[START_REF] Kassar-Duchossoy | Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice[END_REF][START_REF] Rudnicki | MyoD or Myf-5 is required for the formation of skeletal muscle[END_REF]. Despite this uniformity in the acquisition of myogenic cell fate, an underlying heterogeneity characterises the embryonic cells that emerge in different anatomical locations. In the trunk, the paralogous transcription factors Pax3 (Paired Box 3) and Pax7 (Paired Box 7) regulate the emergence of muscle stem (Pax3+ or Pax7+) and committed (Myf5+, Mrf4+ or Myod+) cells [START_REF] Buckingham | PAX3 and PAX7 as upstream regulators of myogenesis[END_REF]Comai and Tajbakhsh, 2014;[START_REF] Relaix | Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage[END_REF]. Notably, somite-derived myogenic cells are eventually lost by apoptosis from mid-embryogenesis after myogenesis has initiated in Pax3;Pax7 double-mutant mice [START_REF] Relaix | A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[END_REF]. In contrast, craniofacial muscles such as EOMs, facial, mastication and subsets of neck muscles, are derived from cranial mesoderm and do not express Pax3 [START_REF] Grimaldi | Diversity in cranial muscles: Origins and developmental programs[END_REF][START_REF] Schubert | To roll the eyes and snap a bite -function, development and evolution of craniofacial muscles[END_REF][START_REF] Tzahor | Head muscle development[END_REF][START_REF] Ziermann | Neural crest and the patterning of vertebrate craniofacial muscles[END_REF]. The early head mesoderm harbors instead a complementary set of upstream markers including Tbx1, Pitx2 and the Six gene family [START_REF] Harel | Distinct origins and genetic programs of head muscle satellite cells[END_REF][START_REF] Kelly | The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF][START_REF] Wurmser | Overlapping functions of SIX homeoproteins during embryonic myogenesis[END_REF]. Notably, Tbx1 (T-Box Transcription Factor 1), is an upstream regulator for the facial, jaw, neck, laryngeal and esophagus muscles, but not EOM [START_REF] Kelly | The del22q11.2 candidate gene Tbx1 regulates branchiomeric myogenesis[END_REF][START_REF] Lescroart | Emergence of heart and branchiomeric muscles in cardiopharyngeal mesoderm[END_REF][START_REF] Sambasivan | Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration[END_REF][START_REF] Schubert | To roll the eyes and snap a bite -function, development and evolution of craniofacial muscles[END_REF]. Instead, Pitx2 (Paired Like Homeodomain 2), which is also expressed in head muscles, plays a critical role as an upstream regulator of EOM development, as these muscles are lacking in Pitx2 null mice [START_REF] Gage | Dosage requirement of Pitx2 for development of multiple organs[END_REF][START_REF] Kitamura | Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra-and periocular mesoderm and right pulmonary isomerism[END_REF], with phenotypes being dependent on the gene dosage [START_REF] Diehl | Extraocular muscle morphogenesis and gene expression are regulated by Pitx2 gene dose[END_REF] and timing of deletion [START_REF] Zacharias | Pitx2 is an upstream activator of extraocular myogenesis and survival[END_REF].

In contrast to the diversity in upstream regulators, Pax7 marks all adult MuSCs and their ancestors from mid-embryogenesis (Comai and Tajbakhsh, 2014;[START_REF] Kassar-Duchossoy | Pax3/Pax7 mark a novel population of primitive myogenic cells during development[END_REF][START_REF] Relaix | A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF]. In the trunk, Pax7+ myogenic cells are primed by prior Pax3 expression during embryonic development [START_REF] Hutcheson | Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for betacatenin[END_REF]. However, the mechanisms that govern the emergence and maintenance of cranial myogenic cells are poorly understood. As Pax3 is absent in the EOMs, and Pax7 is expressed after the MRFs [START_REF] Horst | Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis[END_REF][START_REF] Nogueira | The emergence of Pax7-expressing muscle stem cells during vertebrate head muscle development[END_REF], it is possible that Pitx2 or other transcription factors such as the Six family members [START_REF] Maire | Myogenesis control by SIX transcriptional complexes[END_REF][START_REF] Wurmser | Overlapping functions of SIX homeoproteins during embryonic myogenesis[END_REF] specify the Pax7+ population in this location. Yet, as no lineage-specific deletion of Pitx2 has been performed in EOM MuSCs, it is unclear: i) to what extent Pitx2 is required temporally for emergence and maintenance of the MuSC population, and its relative function compared to the MRFs and Pax7; ii) if Pitx2 is continuously required for maintenance of the adult stem cell population. Further, global deletion of Pax7 in trunk and limb muscles leads to an extensive loss of MuSCs during early postnatal development [START_REF] Kuang | Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[END_REF][START_REF] Lepper | Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[END_REF][START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF][START_REF] Relaix | Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF]. Deletion of Pax7 in adult MuSCs results in reduced MuSC numbers and impaired regeneration following muscle injury [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF]. While Pax7 is dispensable for EOM formation during embryonic development [START_REF] Zacharias | Pitx2 is an upstream activator of extraocular myogenesis and survival[END_REF], the presence of MuSCs in the EOM of postnatal Pax7 null mice and the relative roles of Pax7 and Pitx2 in maintaining EOM cells remain unresolved. Finally, several reports showed that Pitx2 is expressed in postnatal EOM MuSCs at higher expression levels than in limb muscles (Di Girolamo et al., 2023;[START_REF] Evano | Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation[END_REF][START_REF] Taglietti | Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy[END_REF]. As high levels of Pitx2 are retained in dystrophic and ageing mouse EOM [START_REF] Hebert | The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles[END_REF][START_REF] Taglietti | Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy[END_REF] and overexpression of Pitx2 was reported to enhance the regenerative potential of dystrophic skeletal MuSCs [START_REF] Vallejo | PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells[END_REF], Pitx2 could potentially contribute to the sparing of EOMs in muscular dystrophies. However, the functional role of Pitx2 in EOM MuSCs in mdx mice has not been formally evaluated.

In this study, we examined the spatiotemporal functional roles of Pitx2 and Pax7 in the emergence and maintenance of the EOM progenitor population using combinations of genetically modified and lineage traced mice. Our study provides a qualitative and quantitative analysis of myogenesis from embryonic to postnatal stages in vivo and identifies differential cycling states, dynamics of commitment, and establishment of stem cell quiescence in EOM compared to limb muscles. We show that Pitx2 is critically required for establishing EOM prior to Pax7 expression but is subsequently dispensable. Moreover, our findings clarify the behavior of EOM MuSC behavior in mdx mice thus providing valuable developmental and clinical insights.

RESULTS

Differential temporal dynamics of EOM and limb myogenic cells during development and postnatally

Previous studies suggested that EOM have more MuSCs per area and per tissue weight than limb muscles such as the Tibialis anterior (TA) [START_REF] Keefe | Muscle stem cells contribute to myofibres in sedentary adult mice[END_REF][START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF]. To determine if the higher density of EOM MuSCs in vivo results from a longer proliferative phase of EOM Pax7+ cells during embryonic and postnatal development, we performed co-immunostaining of Pax7 and the proliferation marker Ki67 on tissues sections from EOM and TA from E (embryonic day) 14.5 to 4 months postnatally (Fig 1A,1B). As expected, the total numbers of Ki67+/Pax7+ cells were progressively reduced during postnatal growth in both muscles. Yet, EOM MuSCs stopped proliferating by P (postnatal day) 14, whereas proliferating Pax7+ MuSCs continued to be observed in TA muscle at P20 (Fig. 1C). Thus, EOM Pax7+ cells exited the cell cycle earlier than those in the TA. In the TA, cycling Pax7+/Ki67+ cells persisted in pre-pubertal muscle (P28) before quiescence was achieved between 7-8 weeks [START_REF] Gattazzo | Distinct Phases of Postnatal Skeletal Muscle Growth Govern the Progressive Establishment of Muscle Stem Cell Quiescence[END_REF] (Fig. 1C). Notably, the density of EOM Pax7+ MuSCs remained higher in the orbital layer, probably due to the smaller fibre size and less myoblast fusion events during the growth phase compared to the global layer (Fig. S1A). Altogether, these data show that the relatively higher number of Pax7+ cells per area in the EOM cannot be accounted for by a longer proliferative phase. Of note, as Pitx2 was expressed in the majority of EOM Pax7+ cells throughout all stages examined (80-90%; Fig. 1D,1E), its expression alone does not seem account for the higher proliferative status of this population.

Pax7 is critical for maintenance of EOM MuSC pool postnatally

To investigate the role of Pax7 in EOM MuSCs compared to limb muscles, we examined Pax7 nGFP/nGFP knock-out mice (Pax7 KO) [START_REF] Sambasivan | Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF] where Pax7-null and heterozygous cells are GFP+ (Fig. 2A,2B). At P20, the number of GFP+ cells/area was significantly reduced both in EOM and TA of Pax7 KO mice compared to heterozygous controls, although to a greater extent in the EOM (Fig. 2C). Assessment of the number of GFP+ cells/100 fibres revealed similar reductions in reduction in the EOM and TA of Pax7 KO mice compared to heterozygous controls (Fig. S2 A-C). Interestingly, Pax7 KO mice showed a reduction in GFP+ cells principally in the orbital (outer) EOM layer, yet this was relatively unchanged in the global layer, thereby pointing to location specific requirements for Pax7 in this muscle (Fig. S2 A,B).

Next, we addressed the mechanisms underlying MuSC loss in Pax7 null EOM. Progressive loss of limb and trunk MuSCs in newborn constitutive Pax7 mutants was suggested to be due to a proliferation defect and cell death [START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF][START_REF] Relaix | A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[END_REF]. Moreover, a greater propensity to differentiate and fuse into myofibres was reported for postnatal Pax7 mutant MuSCs in vivo [START_REF] Lepper | Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[END_REF]. Similarly, adult limb and trunk Pax7 mutant cells displayed reduced proliferation and increased differentiation in vitro [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF]. We performed immunostaining for Myod (commitment marker) and Myogenin (Myog, differentiation marker) in Pax7 EOM and TA muscles of Pax7 KO and control mice (Fig. 2D,E). At P20, a significantly higher fraction of Myod+/Myog+ (MM+) cells in the GFP+ population was observed in Pax7 KO EOM (30% in KO vs 5.8% in control; Fig. 2F), indicative of depletion of the stem cell population. This difference was not notable for TA muscle at P20. We then examined EOM and TA muscle of Pax7 KO E14.5 embryos to assess the role of Pax7 in maintenance of myogenic progenitors in the foetus (Fig. S2D-L). At E14.5, the number of Pax7+ cells per area was similar between EOM and TA of Pax7 KO embryos (Fig. S2F).

However, the fraction of MM+/GFP+ cells was already higher in the EOM of Pax7 KO embryos compared to controls (Fig. S2I). Notably though, the control TA had ~9-fold and ~2-fold more Myod+/Myog+ cells in the GFP+ population compared to the EOM at P20 (Fig. 2F) and E14.5 (Fig. S2I), respectively, indicating a more rapid transition from stem to committed and differentiated cells in vivo in normal conditions. Evaluation of the expression of Calcitonin receptor (CalcR), which is a marker of postnatal quiescent MuSCs [START_REF] Fukada | Molecular signature of quiescent satellite cells in adult skeletal muscle[END_REF], showed that >90% of GFP+ cells in the Pax7 heterozygous were positive for this marker at P20 as expected, whereas all GFP+ cells in the Pax7 KO were CalcR-negative in both EOM and TA muscles, suggesting that in both muscles this residual MuSC population is perturbed (Fig. 2G-I). Altogether, we conclude that Pax7 is required for maintenance of the stem cells population of the EOM, and that invalidation of this gene results in a faster loss of the upstream population compared to the limb.

Given that Pitx2 is a major upstream regulator of the EOM lineage [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF][START_REF] Zacharias | Pitx2 is an upstream activator of extraocular myogenesis and survival[END_REF], we assessed whether Pitx2+ cells would expand in the Pax7 KO. Co-immunostaining for Pax7 and Pitx2 in Pax7 KO mice at embryonic and adult stages showed that the number of Pitx2+/GFP+ cells were similar in EOM as well as TA in the Pax7 KO and control mice (Fig. 2J-L and S2J-L).

Moreover, unlike other skeletal muscles, EOMs were reported to contain another population of interstitial stem cells that are Pax7-/Pitx2+ [START_REF] Hebert | The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles[END_REF]. Thus, we examined this interstitial population in mutant versus control EOMs and showed similar proportions of Pitx2+/GFP-cells between two (Fig. S2M). Overall, these results suggest a lack of compensation by Pitx2 in the EOM lineage of Pax7 KO mice.

Deletion of Pitx2 in Myf5+ cells impair establishment of EOM and emergence of Pax7+ cells

Throughout the body, the long-term stem cell population (Pax3+/Pax7+ in the trunk; Pax7+ in head) emerges only after an anlage of differentiated cells has been established during mid-embryogenesis (Comai and Tajbakhsh, 2014). Cranial muscles are specified temporally after those in the trunk [START_REF] Bothe | Dynamic control of head mesoderm patterning[END_REF][START_REF] Gage | Dosage requirement of Pitx2 for development of multiple organs[END_REF][START_REF] Horst | Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis[END_REF][START_REF] Nogueira | The emergence of Pax7-expressing muscle stem cells during vertebrate head muscle development[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF] and Pax7+ stem cells appear at later stages during development, after expression of the myogenic markers Myf5 and to a lesser extent Myod [START_REF] Nogueira | The emergence of Pax7-expressing muscle stem cells during vertebrate head muscle development[END_REF]. However, it is unclear if expression of the MRFs is a prerequisite for the emergence of the Pax7+ cells themselves.

Therefore, we performed whole mount immunofluorescence (WMIF) analysis on Myf5 nlacZ/+ mice, where β-galactosidase serves as a proxy for Myf5 expression [START_REF] Tajbakhsh | Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice[END_REF] (Fig. 3A-C). In the EOM primordium, Pitx2 was expressed broadly, including in periocular mesenchyme and connective tissue cells [START_REF] Comai | Local retinoic acid signaling directs emergence of the extraocular muscle functional unit[END_REF][START_REF] Gage | Dosage requirement of Pitx2 for development of multiple organs[END_REF]. WMIF showed that virtually all Myf5+ cells expressed Pitx2 in the EOM anlage throughout the stages analysed (Fig. 3B). In addition, we observed Pax7+ expression from E11.75, only in Myf5+ cells (Fig. 3C). EOM Pax7+ cells then rapidly increased in numbers and together with Pax7 protein levels from E11.75 (Fig. 3B,3C).

To examine further the emergence of EOM MuSCs, we performed lineage tracing of Pax7+ cells originating from Myf5+ cells [START_REF] Haldar | A conditional mouse model of synovial sarcoma: insights into a myogenic origin[END_REF] using the Myf5 Cre ;R26 mTmG ;Myf5 nlacZ genetic combination (Fig. 3D). This genetic strategy allowed us to first assess the number of Pax7+ cells with a history of Myf5-expression (membrane GFP+), then to detect with high sensitivity, contemporary Myf5-expressing cells (β-gal+) that might not have activated Cre-expression. Consequently, a broader view on the Myf5+ population, giving rise to and residing within Pax7-expressing cells, could be resolved.

Remarkably, this analysis showed that at E11.5 and E14.5, all Pax7+ cells showed a history of Myf5 expression (Fig. 3E).

Based on the dynamics of expression of these transcription factors, we used Myf5 Cre [START_REF] Haldar | A conditional mouse model of synovial sarcoma: insights into a myogenic origin[END_REF] to delete Pitx2 before the onset of Pax7 expression. Immunostaining for Pax7, Pitx2, Myod, Myog and MF20 in Myf5 Cre ; Pitx2 flox/   E14.5 embryos showed no Pax7+ cells, only a few Myod+/Myog+ cells, and no myofibres (Fig. 3F,3G). Analysis at foetal stages confirmed the lack of EOM in the mutant, albeit a small cluster of Pax7+ cells and myofibres were detected in dorsal sections, at the level of the muscle attachment point to the base of the skull (Fig. S3A-C). As these EOM remnants contained Pitx2+/Pax7+ and Pitx2-/Pax7+ cells and correspond to the site of the initiating EOM anlagen, it is possible that Myf5 Cre was less efficient in this location or they were derived from Mrf4+ cells [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF].

Given that Myf5 is expressed briefly in muscle progenitors and downregulated when myoblasts commit to differentiation (Comai and Tajbakhsh, 2014), we used the more downstream commitment marker Myod to abolish Pitx2 expression in myoblasts using Myod iCre [START_REF] Kanisicak | Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD[END_REF]. Interestingly, myogenesis appeared unperturbed in the EOM in this case (Fig. 4A,4B). In addition, and in stark contrast to the Myf5 Cre data, similar numbers of Pax7-expressing cells were present in controls and Myod iCre ;Pitx2 fl/fl foetuses (Fig. 4C), despite recombination taking place in >90% of the cells as per Myod iCre ;R26 Tomato lineage tracing (Fig. S4A,S4B). Taken together, these results show that Pitx2 is required in Myf5+ myogenic progenitors for the specification and emergence of the Pax7+ population and is then dispensable upon activation of Myod.

Temporal requirement for Pitx2 in EOM stem cells

To assess the role of Pitx2 specifically in the Pax7+ cells, we generated Tamoxifen-inducible Pax7 CreERT2 ; Pitx2 flox/flox mice (conditional KO). Tamoxifen was induced twice at E11.5 and E12.5, at the onset of Pax7 expression in developing EOM [START_REF] Horst | Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis[END_REF] (Fig. 3B,3C), and samples were collected at E17.5 (Fig. 5A). Immunostaining for Pax7, Pitx2 and MF20 showed that although EOM myofibres were present in Pax7 CreERT2/+ ; Pitx2 flox/flox foetuses (Fig. 5B), the number of Pax7+ cells was reduced by 31.3% (Fig. 5C), and the proliferating population (Ki67+) was reduced by 16.6% (Fig. 5D) in the conditional KO. No significant perturbations were noted in the HL muscle at this stage. We note that these experiments do not distinguish whether deletion of Pitx2 impairs the continuous emergence of Pax7+ cells from E11.5, or if the reduced number of Pax7+ cells observed in the foetus is due to reduced proliferation of Pax7+/Pitx2-cells.

Next, we investigated the role of Pitx2 in Pax7+ cells postnatally where tamoxifen was injected from the early perinatal (from P1 to P7, condition 1) or weaning (P21, condition 2) stages with additional monthly injections until analysis in the adult (4 months) (Fig. 5E). The rationale was to assure full Pitx2 deletion from the time of induction, and to avoid a potential rescue by non-deleted cells. In both conditions, there was no difference in the number of EOM Pax7+ cells between the conditional KO and controls (Fig. 5F, 5G), despite an efficient deletion (Fig. S5A-D). Of note, a concomitant deletion of Pitx2 in myonuclei was observed when Tamoxifen induction was done at P7 (Fig. S5B, S5B') but not P20 (Fig. S5C,S5C'). This is consistent with our results (Fig. 1C) showing that most of the EOM MuSCs enter quiescence before P20, thus Pax7+ myoblast proliferation and fusion taking place prior to that stage. Therefore, absence of Pitx2 alone does not result in the loss of Pax7+ MuSCs during homeostasis postnatally.

Loss of Pitx2 in MuSCs does not account for sparing of EOM in mdx mice

High levels of Pitx2 in EOM MuSCs were proposed to be responsible, at least in part, for sparing of EOMs in DMD [START_REF] Hebert | The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles[END_REF][START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF]. Therefore, we generated mdx βgeo ; Pax7 CreERT2 ; Pitx2 flox/flox compound mutant mice (dKO), where mdx βgeo is a dystrophin null mouse model [START_REF] Wertz | Dmd(mdx-beta geo): a new allele for the mouse dystrophin gene[END_REF]. Tamoxifen was injected 3 times from P1 to P7, followed by monthly injections (from 2 months) until analysis in the adult (Fig. 6A). Immunostaining for Pax7 and Ki67 allowed evaluation of the density and proliferative fraction of Pax7+ MuSCs in EOM and TA (Fig. 6B-F). The number of Pax7+ cells per area was similarly decreased in the EOM of mdx βgeo and dKO mice compared to controls and cKO (Fig. 6C). Moreover, there were virtually no Ki67+/Pax7+ cells in EOM in mdx βgeo mice and 3.6% in dKO mice (Fig. 6D), suggesting that EOM MuSCs are not activated in either model at the time points analysed. In contrast, TA muscle from mdx βgeo mice and dKO showed a significant increase in the number of Pax7+ cells per area, and percentage of proliferative MuSCs (Fig. 6E,6F) in agreement with the notion that MuSCs undergo activation in mdx mice during cycles of muscle degeneration and regeneration [START_REF] Georgieva | Inactivation of Sirt6 ameliorates muscular dystrophy in mdx mice by releasing suppression of utrophin expression[END_REF].

Constitutive deletion of Pitx2 in myofibres of mdx4cv mice was reported to result in severe dystrophic changes in EOM compared to a mild fibre phenotype observed in the limb [START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF]. Thus, we assessed the number of centrally nucleated myofibres in the EOM of both mdx βgeo and dKO mice (Fig. S6A,S6B). Although in the dKO a large fraction of myonuclei is Pitx2-as they were incorporated from Pitx2-deleted Pax7+ cells after P7 (Fig. S5B), only sparse centrally located myonuclei were observed (2.0% in dKO compared to 0.6% in mdx βgeo Fig. S6A,S6B). This is at odds with the high numbers of centrally nucleated fibres normally observed in mdx βgeo TA muscles (Fig. S6A), which can reach up to ~60% [START_REF] Lau | Automated muscle histopathology analysis using CellProfiler[END_REF]. Next, we calculated the cross-sectional area of EOM myofibres in the 4 recti muscles from WT, mdx βgeo , Pitx2 cKO and dKO and classified them by size (Fig. S6C). While a trend towards larger myofibre size was observed in the EOM of mdx βgeo mice, dKO mice showed some heterogeneity with a slight shift towards smaller and larger size fibres (Fig. S6C). Altogether, Pitx2 appears to play a minor role in EOM MuSCs in a dystrophic background.

Discussion

Pax7 plays a critical role in the maintenance of mouse muscle stem cells from late fetal stages and it is the most widely used marker for adult MuSCs and their ancestors from mid-embryogenesis [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Kassar-Duchossoy | Pax3/Pax7 mark a novel population of primitive myogenic cells during development[END_REF][START_REF] Lepper | Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[END_REF][START_REF] Relaix | Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF]. Pax7 is required for maintenance and function of the MuSC pool from late fetal stages in mouse [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Murphy | Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF]. Adult MuSCs in the head also express Pax7, yet they also retain the expression of the early mesodermal markers characteristic of each location [START_REF] Evano | Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation[END_REF][START_REF] Harel | Distinct origins and genetic programs of head muscle satellite cells[END_REF][START_REF] Ono | Gene expression profile of mouse masseter muscle after repetitive electrical stimulation[END_REF][START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF]. In this study, we investigated the functional relationships between Pax7, Pitx2 and Myf5 in the emergence and maintenance of MuSCs. Our data reveal different temporal requirements for Pitx2 during prenatal and postnatal stages, and during lineage progression, for the emergence of the EOM Pax7+ stem cells.

Interplay between Pitx2, Myf5 and Pax7 in emergence of EOM stem cells

In the trunk, both at the dermomyotome and limb levels, expression of Pax3 and Myf5 precede the expression of Pax7 (Comai and Tajbakhsh, 2014;[START_REF] Jostes | The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system[END_REF][START_REF] Kassar-Duchossoy | Pax3/Pax7 mark a novel population of primitive myogenic cells during development[END_REF][START_REF] Tajbakhsh | Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD[END_REF], with Pax3 expression being then rapidly downregulated during myogenic cell commitment (Myod+ cell). Following the establishment of a muscle anlagen (uncommitted and differentiated cells), Pax7 is expressed from mid-embryogenesis in the upstream undifferentiated population, and it persists to give rise to adult MuSCs [START_REF] Kassar-Duchossoy | Pax3/Pax7 mark a novel population of primitive myogenic cells during development[END_REF][START_REF] Relaix | A Pax3/Pax7-dependent population of skeletal muscle progenitor cells[END_REF]. In the head of vertebrate models analysed, Pax7+ myogenic cells were reported to emerge de novo, as Pax3 is not expressed in head MuSCs [START_REF] Horst | Comparative expression analysis of Pax3 and Pax7 during mouse myogenesis[END_REF][START_REF] Nogueira | The emergence of Pax7-expressing muscle stem cells during vertebrate head muscle development[END_REF]. Instead, and like the role of Pax3 in the trunk and limbs, Pitx2 is critically required cell-autonomously to maintain myogenic progenitors in an immature state, ensure their survival, and to activate the MRFs [START_REF] Zacharias | Pitx2 is an upstream activator of extraocular myogenesis and survival[END_REF]. Differently to the role of Pax3 in the trunk, Pitx2 expression persists following activation of the MRFs and myogenic commitment in the developing EOMs, and it is required for myogenic cell survival until the end of the embryonic period [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF]. The expression of Pitx2 also in adjacent mesenchymal cells [START_REF] Comai | Local retinoic acid signaling directs emergence of the extraocular muscle functional unit[END_REF][START_REF] Gage | Dosage requirement of Pitx2 for development of multiple organs[END_REF] has complicated the analysis and interpretation of the cell-autonomous roles of this gene in EOM development. In addition, conditional inactivation of Pitx2 in neural-crest-derived cells does not affect the specification of the EOM primordia, but it results in severe muscle patterning defects [START_REF] Evans | Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development[END_REF].

We demonstrated herein a temporal requirement for Pitx2 during lineage progression: deletion of this gene in muscle progenitors (Myf5 Cre ) results in the absence of myofibres and upstream cells in the embryo, while deletion in myoblasts (Myod iCre ) does not. Further, deletion of this gene in Pax7+ cells led to a reduction in the number of EOM MuSCs in the foetus, whereas Pitx2 function appears to be dispensable for EOM MuSC maintenance in the adult. In this muscle group, Myf5 and Mrf4 also play critical roles as EOMs and the upstream population are absent in Myf5;Mrf4 double mutants [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF][START_REF] Tajbakhsh | The birth of muscle progenitor cells in the mouse: spatiotemporal considerations[END_REF]. Collectively, these observations indicate that EOM specification and differentiation has evolved a unique gene regulatory network where EOM progenitor survival as well as activation of the downstream myogenic factors are dependent on either Myf5 or Mrf4, and that Pitx2 alone cannot override their role. Therefore, it is possible that the Pitx2 survival function is progressively relayed onto Myf5 and/or Pax7. A double Pax7;Pitx2 KO in the MuSC lineage would be required to formally address this hypothesis. Moreover, while Pitx2 binds directly to the promoters of Myf5 and Myod [START_REF] Zacharias | Pitx2 is an upstream activator of extraocular myogenesis and survival[END_REF], it is unclear whether it can directly activate Pax7, or if other TFs are necessary. Of note, sc-RNAseq data from our lab [START_REF] Grimaldi | Identification of bipotent progenitors that give rise to myogenic and connective tissues in mouse[END_REF] revealed several novel TFs that are specifically expressed in EOM progenitors. Whether those factors are required for the emergence of the myogenic population in the EOM requires further investigation.

Moreover, a recent study showed that Six1 and Six2 genes are required for craniofacial myogenesis by controlling the engagement of unsegmented cranial paraxial mesodermal cells in the myogenic pathway [START_REF] Wurmser | Overlapping functions of SIX homeoproteins during embryonic myogenesis[END_REF]. It remains unclear if expression of these genes in adult MuSCs would be required for Pax7 maintenance in the EOM.

Adult EOM MuSCs do not proliferate during homeostasis despite coexpression of Pax7 and

Pitx2

Activated EOM MuSCs have greater proliferative and self-renewal abilities in vitro compared to those in the limb (Di Girolamo et al., 2023;[START_REF] Stuelsatz | Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency[END_REF]. Moreover, expression of Pitx2 in postnatal EOM myogenic cells (CD34+/Sca1-/CD31-/CD45-lineage), at higher levels than limb MuSCs, was reported to contribute to the proliferative and stress resistance properties of EOM MuSCs in vitro [START_REF] Hebert | The role of Pitx2 in maintaining the phenotype of myogenic precursor cells in the extraocular muscles[END_REF][START_REF] Kallestad | Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis[END_REF]. However, with respect to the proliferative status of EOM MuSCs in vivo, we note several discrepancies. In some species, EOM MuSCs were suggested to chronically proliferate in vivo [START_REF] Kallestad | Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis[END_REF]McLoon andWirtschafter, 2002, 2003), but these studies generally lacked co-immunostainings for Pax7 and proliferation markers. In addition, EOM MuSCs appeared to contribute to new myofibre myonuclei in homeostasis at higher frequency than that observed for limb muscles [START_REF] Keefe | Muscle stem cells contribute to myofibres in sedentary adult mice[END_REF][START_REF] Mcloon | Activated satellite cells are present in uninjured extraocular muscles of mature mice[END_REF][START_REF] Pawlikowski | Pervasive satellite cell contribution to uninjured adult muscle fibers[END_REF].

In this study, we performed co-immunostaining for Pax7 and Ki67 and found that EOM MuSCs entered quiescence earlier than those in TA muscle, and we did not observe proliferative EOM MuSCs during adult homeostasis. These observations agree with a previous study [START_REF] Stuelsatz | Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency[END_REF] showing that freshly isolated mouse EOM MuSCs are not proliferative. While the origin of the discordance with lineage tracing studies is unclear, one possibility could be that EOM MuSCs contribute to myofibres without cell cycle entry and/or the genetic constructs used as Cre drivers are unexpectedly expressed in EOM myofibres.

It was also proposed that Pitx2+ myogenic cells do not co-express Pax7 but express Myod in human EOM, and these cells could then represent a second progenitor population involved in EOM remodeling, repair, and regeneration [START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF]. Given our observation that more than 92.5% of Pax7+ MuSCs coexpress Pitx2, this might highlight species specific differences or antibody sensitivity and tissue processing issues in the different studies.

Deletion of Pax7 results in loss of the EOM MuSC population

Several studies analysed the limb and trunk phenotypes of constitutive [START_REF] Kuang | Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[END_REF][START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF][START_REF] Relaix | Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF] and inducible [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Lepper | Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF] Pax7 mutants. While MuSC progenitors are initially specified in homeostatic conditions [START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF], the progressive loss of MuSCs in newborn mice appears to be due to proliferation defects of MuSCs and cell death [START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF][START_REF] Relaix | Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[END_REF]. Elimination of Pax7 in adult MuSCs results in MuSC loss likely due to enhanced differentiation and reduced heterochromatin condensation in the remaining cells [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF] and severe deficits in muscle regeneration [START_REF] Gunther | Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells[END_REF][START_REF] Kuang | Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[END_REF][START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF]. The phenotypes of Pax7 null cells during in vitro activation were somewhat more divergent, depending on the system used (isolated myoblasts, myofibers, clonogenic assays) including absence or reduction in the number of myoblasts derived from Pax7KO cells together, increased or reduced differentiation and/or commitment to alternative fates [START_REF] Kuang | Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[END_REF][START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF][START_REF] Relaix | Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells[END_REF][START_REF] Seale | Pax7 is required for the specification of myogenic satellite cells[END_REF][START_REF] Von Maltzahn | Pax7 is critical for the normal function of satellite cells in adult skeletal muscle[END_REF].

As EOMs are not amenable to in vivo muscle injury, in this study we examined the in vivo phenotype of Pax7 nGFP/nGFP null EOM compared to heterozygous controls at E14.5 and P21. The perdurance of the GFP allowed us to trace the progenitors and more differentiated cells. We highlight several differences between the EOM and TA muscles. First, in P21 control samples, EOM displayed a higher number of Pax7+ cells per area, but not per fibre compared to TA. This agrees with previous data showing higher number of MuSCs per volume unit in the EOM [START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF], which might be related to their smaller fibre size. Second, a greater reduction of GFP+ cells per area was noted in Pax7 null EOM compared to TA, suggestive of a more relevant role for Pax7 in the maintenance of MuSCs without a history of Pax3 expression. Notably, no differences in the number of GFP+ cells per area neither in the proportion of Pitx2+GFP+ cells were observed in the fetal period between control and mutant EOMs, suggesting that other factors different than Pax3 and Pitx2 may ensure the specification of this cell population. Previous studies showed that Pax7 is required for in limb MuSC precursors to express genes normally associated with functional MuSCs such as Syndecan4 and CD34 [START_REF] Kuang | Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis[END_REF][START_REF] Oustanina | Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification[END_REF]. Here, we further show that expression of CalcR, another MuSC marker [START_REF] Fukada | Molecular signature of quiescent satellite cells in adult skeletal muscle[END_REF], was fully impaired in EOM and TA from Pax7 null. In addition, while the fraction of committed GFP+ cells in Pax7null EOM display a marked increase compared to controls, no significant changes were observed in mutant and control TA at P21. This observation probably reflects differences on the pace of lineage progression between these muscles in vivo. It would be of interest in future studies to assess whether the downstream targets of Pax7 in MuSCs of cranial and somite origin are similar and to identify complementary TFs that confer these distinct MuSC phenotypes.

Absence of proliferation in EOM of mdx and mdx;Pitx2 KO mice

Dystrophin protein is expressed in differentiated myofibres where it is required for sarcolemmal integrity, but it is also expressed in activated MuSCs, where it regulates MuSC polarity and the mode of cell divisions [START_REF] Dumont | Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division[END_REF][START_REF] Wang | EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions[END_REF][START_REF] Weng | Single-Cell Transcriptomics Uncovers Glial Progenitor Diversity and Cell Fate Determinants during Development and Gliomagenesis[END_REF]. Moreover, several mechanisms of MuSC dysfunction have been described in DMD [START_REF] Filippelli | Empowering Muscle Stem Cells for the Treatment of Duchenne Muscular Dystrophy[END_REF], including severe proliferation defects and premature senescence of chronically activated MuSCs [START_REF] Blau | Defective myoblasts identified in Duchenne muscular dystrophy[END_REF][START_REF] Chang | Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes[END_REF][START_REF] Sugihara | Cellular senescence-mediated exacerbation of Duchenne muscular dystrophy[END_REF][START_REF] Taglietti | Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy[END_REF]. Notably, in a preclinical rat model of DMD, expression of thyroid-stimulating hormone receptor (Tshr) was shown to protect EOM MuSCs from entering senescence and for them to remain in a proliferative state [START_REF] Taglietti | Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy[END_REF].

In this study, we found that the number of Pax7+ cells/area were reduced in EOM of mdx and mdx;Pitx2 KO mice, and that these cells were not proliferative. The mdx mice have a relatively normal lifespan and have a milder phenotype compared to rats, dogs, and human [START_REF] Mcgreevy | Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy[END_REF][START_REF] Taglietti | Duchenne muscular dystrophy trajectory in R-DMDdel52 preclinical rat model identifies COMP as biomarker of fibrosis[END_REF]. Thus, the absence of proliferation of mdx EOM MuSCs might reflect a complete sparing of these muscles in the murine model. Alternatively, it might reflect activation and fusion of the MuSC population without prior entry in the cell cycle given that the number of Pax7+ cells/area were reduced in mdx and mdx;Pitx2 mice compared to control EOMs. Moreover, we observed similar reductions in the number of MuSCs in the EOM of mdx and mdx;Pitx2 mice. As the Pax7 CreERT2 ;Pitx2 fl/fl EOMs were unaffected when Pitx2 was deleted postnatally, these data suggest that the reduction in the number of EOM MuSCs is mdx but not Pitx2 dependent.

Finally, some insights into the potential role of Pitx2 in the sparing of EOMs in Dmd come from a preliminary study showing that in the absence of Pitx2 expression in myofibres of mdx4cv mice, the EOMs succumb to dystrophic changes that are even more severe than those seen in the limb muscles of the same mice [START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF]. In the present study, we deleted Pitx2 in MuSCs at an early postnatal stage (<P7), where considerable fusion is ongoing and thus MuSC-derived myoblasts actively contribute to new myonuclei. While this resulted in a small percentage of centrally nucleated fibres and a more heterogeneous distribution of fibre sizes (shift towards smaller and larger fibres), the resulting phenotype was less severe than when Pitx2 was deleted with a constitutive myofibre Cre driver [START_REF] Verma | Extraocular Muscle Repair and Regeneration[END_REF]. However, the latter study was performed with 18 months old mice, therefore a long-term phenotype upon deletion of Pitx2 in MuSCs cannot be excluded. Performing deletion of Pitx2 prenatally in MuSCs or timed deletions within myofibres using a conditional fibre Cre driver mouse would help elucidate the relative and roles of Pitx2 in each compartment.

In summary, how some muscles are spared in myopathies, such as the EOM in dystrophic mice, remains unresolved and under debate. Our studies on the prenatal and postnatal development of susceptible and spared muscles provides insights into this process regarding the roles of key transcription factors that specify and maintain the muscle stem cell populations in these muscles.

MATERIALS AND METHODS

Mouse strains

Animals were handled as per European Community guidelines and the ethics committee of the Institut Pasteur (CETEA) approved protocols (APAFIS#41051-202302204207082). The following strains were previously described: mdx-βgeo [START_REF] Wertz | Dmd(mdx-beta geo): a new allele for the mouse dystrophin gene[END_REF], Myf5 Cre [START_REF] Haldar | A conditional mouse model of synovial sarcoma: insights into a myogenic origin[END_REF], Myf5 nlacZ [START_REF] Tajbakhsh | Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice[END_REF], Pax7 CreERT2 [START_REF] Murphy | Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration[END_REF], Myod iCre [START_REF] Kanisicak | Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD[END_REF], mdx βgeo [START_REF] Wertz | Dmd(mdx-beta geo): a new allele for the mouse dystrophin gene[END_REF], Pax7 nGFP [START_REF] Sambasivan | Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates[END_REF] and Pitx2 flox [START_REF] Gage | Dosage requirement of Pitx2 for development of multiple organs[END_REF], in which the DNA binding homedomain region common to Pitx2a/b/c isoforms is flanked with LoxP sites.

Mice were kept on a mixed genetic background (B6D2F1, Janvier Labs). Mouse embryos and foetuses were collected at embryonic day (E) E14.5 and E17.5, with noon on the day of the vaginal plug considered as E0.5. To generate conditional KOs, Pax7 CreERT2/CreERT2 ; Pitx2 flox/+ , Myf5 Cre/+ ; Pitx2 flox/+ and Myod iCre/+ ; Pitx2 flox/+ males were crossed with Pitx2 flox/flox or Pitx2 flox/+ females. The cross with Myf5 Cre/+ ; Pitx2 flox/+ male and Pitx2 flox/flox or Pitx2 flox/+ females generated Myf5 Cre/+ ; Pitx2 flox/ fetuses as conditional KOs. To generate dKO (Pax7 CreERT2/+ ; Pitx2 flox/+ ; mdx βgeo ), Pax7 CreERT2/CreERT2 ; Pitx2 flox/+ male was crossed with Pitx2 flox/flox ; mdx βgeo/+ or Pitx2 flox/+ ; mdx βgeo/+ females.

Immunofluorescence

Embryonic tissues for cryosections were fixed for 2h in 4% paraformaldehyde (PFA) in PBS (Electron Microscopy Sciences, Cat #:15710) with 0.5% Triton X-100 at 4°C. Adult muscles for tissue sections were fixed for 2h in 1% PFA in PBS with 0.1% Triton X-100 at 4°C. After PBS washes for a few hours, samples were equilibrated in 30% sucrose in PBS overnight at 4°C then embedded in OCT compound (Sakura Fineteck,Cat #:4583). Cryosections (16μm) were permeabilized with 0.5% Triton in PBS for 5 min at RT and blocked for 1h at RT in Blocking solution (10% Goat serum, 1% BSA and 0.5% Triton X-100 in PBS). Primary antibodies used are: chicken polyclonal anti-GFP (Abcam, Cat#: ab13970, dilution 1:1000), mouse monoclonal anti-Myod (BD-Biosciences, Cat#: 554130, dilution 1:500), mouse monoclonal anti-Pax7 (DSHB, Cat# Pax7, dilution 1:20), mouse monoclonal anti-Myog (DSHB, Cat# F5D, dilution 1:20), rabbit polyclonal anti-Pitx2 (Abcam, Cat# ab221142, dilution 1:750), rabbit polyclonal Anti-Ki67 (Abcam, Cat# ab16667, dilution 1:500), mouse monoclonal anti-MF20 (DSHB, Cat# MF 20, dilution 1:20) rabbit polyclonal anti-Calcitonin Receptor (Bio-RAD, Cat# AHP635, dilution 1:2000), chick anti-GFP (abcam, Cat# 13970, dilution 1:1000), rabbit β-gal antibody (MP 559761, dilution 1/500), rabbit anti DsRed (clontech 632496, dilution1:200) for tdTomato detection and rabbit polyclonal anti-Laminin (Sigma-Aldrich, Cat# L9393, dilution 1:1000). Following washes in PBS-T (0.05% Tween20 in PBS), fluorescent secondary antibodies (dilution 1:500) and Hoechst (Thermo Scientific, Cat. #:H3570, dilution 1:10000) were diluted in Blocking solution and incubated for 1h at RT. Images were acquired by Spinning disk Ti2E (Nikon). Whole Mount immunostaining and clearing were performed as described [START_REF] Comai | Local retinoic acid signaling directs emergence of the extraocular muscle functional unit[END_REF]. Images were acquired with a confocal microscope (LSM700) and 3D reconstructions performed on Imaris.

Tamoxifen injection

Tamoxifen injections were done to induce Cre ERT2 -mediated recombination in Pax7 CreERT2 ;Pitx2 flox/flox mice. A solution of 15-20mg/ml Tamoxifen (Sigma-Aldrich, Cat# T5648) in 5% ethanol in sunflower oil was prepared by vortexing and rolling at 4°C in the dark and kept for up to one week at 4°C. For the induction at embryonic stages, pregnant female mice were treated with 150μl tamoxifen solution (20 mg/ml) by gavage at the indicated timepoints. For induction of before P7, pups were injected subcutaneously with 10μl tamoxifen solution (15mg/ml). For induction of at P20, pups were injected intraperitoneally with 50μl tamoxifen solution (20mg/ml). Adult mice were treated with 150μl tamoxifen solution (20 mg/ml) by gavage monthly from 2 months until the time of analysis. Tamoxifen was injected in all animals regardless of their genotypes (Control, MDX, cKO and dKO). (medial, lateral, dorsal, ventral) and 2 oblique (superior, inferior) muscles [START_REF] Comai | Local retinoic acid signaling directs emergence of the extraocular muscle functional unit[END_REF][START_REF] Suzuki | Comparative morphology and development of extra-ocular muscles in the lamprey and gnathostomes reveal the ancestral state and developmental patterns of the vertebrate head[END_REF]. (F) Immunostaining of E14.5 EOM sections from Myf5 Cre ;Pitx2 flox/+ (control) and Myf5 Cre ;Pitx2 flox/ (KO) at for Pax7 (magenta), Pitx2 (green) and MF20 (cyan). Bottom panels, higher magnification views of the area delimited with dots. Periocular mesenchyme (pom).

Image analysis and statistics

(G) Immunostaining of E14.5 EOM sections from Myf5 Cre ;Pitx2 flox/+ (control) and Myf5 Cre ;Pitx2 flox/ (KO) for Myod/Myogenin (MM) (magenta), Pitx2 (green) and MF20 (cyan). Bottom panels, higher magnification views of the area delimited with dots. Periocular mesenchyme (pom). (Control,and MDX,n=3;cKO,n=4;dKO,n=4).

(E) Number of Pax7+ cells per area on TA sections from immunostaining in (B) (Control,and MDX,n=3;cKO,n=4;dKO,n=6).

Figure 1 .

 1 Figure 1. Postnatal EOM myogenic cells exit the cell cycle earlier than those in TA muscle.

( C )

 C Percentage of Ki67+/Pax7+ cells in EOM and TA muscles at each stage (n=3 per stage). (D) Percentage of Pitx2+/Pax7+ cells over total Pax7+ stem cells at indicated stages from E14.5 to 4 months (4M). Note that Pitx2 is expressed expresses in about 80~90% of myogenic cells in EOM throughout all stages (n=3 each stage). (E) Immunostaining for Pax7(magenta) and Pitx2(green) on EOM sections at indicated stages. White arrowheads point to Pitx2+/Pax7+ cells. Bottom panels, higher magnification views of the area delimited with dots. Scale bars: 100μm (A), (B) and (E). Error bars represent SEM. ns, non-significant, *P<0.05, ***P<0.001, ****P<0.0001.

Figure 2 .

 2 Figure 2. Pax7 is critical for the maintenance of EOM MuSC pool postnatally.

Figure 3 .

 3 Figure 3. Deletion of Pitx2 in the Myf5 positive cells impairs EOM establishment.

  Scale bars: 100μm (D) and (B), 40μm (C)(i') and (C)(ii'), 50μm (C)(iii') and (C)(iv') 500μm (F) and (G). Error bars represent SEM. ****P<0.0001.

Figure 4 .

 4 Figure 4. Normal EOM formation following deletion of Pitx2 in Myod-expressing cells.

Figure 5 .

 5 Figure 5. Timing of inactivation of Pitx2 differently impairs EOM MuSCs.

Figure 6 .

 6 Figure 6. Absence of proliferation in EOM of mdx and mdx;Pitx2 KO mice.

(F)

  Percentage of Ki67+/Pax7+ cells over Pax7+cells on TA sections from immunostaining in (B)(Control, cKO and MDX, n=3; dKO, n=5). 4 months old (Control, MDX and dKO) and 1 year old (dKO and cKO) animals were littermates. 8month-old mice, dKO and control mice were littermates while MDX were generated in independent litters. All mice were treated with Tamoxifen. Scale bars: 100μm (B) Error bars represent SEM. ns, nonsignificant, *P<0.05.

  Figure 1

Acknowledgements

We acknowledge funding support from the Institut Pasteur, Agence Nationale de la Recerche (Laboratoire d'Exellence Revive, Investissement d'Avenir; ANR-10-LABX-73 to ST and ANR-21-CE13-0005 MUSE to GC), Association Française contre les Myopathies (Grant #20510 to ST and #23201 to GC), and the Centre National de la Recherche Scientifique. M.K. was supported by a Post-Doctoral Fellowship from Uehara Memorial Foundation (Grant# 202231020). We gratefully acknowledge the UtechS Photonic BioImaging (Imagopole), C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging; ANR-10-INSB-04; Investments for the Future) for support in conducting this study. We thank Dounia Bouragba and Sayna Miri for initial contributions to this study.

Quantifications were performed using Fiji (https://imagej.net/software/fiji/). Barplots were generated using Prism (https://www.graphpad.com/features). Fibre sizes and numbers were measured automatically by Cellpose (https://cellpose.readthedocs.io/en/latest/) and Fiji. All data are presented as the mean±s.d. with n (number of animals analysed) ≥3. Statistical significance was assessed using Student's t-test.

Competing interests

The authors declare no competing or financial interests.

Author contributions

GC and ST conceived the study. MK and GC performed experiments and analysed data. GC, MK, and ST wrote the manuscript. GC and ST provided funding.

SUPPLEMENTARY FIGURE LEGENDS

Supplementary


domain: spi phys

text:
Francis Feaugas

Tommaso Nicolini

Gilles H Roche

Lionel Hirsch

Olivier Dautel

Guillaume Wantz

Homojunction Doping for Efficient Hole Extraction in Polymer Solar Cells

Keywords:

Hole transporting layers (HTL) in polymer solar cells remains a subject of importance to enable enhanced efficiency and stability compared to the benchmark PEDOT:PSS. We report here the design of an interlayer based on the same polymer as the one used in the bulk heterojunction. In this HTL the polymer is doped, thus forming a so called homojunction. The conductivity of PTQ10 doped with Magic Blue was optimized varying dopant concentration. Resulting solar cells showed competing power conversion efficiency as the widely used PEDOT:PSS and an improved stability. This strategy opens the route towards the development of deep lying work function HTL and is promising for future bulk heterojunction materials with deep lying HOMO polymers.

In recent years, progress in improving the performance of organic photovoltaic (OPV) cells has been considerable, especially thanks to the development of novel organic semiconductors (OSC) [1], [2], [3] . At the same time, the interest in doping OSCs has never stopped growing with versatile applications from energy storage [4] , thermoelectrics [5] and more generally for organic electronics [6], [7], [8] . For OPV solar cells, doping is also attracting attention. Several recent records of power conversion efficiency (PCE) have been achieved by introducing either n-type dopants [9], [10] or p-type dopants [11], [12], [13], [14] directly into the device bulk heterojunction (BHJ) layer, leading to a maximum PCE of 18.3% [9] . Moreover, as the improvement of performance and stability in OPVs has also been possible thanks to interfacial engineering, OSC doping of interlayers can be used as a strategy.

While the selection of materials used as electron transporting Layers (ETL) is quite wide [15] , this is not the case for hole transporting layer (HTL) materials, where the conducting polymer poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) has established itself as the most popular material due to its relatively high transparency, high conductivity and relatively high work function [16] . However, PEDOT:PSS has been shown to be responsible for several stability issues in devices, especially due to its strong acidity that can cause material degradation [17] , including ITO etching [18] . PEDOT:PSS is also known to form a mechanically poor interface with the bulk heterojunction active layer inducing premature delamination of flexible solar cells, when subjected to mechanical strains, especially for industrial fabrication processes such as roll-to-roll technique [19] . These drawbacks push for the need to develop novel hole extraction strategies and materials for OPV solar cells.

In the growing field of hybrid perovskite p-i-n solar cells (PSC), p-doped semiconducting polymers like tris(pentafluorophenyl)borane-doped poly[bis(4-phenyl)(2,4,6-trimethylphenyl)amine (PTAA) [20] can be easily used as an efficient HTL due to the solvent orthogonality when depositing the hybrid perovskite layer onto PTAA. Such interlayer cannot be used for conventional OPV cells (p-i-n) because most of the organic solvents used to deposit the bulk heterojunction are also good solvents for the doped-PTAA, with the risk of dissolving and degrading the bottom layer. To overcome this issue, Shu et al. [21] developed in 2013 a method to stack two layers of polymer by deposing them on two different substrates prior to lamination. This technique enabled to produce conventional OPV structures using poly(3-hexylthiophene) (P3HT) doped with Mo(tfd-CO 2 Me) 3 in homojunction with a P3HT:PC 61 BM active layer, showing similar performances to those made with the PEDOT:PSS HTL [22] . The term "homojunction" here stands on the fact of using the same polymer in the HTL and the active layer, as the same p-type polymer is used in both. Another alternative to classic heterojunction HTLs in OPV is to employ sequential doping of the active layer in inverted devices (n-i-p). By doping the top of the active layer, efficient hole collection is achieved at the anode without the use of a dedicated additional HTL. In 2014, Guillain et al. successfully solution-deposited a F4TCNQ layer onto a P3HT:PCBM active layer [23] , sparking interest for this promising approach. However, this process cannot be easily implemented at the industrial scale for large area printing. Moreover, F4TCNQ is not necessarily an efficient dopant for some recent semiconducting polymers with deep-lying HOMO levels. Furthermore, controlling the depth of penetration of the dopant molecules into the active layer seems to be a major issue in the fabrication of such devices [24] . Several other recent attempts to replace PEDOT:PSS have shown promising results. In 2019, Lin et al. successfully improved the PCE of devices with several active layers by using a liquid exfoliated transition metal dichalcogenide WS 2 [25] . At the same time, an interest appeared in the use of Self-Assembled Monolayers (SAM) in OPVs, that allowed to achieve high PCE of 18.4%. However, the stability of such devices have not yet been fully investigated [26] . Finally, Xu et al. have reported devices exhibiting better performances and stability using a new, less acidic and solution processable polymer called PCPDT-2Ph-H [27] .

Here we demonstrate the fabrication of solution-processed conventional (p-i-n) OPV structure embedding an HTL consisting of the same polymer used in the active layer: Poly[[6,7-difluoro[(2hexyldecyl)oxy]-5,8-quinoxalinediyl]-2,5-thiophenediyl] (PTQ10, Figure 1a), a recent major polymer candidate for OPV with simplified chemical structure and lower cost synthesis [28] . Against the rules of solvent orthogonality, we show that coating the polymer:NFA (Non-Fullerene-Acceptor) bulk heterojunction on top of the HTL consisting of doped PTQ10 is feasible. The well-known p-type dopants such as F4TCNQ cannot efficiently dope the deep lying HOMO donor polymer PTQ10 (-5,54 eV [28] ) because they do not display a deep enough LUMO level (-5.24 eV [29] ). Thus, the chosen dopant is the strong oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, shown in Figure 1a, also known as Magic Blue (MB) for its intense dark blue color. It has been recently used to dope polymers with deep lying HOMO level down to -5.8 eV [30] . MB-doped PTQ10 shows improved electrical conductivity and work function (WF) allowing an efficient extraction of charges. This layer is used in conventional OPV architectures with a PTQ10: ITIC-4F active layer. ITIC-4F (3,9-bis(2methylene-((3-(1,1-dicyanomethylene)-6,7-difluoro)-indanone))-5,5,11,11-tetrakis(4-hexylphenyl)dithieno [2,3- 1a) is a recognized Non Fullerene Acceptor (NFA) exhibiting good performances when mixed with a fluorinated donor polymer [31], [32] including PTQ10 [33] . The resulting solar cells performances are competing with those obtained with the reference material PEDOT:PSS. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) measurements showed that the vertical composition of the device is preserved despite forming the homojunction between the active layer and the HTL. No unwanted diffusion of species between layers is observed, suggesting that such solar cells present promising stability.

d:2',3'-d']-s-indaceno[1,2-b:5,6-b']dithiophene, Figure

Materials & Methods

PTQ10 and ITIC-4F were purchased from Brillant Matters. PDINO was purchased from 1-Material. PEDOT:PSS Clevios PH was purchased from Heraeus. MB was purchased from Sigma Aldrich. Thicknesses of all layers > 30 nm were measured with the Alpha-Step IQ surface profilometer from KLA-Tencor. Thickness of ultra-thin HTL films was estimated by UV-vis absorbance spectroscopy comparing their absorbance with the absorbance of a thicker film made of the same material. ITO coated glass (10 Ω/sq) were successively ultrasonically cleaned with a basic solution, deionized water, and isopropanol. An additional 15min UV-Ozone treatment was applied only to the reference PEDOT:PSS devices. For MB-doped PTQ10 deposition, ITO substrates did not require any UV-ozone treatment to be well-covered by the solution. PEDOT:PSS was spin-coated at 3000 rpm -1000 rpm/s during 45 s and subsequently annealed at 130 °C during 30 min to provide 43 ± 3 nm thick layers. Magic Blue solutions with various concentrations in acetonitrile (ACN) were introduced in PTQ10 solutions in cyclopentyl methyl ether (CPME) with a ACN:CPME ratio of (1:7.5). The doped solutions were spin-coated at 3000 rpm during 60s and dried 5 min at 110 °C to obtain 7 ± 1 nm thick layers. Active layer composed of PTQ10:ITIC-4F (1:1) at 16 mg/mL in chloroform was spin-coated at 1400 rpm during 60 sec then annealed 5min at 80 °C. PDINO at 1 mg/mL in methanol was spin-coated at 3500 rpm during 60 s. 100 nm-thick Al electrodes were thermally evaporated under secondary vacuum (10 -6 mbar). An active area of 10.5 mm² was defined through the use of a shadow mask for the evaporation. All devices performance measurements were characterized at room temperature under nitrogen atmosphere using Keithley 2400 under AM 1.5G illuminations (1000 W/m²) provided by a LCS-100 solar simulator from Oriel and calibrated using a Silicon KG5 cell. The performances were measured for 8 different devices for all samples, and 16 samples for the devices made with PTQ10 doped with 20%mol of MB (estimated vs PTQ10 monomer). UV-Vis NIR absorbance spectra were obtained using a Shimadzu UV-3600 Plus spectrophotometer. AFM images were obtained in tapping mode using a Innova Microscope from Bruker. UV-Vis-NIR spectroelectrochemical conductance measurements were performed according to a method recently proposed [34] . Two PalmSense4 potentiostats were used to perform electrochemical cycling and conductance measurements while a Black Comet CSR-XR compact spectrophotometer equipped with optical fibers and collimating lenses were used to record the evolution of PTQ10 conductivity upon electrochemical doping. Conductivity of Magic Blue-doped PTQ10 was measured between 2 gold electrodes of 5 mm with a gap of 0.5 mm. Electrodes were thermally evaporated on top of the film and 6 different IV measurements were realized and averaged. Kelvin Probe measurements were performed using a Kelvin Control 07 from Besocke Delta Phi and a gold cantilever and Highly Oriented Pyrolytic Graphite (HOPG) as a calibration sample (with an assumed work function of 4.6 eV) [35] . ToF-SIMS measurements were performed using a ToF-SIMS 5 from IonTof, with negative ion depth profiles, using a 500 eV O 2 source on a 300 x 300 µm² surface for sputtering and a 30 kV Bi+ source on a 100 x 100 µm surface for analysis. Figure 1 shows photographs of the undoped PTQ10 solution and PTQ10 with 20%mol of MB. As can be seen, upon the introduction of Magic Blue into the PTQ10 solution, the solution turns from light purple to dark purple. UV-Vis-NIR absorption spectra of corresponding thin films are shown in Figure 2a. Upon addition of MB, two distinct polaronic signatures are observed. The PTQ10 absorption feature at 500-580 nm progressively disappears as dopant concentration is increased. The polaronic absorption peaks are described as a wide peak between 700 and 920 nm and a well-delocalized broad feature in the infrared region. Those optical changes upon the introduction of Magic Blue are typical of Pi-conjugated polymer doping and similar to those observed for the sequential doping of others polymers [30] . At large dopant concentration, the spectra attest to the oxidation of a large fraction of the oxidizable sites of PTQ10. The UV-Vis-NIR spectra of an ACN solution of pure MB is also shown in Figure 2a (blue curve). Its absorption band localized at 780 nm in the same area of the polaronic peak of the doped PTQ10 thin film could lead to consider the band observed in the film as the result of the presence of a high amount of MB instead of a polaronic peak of the doped PTQ10. To determine whether the peak between 700 and 920 nm is due to the presence of MB, or characteristic of polaron formation, we performed spectroelectrochemical UV-Vis-NIR spectroscopy of the doped PTQ10 films. Using a transparent solution of tetrabutylammonium perchlorate (TBAP), we applied a potential on the film versus a silver pseudo reference and measured the transmittance variation while performing cyclic voltammetry. In Figure 2c, the optical density variation, OD, across the UV-Vis-NIR spectrum, for the polymer in neutral, partially oxidized, and highly oxidized is reported. Those measurements clearly show decrease of transmittance between 500 nm and 650 nm and the concurrent appearance of a new band between 650 nm and 950 nm, corresponding respectively to the collapsing of the neutral absorption band of the polymer and the creation of the polaronic band visible on the Figure 2a. Figure 2b examines the dopant concentration threshold for which a significant contribution is observable on the spectroscopic signature of polarons. As it can be seen following the evolution of the normalized absorbance of the neutral state peak (580 nm) and the polaronic peaks (850 nm and 1465nm), the doping effect becomes significantly visible in UV-Vis spectroscopy for doping ratios higher than 10%mol. The impact of doping on the morphology of the PTQ10 films was studied by Atomic Force Microscopy (AFM). The Figure 1c and Figure 1d shows the height profile of pristine and 20%mol doped PTQ10. There is a clear formation of aggregates upon the introduction of dopant in high amount. This phenomenon, inherent to bulk doping, was a limit to the processing of PTQ10 solutions with a doping ratio over 50%mol and has previously been reported as a possible limit of bulk doping at high concentrations [36], [37] .

Results and discussion:

The Figure 3a shows the electrical conductivity of doped PTQ10 films from 10%mol to 50%mol. Due to our measurement device sensibility limited to approximatively 10 -6 S/cm, attempts to measure the conductivity of pristine PTQ10 and doped PTQ10 at low concentration from 0.01%mol to 1%mol was not successful. This result confirms that the polymer, as received, is well purified and not significantly doped by any impurity from the chemical synthesis [38] . The maximum conductivity of 0.13 S/cm is achieved for 30%mol MB concentration which represents an increase of the conductivity of at least five orders of magnitude compared to pristine PTQ10. In order to estimate the efficiency of chemical doping obtained with Magic Blue from solution processing, we performed electrochemical conductance measurements on pristine PTQ10 films on interdigitated electrode as the working electrode of an electrochemical cell. This doping process occurs through the oxidation of PTQ10, which is accompanied by anion injection and concurrent swelling of the polymer structure. We therefore expect to obtain the maximum achievable doping of PTQ10 and conductivity in the explored potential window, limited only by the electrochemical stability of the electrolytic solution and of the polymer. The evolution of the conductivity during electrochemical doping is shown on Figure 2d. The achieved maximum conductivity of electrochemically doped PTQ10 saturates at around 3 S/cm above applied potential difference of 1.5 V, suggesting that all sites contributing to electrical charge transport are efficiently doped. This value is relatively higher than the 0.13 S/cm obtained with 20%mol doping ratio. Indeed, the 3 S/cm value may represent a maximum conductivity value of electrochemically doped PTQ10 which is not easily achievable through bulk doping by molecular dopants through solution processing. In fact, for doped PTQ10 films with doping ratios higher than 20%mol, the conductivity saturates and eventually decreases. This phenomenon, not in line with the polaronic spectroscopic data, could be due to the growth of aggregates at high dopant concentration, as observed on AFM images, hindering charge transport through the doped polymer film. Similar behavior has already been reported for solution doping cases with different polymers and dopants, like PTBTTT-c doping by Mo(tfd-COCF 3 ) 3 [37] or P3HT doping by F4TCNQ [39] . It is also important to mention that the new HTL created by doping the PTQ10 does not necessarily need to show record conductivity values when it comes to the development an efficient HTL for OPV solar cells. It is related to the HTL layer thickness which, in general, remains low to enable maximized light transmission. For example, Fu et al. modified the PEDOT:PSS, resulting in modifications of its conductivity, and obtain good performances for PEDOT:PSS with conductivities of 7.85 x 10 -4 S/cm [40] . Moreover, an anodic current was recorded, corresponding to the oxidation of the polymer, while the increase of the conductivity occurred. It confirms that positive charges are injected into the film and then, that p-type doping occurred without a molecular dopant.

Kelvin Probe was used to measure the work function (WF) at the surface of the doped PTQ10 films deposited on ITO coated glass substrates. The results on Figure 3b shows significant increased WF values upon doping. For the 20%mol MB doped PTQ10, it reaches the value of 5.23 eV, matching the reference value of PEDOT:PSS, also measured internally. WF of ITO coated with highly doped PTQ10 surpasses PEDOT:PSS-coated ITO up to 5.58 eV. Such values are very promising for the OPV HTL application since the highest WF difference between the anode and the cathode, is a pre-requisite for maximizing the open-circuit voltage (V oc ) of solar cells [41] . Although it might seem unusual to deposit an active layer on an interlayer without respecting the solvent orthogonality, functional devices were obtained. Indeed, after having carefully evaporated the CPME used to deposit the doped PTQ10, thanks to a thermal annealing at 110 °C for 5 min, the active layer is deposited from chloroform, a highly volatile solvent, in order to avoid its penetration in the HTL. The expected structure of the resulting device is shown in Figure 4a.

To ensure that dopants from the HTL were not washed away from the polymer during the deposition process of the active layer or that the whole HTL deposited remained, ToF-SIMS measurements were performed on the ITO/PTQ10:20%mol Magic Blue/BHJ stack deposited in the same conditions including thermal annealing before and after the deposition of the BHJ layer and are shown in Figure 4b. Monitoring the signals for bromine and chlorine ions (present in the cation and anion of Magic Blue, respectively) allows to localize the dopant in the thin film layer structure. As shown on the figure, the dopants are clearly localized into the HTL. No diffusion of dopant into the active layer is observed even when the stack has been annealed. Consequently, the actual vertical composition of the device is close to the expected one, drawn on Figure 4a. From this data, we cannot however exclude the possible diffusion of the NFA acceptor into the HTL, which is not traceable with this characterization method considering that PTQ10 and ITIC-4F contain the same elements. However, if a significant amount of NFA were to migrate into the HTL it may hindered the solar cells from working properly, which is not the case here.

The apparent stability of the stacked layer structure could be explained by the quick deposition and evaporation of the BHJ solution coupled with the phenomenon of increased pi-stacking occurring between the polymer chains upon doping. This effect was previously reported on polymers such as P3HT [42] , and more recently exploited by Zokaei et al. to increase the elastic modulus of p(g42T-T) and P3DDT upon doping [43] . Moreover, dopant diffusion in a polymer matrix is a major cause of doping instability as reported for the Mo(tfd-CO 2 Me) 3 doping of P3HT [44] . We performed similar ToF-SIMS measurements realized in the exact device fabrication conditions with thermal annealing after BHJ deposition at 80 °C for 5 min. Results were compared on the same device stack, without thermal annealing and after a higher thermal annealing of 110 °C during 10 min (see Figure S1 and Figure S2). No significant changes neither in the position nor the thickness of the Magic Blue signals were observed. We conclude that the vertical diffusion of the dopant seems to be kinetically impaired under the explored conditions in such devices. This could be due to a blocking effect of the acceptor at the interface between HTL and active layer as mentioned in a previous study [44] . After having successfully realized PTQ10-doped HTL with increased work function, improved conductivity and having successfully stacked the BHJ on top of the HTL, complete OPV devices were prepared in conventional (p-i-n) architecture. Devices with PTQ10 doped from 0.01% to 50%mol were compared with reference devices with PEDOT:PSS. The HTL layer thickness was first carefully optimized to minimize light absorption by PTQ10. Device performances as a function of HTL thickness are shown on Figure S3. The short-circuit current density (J SC ) is strongly affected by the thickness of the HTL. The optimal thickness of the doped PTQ10 layer was found to be between 6 nm and 8 nm and it was kept constant for the following investigations. Figure 5 shows the resulting PV parameters as a function of the dopant concentration. HTLs with low dopant concentration lead to inefficient devices suffering from low open-circuit voltage (V oc ) and low fill factor (FF). Inadequate HTL work function , i.e., not deep enough with respect to the HOMO of PTQ10, is penalizing the V oc . Insufficient HTL conductivity leads to increased series resistances, inducing low FF. Increasing dopant concentration gradually, both the V oc and the FF increase. For a dopant concentration of 20%mol, the performances are maximized. The V oc reaches 0.94 V which is comparable to the V oc for PEDOT:PSS reference sample. Such V oc is consistent with the state-of-the-art for PTQ10:NFA based solar cells [33] . The J sc of 20 mA/cm² shows efficient charge extraction. Combined with a FF of 0.58 ± 0.02, the optimized PCE is 11.3 %. Such efficiency is competing with the PEDOT:PSS-based reference cells as can be seen on Figure 6a comparing the J-V curves of both devices. Table 1 summarizes the device performances. Magic blue containing devices show good reproducibility despite the nonorthogonality of solvents during the fabrication process. For doping ratio higher than 20%mol, much larger standard deviations are observed especially on the short-circuit currents. The proposed explanation is that the rough morphology of the over-doped layers is detrimental to device performance, either promoting charge recombination in the HTL or presenting of defects at the homojunction interface between the HTL and the active layer. This is consistent with the inhomogeneity of layers observed by AFM (Figure 1d). The stability of devices was examined in dark conditions in inert atmosphere and is shown on Figure 6b. After 340 h, the doped-PTQ10 based devices retain almost 95% of their initial performances. They are more stable than devices containing PEDOT:PSS whose performance decreases to 82% over the same period. According to the stability evolution of V oc , J sc and FF shown in Supplementary Informations (Figure S4, S5, S6), this PCE stability improvement is due to a simultaneous slight improvement of the stability of the three PV parameters.

Conclusions

A novel and efficient alternative to PEDOT:PSS in conventional architecture organic solar cells is reported. This is the first time a hole transport layer is designed based on doping the same polymer as the one used in the bulk heterojunction thus forming a so called homojunction. PTQ10 doped by Magic Blue was optimized to tune its conductivity and significantly increase its work function.

Resulting solar cells showed competing power conversion efficiency as the widely used PEDOT:PSS and an improved stability. This homojunction strategy opens the route to be applied to future polymers to come in the OPV field. Especially. with the actual tendency to use deep lying HOMO polymers. classical HTL like PEDOT:PSS may not be appropriate to maximize the open-circuit voltage because of its work function. The homojunction doping strategy of using the same polymer in the HTL and in the active layer enables to insure an HTL with a deeper work function than the HOMO level of the polymer. As a consequence. this strategy may become a versatile method for future high efficiency polymer solar cells.

Figure 1 :

 1 Figure 1: a) Molecular structures of materials; b) Energy levels of materials, note that doped PTQ10 values are experimental from Kelvin probe measurements performed in the present study; AFM images of c) Pristine PTQ10 and d) PTQ10 doped with 20%mol of Magic Blue (inset show photographs of undoped and doped PTQ10 solutions).

Figure 2 :

 2 Figure 2: a) Absorption spectra of pristine and doped PTQ10 films for various MB concentrations. b) Evolution of the normalized absorbance at 580, 850 and 1465nm as a function of doping concentration c) UV-Vis electrochemical spectroscopy spectra d) C-V cyclic curve and evolution of conductivity during electrochemical cycling of PTQ10.

Figure 3 :

 3 Figure 3: a) Conductivity of doped PTQ10 films versus doping molar ratio. b) Work function of ITO coated by doped PTQ10 films versus doping molar ratio.

Figure 4 :

 4 Figure 4: a) Schematic representation of the obtained architecture of the cell. b) ToF-SIMS measurements of the HTL and active layer stack deposited on ITO.

Figure 5 :

 5 Figure 5: Solar cell parameters as a function of doping concentration in the PTQ10 interlayer. a) Short-circuit current density Jsc b) fill factor FF c) open-circuit voltage V oc and d) power conversion efficiency PCE -blue mark is a guideline for the eye representing the reference PEDOT:PSS based solar cells.

Figure 6 :

 6 Figure 6: a) J-V curve of best device compared with Ref. PEDOT:PSS device. b) Device stability under inert atmosphere

Table 1 : Photovoltaic parameters (maximum and average values) as function of HTL dopant concentration, compared with PEDOT: PSS reference devices

 1

	Sample	Voc	max (Voc	avg ) [V]	Jsc	max (Jsc	avg ) [mA/cm²]	FF	max (FF	avg )	PCE	max (PCE	avg ) [%]
	PTQ10 : 0.01% MB	0.31 ( 0.26 ± 0.04 ) 15.53 ( 14.38 ± 0.90 ) 0.36 ( 0.33 ± 0.01 )	1.69 ( 1.26 ± 0.29 )
	PTQ10 : 0.1% MB	0.29 ( 0.25 ± 0.04 ) 15.24 ( 14.54 ± 0.83 ) 0.34 ( 0.33 ± 0.01 )	1.52 ( 1.23 ± 0.29 )
	PTQ10 : 1% MB	0.43 ( 0.40 ± 0.03 ) 18.53 ( 17.71 ± 0.87 ) 0.39 ( 0.39 ± 0.01 )	3.04 ( 2.71 ± 0.31 )
	PTQ10 : 10% MB	0.81 ( 0.76 ± 0.04 ) 19.83 ( 19.07 ± 0.54 ) 0.51 ( 0.48 ± 0.02 )	8.06 ( 6.99 ± 0.79 )
	PTQ10 : 20% MB	0.95 ( 0.94 ± 0.01 ) 19.81 ( 18.90 ± 0.78 ) 0.61 ( 0.58 ± 0.02 )	11.25 ( 10.38 ± 0.75 )
	PTQ10 : 30% MB	0.94 ( 0.90 ± 0.05 ) 21.00 ( 18.11 ± 2.38 ) 0.54 ( 0.52 ± 0.02 )	8.92 ( 8.37 ± 0.45 )
	PTQ10 : 40% MB	0.93 ( 0.90 ± 0.03 ) 12.78 ( 8.83 ± 3.01 ) 0.53 ( 0.49 ± 0.03 )	6.27 ( 3.93 ± 1.63 )
	PTQ10 : 50% MB	0.93 ( 0.86 ± 0.05 ) 13.74 ( 8.14 ± 4.23 ) 0.53 ( 0.49 ± 0.04 )	6.54 ( 3.53 ± 2.08 )
	Ref. PEDOT:PSS	0.94 ( 0.94 ± 0.00 ) 19.75 ( 19.08 ± 0.48 ) 0.58 ( 0.56 ± 0.01 )	10.64 ( 10.06 ± 0.43 )