Oncogenic MAPK pathway activation disrupts Schwann cell fate commitment, inducing congenital and progressive neuropathy in mice

Oncogenic MAPK pathway activation disrupts Schwann cell fate commitment, inducing congenital and progressive neuropathy in mice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Oncogenic MAPK pathway activation disrupts Schwann cell fate commitment, inducing congenital and progressive neuropathy in mice View ORCID Profile Elise Marechal , View ORCID Profile Daniel Aldea , View ORCID Profile Patrice Quintana , View ORCID Profile Grégoire Mondielli , View ORCID Profile Nathalie Bernard-Marissal , View ORCID Profile Mathias Moreno , View ORCID Profile Claire El Yazidi , View ORCID Profile Natacha Broucqsault , View ORCID Profile Valérie Delague , View ORCID Profile Lauren A. Weiss , View ORCID Profile Anne Barlier , View ORCID Profile Heather C. Etchevers doi: https://doi.org/10.1101/2024.04.24.590951 Elise Marechal 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elise Marechal Daniel Aldea 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel Aldea Patrice Quintana 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrice Quintana Grégoire Mondielli 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Grégoire Mondielli Nathalie Bernard-Marissal 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nathalie Bernard-Marissal Mathias Moreno 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mathias Moreno Claire El Yazidi 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Claire El Yazidi Natacha Broucqsault 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Natacha Broucqsault Valérie Delague 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Valérie Delague Lauren A. Weiss 2 Department of Psychiatry and Behavioral Sciences, University of California , San Francisco, San Francisco, CA 94143, USA 3 Institute for Human Genetics, University of California , San Francisco, San Francisco, CA 94143, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lauren A. Weiss Anne Barlier 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France 4 AP-HM, MMG, MarMaRa Institute, La Conception Hospital Laboratory of Molecular Biology , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anne Barlier Heather C. Etchevers 1 INSERM, Marseille Medical Genetics, U1251, MarMaRa Institute, Aix Marseille University , Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Heather C. Etchevers For correspondence: heather.etchevers{at}inserm.fr Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract RASopathies, rare congenital syndromes affecting multiple organ systems, often include peripheral neuropathy of unknown origin. While RASopathy-associated gene variants are proto-oncogenic, the impact of timing and mosaicism on pathogenicity remains poorly understood. Here, we investigate the links between Braf, a key mitogen-activated protein kinase (MAPK) effector, and peripheral neuropathy. By targeting Braf p.V600E, an oncogenic variant found in mosaic RASopathies, to embryonic Mpz-expressing cells in mice, we induced a congenital Charcot-Marie-Tooth-like degenerative neuropathy. This phenotype was characterized by hyperplastic nerves, hindlimb weakness, and unexpectedly reduced body size. Constitutively active Braf expanded a Jun+ Schwann cell repair state, impairing myelination and nerve homeostasis. To examine relevance to RASopathies, we differentiated patient-derived stem cells bearing the cardio-facio-cutaneous syndrome-associated BRAF p.Q257R variant into Schwann cells. Compared to wild-type controls, CFC-derived cells failed to acquire mature phenotypes, instead exhibiting progenitor or repair-type transcriptional profiles. Our findings implicate somatic mosaicism in the unresolved genetic heterogeneity of neuropathies and expand the candidate gene list for peripheral nerve disorders. Moreover, they reveal a MAPK-dependent mechanism linking neural crest-derived Schwann cell dysfunction to both body growth and nerve homeostasis, providing new insights into the mechanisms in RASopathy-associated neuropathy and potential therapeutic targets. Introduction Many cancers and congenital diseases co-opt and deploy the molecular mechanisms of normal organogenesis in inappropriate contexts. Activation of the mitogen-activated protein kinase (MAPK) intracellular signaling pathway drives dozens of cancers in somatic tissues 1 . In contrast, RASopathies are heterogeneous developmental syndromes that preferentially affect epidermis and pigmentation; cardiovascular, craniofacial and skeletal systems; and neurological or endocrine functions impacting body size and fertility 2 – 5 . Pathogenesis results from variants in at least 24 genes that sustain MAPK signaling to, and nuclear translocation of, the extracellular signal-regulated kinases (ERK1/2) 6 . Activating variants may be expressed constitutionally or as mosaic lineage-specific (somatic) RASopathies 7 . Most constitutional and somatic RASopathies associate with specific cancer predispositions, particularly when syndromic 8 , 9 . A limited number of MAPK pathway variants engender this array of overlapping clinical outcomes. Oncogenic variants in BRAF , particularly encoding changes to valine (p.V)600, drive aggressive forms of melanoma as well as many other cancers 1 , 10 . BRAF p.V600E has never been observed in germline disease because of early embryonic lethality, modeled in mice 11 . BRAF p.V600E yields an active, monomeric kinase, decoupled from RAS-mediated transduction 12 , 13 . In contrast, less-activating BRAF variants such as p.Q257R underlie a growing list of congenital RASopathies 8 , 14 . These include the germline disorders cardio-facio-cutaneous syndrome (CFC; ORPHA:1340) and Noonan syndrome with (ORPHA:500) or without (ORPHA:648) multiple lentigines. Their common clinical features include reduced postnatal growth, dysmorphic facial features, melanocytic nevi or other pigmented lesions, congenital heart defects, and tumor predispositions 15 – 17 . The mechanisms and triggers of neuropathies in the context of RASopathies remain unexplored. However, recent reports describe CFC and Noonan syndrome patients with early-onset neuropathies characterized by pain, sensory loss and/or muscle wasting 18 – 20 , resembling severe, early-onset Charcot-Marie-Tooth (CMT) syndromes 21 , 22 . Peripheral nervous system (PNS) function relies entirely on Schwann cells (SC), which provide physical and trophic nerve support while insulating large, fast-conduction axons 23 – 25 . The PNS — including SC, perineurial stroma and sensory/autonomic neurons — derives from multipotent embryonic neural crest (NC) cells, which also generate craniofacial and cardiovascular connective tissues, neuroendocrine cells and melanocytes 25 – 28 . Mek1, the primary MAP2K activating Erk1/2, is essential for nerve myelination during maturation, but its sustained overactivation in adult SC induces murine neuropathy 29 – 32 . Despite broad CMT genetic heterogeneity, with dozens of identified genes, no MAPK effectors have been implicated; however, approximately 20% of CMT patients of all subtypes lack a molecular diagnosis 33 . Given the shared NC origin of affected cell types in RASopathies, we hypothesized that multipotent NC-derived lineages may be vulnerable to prenatal MAPK hyperactivation, compromising their ability to instruct fate or regulate tissue homeostasis over time. The goal of this study was to assess systemic and cell-autonomous effects of constitutively active BRAF signaling on NC-derived progenitor differentiation. We found that conditional expression of Braf p.V600E in mouse cells expressing the SC gene Mpz 34 (encoding a homophilic adhesion molecule, myelin protein zero [P0]) led to a peripheral neuropathy in association with induction and maintenance of an ineffective repair SC phenotype. Although nerve myelination initiated after birth as usual, mutant peripheral glia rapidly misregulated hundreds of genes associated with mature SC function, including myelination, and nerves underwent active myelin destruction. Unexpectedly, although endocrine tissues had not been intentionally targeted in this model, mice also underwent developmental delay and remained sexually ambiguous or immature. Furthermore, human induced pluripotent stem cells derived from two teenaged CFC syndrome donors, carrying the most frequent BRAF missense variant, p.Q257R, were also incapable of terminal SC differentiation in vitro , in contrast to cells from wildtype donors, after upregulating repair SC-specific transcripts. These results suggest that molecularly undiagnosed cases of RASopathies, or endophenotypes such as CMT type 1 or idiopathic cryptorchidism 35 , may be imputable to somatic mosaicism for oncogenic MAPK pathway gene variants in the NC lineage. Results Sustained Schwann cell MAPK signaling in causes juvenile locomotor and growth deficits In order to model constitutive Braf-mediated signaling in progenitors of Schwann cells in vivo , we used Mpz-Cre +/° mice, which transcribe Mpz (encoding myelin protein zero, or P0) as early as embryonic day 12 in neural crest cells 36 , 37 . Crossed to a floxed Braf hotspot V600E allele ( Braf V600E ) ( Supplemental Figure S1a ), resulting Mpz -Cre +/° ; Braf V600E/+ (Mpz- Braf ) mice were born at expected Mendelian ratios, remaining asymptomatic for the first week. From postnatal day (P)7, all Mpz- Braf mutants developed signs of neuropathy including tremors, abnormal hindlimb postural reflexes and trailing legs ( Fig 1a ). They nonetheless showed age-appropriate activity in exploration, feeding and grooming. Correlating with phenotypic onset, mutant sciatic nerves exhibited significantly more Erk1/2 phosphorylation than controls, increasing from early (P5; Fig 1c, e ) to later (P21; Fig 1d, e ) stages of SC-mediated myelination, demonstrating congenital and persistent MAPK activation in peripheral glia. Download figure Open in new tab Figure 1. Constitutive MAPK activation mediated by a single copy of Braf V600E in murine Schwann cell progenitors induces early peripheral neuropathy and small body size. a) By postnatal day (P)21, Mpz- Braf mice displayed abnormal hindlimb postural reflexes and ability to keep hindfeet on the bars of a wire cage lid (arrowheads), indicating a functional peripheral nerve defect. b, c) Western blot analysis of total and phosphorylated (p)Erk1/2 signaling performed on sciatic nerve lysates from (b) P5 and (c) P21 control and Mpz- Braf mice. Gapdh was detected as a loading control. d) Protein densitometry in control (n=4) vs. mutant (n=5) sciatic nerve lysates at P5, and control (n=13) vs. mutant (n=15) lysates at P21. e ) Body weight of both sexes was monitored at P5, P10, P21, P35 and P42 in control and Mpz- Braf mice, respectively (n = 28 vs. 8 at P5, n = 15 vs. 5 at P10, n = 54 vs. 45 at P21, n = 14 vs. 10 at P35, n = 5 vs. 5 at P42). f ) Quantitative PCR analysis of Ghr and Igf1 expression in control (n=5) vs. mutant (n=5) livers at P21. Statistical significance from unpaired Mann-Whitney test in d ), two-way ANOVA followed by Šidák’s post-hoc test in f ), and Student’s t-test in g ): ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. After P10, mutant mice were also visibly smaller than control littermates, with significantly reduced but proportional nose-to-tail length and weight ( Fig 1f ). Since activating BRAF mutations have been described in the context of human hypopituitarism 2 , we looked for a possible central deficiency in growth hormone (Gh) by comparing the transcriptomes of pituitaries at P5 between control and Mpz- Braf mice with bulk RNA-sequencing (RNAseq). Gh-releasing hormone receptor Ghrhr or Gh transcripts, along with all other known coding genes, were not significantly altered in mutants at this stage ( Supplemental Figure S1b ; Supplemental Table S1 ). Gh binds to peripheral Ghr, a type I cytokine receptor, to regulate production of its effector, insulin-like growth factor 1 (Igf1) in the liver and other sites, exerting thereby anabolic effects on bone and muscle 38 . We therefore compared the transcription of Ghr and Igf1 by RT-qPCR in the livers of P21 Mpz- Braf and control littermate mice. While Ghr was upregulated on average by 3.3-fold in mutant livers, Igf1 was far more significantly diminished, by 211-fold ( Fig 1g ). The juvenile Mpz- Braf growth deficiency was the likely consequence of a near-complete loss of peripheral Igf1 production. Braf V600E/+ induces widespread peripheral nerve demyelination and axonal loss Strikingly broad nerves were visible at autopsy in all Mpz- Braf mice at P5 or P21, lacking the structural bands of Fontana 39 ( Fig 2a ; Supplemental Figure S1 ). Despite their smaller body size, mutant sciatic nerves were significantly wider at P10 (0.48 ± 0.07 vs. 0.31 ± 0.05 mm diameter in controls; Fig 2b ). This discrepancy stabilized after weaning, with mutant nerves measuring 0.74 ± 0.09 mm at P21 ( vs. 0.39 ± 0.05 mm in controls), and 0.78 ± 0.05 mm at P35 ( vs. 0.50 ± 0.04 mm). Nerve enlargement was consistent across all examined peripheral nerves in P21 Mpz- Braf mice, including sensorimotor (peroneal, tibial, sural, trigeminal, ophthalmic) and sensory (saphenous, maxillary) nerves ( Supplemental Figure S1c-e ). Download figure Open in new tab Figure 2. Myelination initiation is not impaired in Braf -mutant sciatic nerves, while maintenance is compromised. a) Macroscopic examination of control and mutant sciatic nerve at P5 and P21. Arrows indicate Fontana bands, present only in control nerves. b) Sciatic nerve diameter measurements at P5, P10, P21, and P35 in both Mpz- Braf and control mice (n = 11 vs. 6 at P5, n = 6 vs. 4 at P10, n = 15 vs. 10 at P21, and n = 5 vs. 5 at P35; in control vs. mutant nerves, respectively). Two-way ANOVA followed by Šidák’s post-hoc test; ns, not significant; ** p < 0.01; **** p < 0.0001. c) Representative transmission electron micrographs of sciatic nerve cross-sections at P5 and P21 from control and mutant mice. Bars = 10 µm except for magnified axons at P28, 2 µm. Arrows, myelin aberrations; c, extracellular collagen; f, fibroblast; nm, non-myelinating Schwann cell; sc, myelinating Schwann cell;. To assess myelination dynamics and ultrastructure, we analyzed mutant sciatic nerves cross-sections using transmission electron microscopy (EM) at P5, P21 and P28. Over time, mutant nerves exhibited degenerating axons, extracellular collagen, fibroblasts, macrophages and abundant SC nuclei. By P21, myelin aberrations, including tomaculae, infoldings, onion bulbs degenerated axons, and focal hypermyelination of small-caliber axons, were frequent ( Fig 2c ; Supplemental Figure S2a-d ). These anomalies were interspersed with abundant extracellular collagen fibrils and tentacular fibroblasts ( Fig 2c ). By P42, axon-associated macrophages containing myelin debris were also evident ( Supplemental Figure S2e-f ). At P5, no significant differences were observed in average axonal density (52 ± 2 axons/mm 2 in Mpz- Braf vs. 48 ± 3 axons/mm 2 in controls) or diameter (1.447 ± 0.026 µm vs. 1.476 ± 0.072 µm; Fig 3a ; Supplemental Figure S3a-b ). Likewise, immune infiltration was absent, and myelin thickness as quantified by the g -ratio, inner/outer axonal myelin daimeter) 40 , remained unchanged (0.732 ± 0.019 in mutants vs. 0.708 ± 0.028 in controls; Fig 3b and Supplemental Figure S3c-d ). Radial sorting and myelination initiated similarly in mutants and controls, with only occasional myelin aberrations at this stage ( Fig 2c ). Download figure Open in new tab Figure 3. Braf V600E in Schwann cells triggers demyelination and degeneration of large-caliber axons a) Mean axon diameter at P5 (n = 3 per genotype, not significantly [ns] different). b ) MyelTracer-obtained g -ratios as a function of axon diameter at P5, to assess myelin thickness in control (n = 803) vs. mutant (n=811) myelinated fibers (n=3 per genotype). The slopes of a simple linear regression do not vary significantly across P5 genotypes. c) Axon density decreases physiologically between P21 and P28 in control sciatic nerves but is significantly reduced to 7-11% of normal levels in mutant nerves at these stages. d ) Mean axon diameter per genotype at P28 (n = 2 per genotype) shows that smaller fibers remain on average. d) Binning analysis of g -ratios as a function of axon diameter at P28. A total of n = 792 vs. 746 myelinated axons were analyzed for control vs. mutant nerves, respectively, from 2 individuals of each group (correlation significance indicated by asterisks). f) g -ratios of axonal diameters at P28, with a significantly different slope (p3 µm), there is modestly significant hypermyelination in small axons but more significant hypomyelination at diameters over 1 µm. Statistical significance was assessed with two-way ANOVA followed by Šidák’s post-hoc test; ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. By P21, axon density had declined dramatically in Mpz- Braf mutants (4,060 ± 1,378 axons/mm 2 vs. 36,172 ± 2,726 in controls; Fig 3c ). At P28, only 7.2% of myelinated axons remained in mutant sciatic nerves (1,841 ± 465 axons/mm 2 vs. 25,703 ± 2,812 axons/mm 2 in controls; Fig 3c ). Surviving mutant axons were significantly smaller (1.89 ± 0.23 µm vs . 2.64 ± 0.07 µm in controls; Fig 3d-e ). To determine which axon calibers were affected, we analyzed 792 mutant and 746 myelinated axons at P28 ( Fig 3f ). Large-diameter axons (>3 µm) were disproportionately lost post-weaning (8.7 ± 6.7% in Mpz- Braf vs . 33.3 ± 5.6% in controls; Fig 3e-f ; Supplemental Figure S3e ). Residual axons exhibited 12% higher g -ratios, indicating thinner myelin sheaths ( g -ratios: 0.66 ± 0.02 in mutants vs. 0.59 ± 0.00 in controls; Fig 3f ; Supplemental Figure S3f ). When binned by caliber into small (<1 µm), medium (1-3 µm) and large (3 µm) diameters, medium- and large-caliber axons showed significantly thinner myelin in mutants ( g -ratios: 0.67 ± 0.03 vs. 0.59 ± 0.01 for medium; 0.75 ± 0.00 vs. 0.60 ± 0.01 for large; Fig 3g ). In controls, myelin thickness increases with axon size to plateau at axon diameters ≥4 µm 30 . Conversely, small-caliber axons exhibited a modest (10.6%) but significant increase in myelination ( g -ratios: 0.47 ± 0.01 in mutants vs. 0.52 ± 0.02 in controls; Fig 2c ; Supplemental Figure S2d ). Fast-conduction motoneuron axons 41 were thus most severely affected in mutants, while small axons were often hypermyelinated. Myelin component mislocalization and SC progenitor expansion To extend our structural findings, we examined myelinated axon components by teasing fixed fibers from P5 and P21 sciatic nerves. Immunofluorescence for contactin-associated protein 1 (Caspr/paranodin) revealed that, unlike controls ( Fig 4a ), mutant axons displayed irregularly spaced, diffuse or absent myelin internodes ( Fig 4b, d-g ). At P5, longitudinal views already showed early myelin breakdown, with F-actin labeling highlighting beaded myelin ovoids along some mutant fibers ( Fig 4b-d ). By P21 ( Fig 4e ), Mpz- Braf mice alone displayed demyelinated segments lacking myelin basic protein (Mbp) ( Fig 4f ). The presence of multiple adherent nuclei between residual Caspr+ nodes ( Fig 4f ), rather than the single SC expected after normal radial sorting 42 , suggests persistent immature SCs in mutants. Download figure Open in new tab Figure 4. Segmental demyelination is preceded by myelin disassembly in Mpz- Braf mice. Teased fibers from sciatic nerves of control ( a, e ) and Mpz- Braf mice ( b-d , f-g ) at P5 (top panels) and P21 (bottom panels) were immunostained with antibodies against Mbp and Caspr and Alexa Fluor-488-conjugated phalloidin. Mpz- Braf fibers had degenerating myelin ovoids (white arrowheads), fewer apparent nodes of Ranvier with diffusing paranodal Caspr (orange arrowheads), and segmental demyelination, not observed in controls. Nuclear DNA was stained with DAPI (grey). Maximum intensity z-projection images were generated in b, e and f with Nikon NIS-Elements Viewer software. Scale bars: 50 µm ( a, b, f ) or 10 µm ( c, e, g ). To assess proliferating SCs, we examined the co-expression of Ki67, a cell cycle progression marker 43 , 44 , and Sox10 (SRY-box transcription factor 10), present throughout the SC lifespan 45 , by immunostaining sciatic nerve sagittal sections at P42. While control nerves showed sparse Sox10 + /Ki67 + nuclei ( Supplemental Figure S4a ), mutant hyperplastic nerves exhibited densely, irregularly arranged double-positive nuclei ( Fig. S4b ). Notably, all mutant Sox10 + nuclei were strongly Ki67 + in mutant nerves, interspersed with additional Ki67 + Sox10 - cells. These findings indicate nerve hyperplasia rather than hypertrophy in mutants, as sustained SC proliferation is concomitant with inflammation and fibrosis. Braf V600E/+ glia in vivo downregulate myelin sheath gene transcription To identify global transcriptional changes associated with myelin loss after MAPK pathway activation, we performed bulk RNAseq on Mpz- Braf and control sciatic nerves dissected from four mice each at P5 and P21 ( Fig 5 and Supplemental Table S2 ). Unlike P5 pituitaries, 2,540 differentially expressed genes (DEGs; padj< 0.05) were identified at P5 between mutant and control nerves. By P21, this rose to 10,409 DEGs ( Fig 5a ), indicating early, large-scale transcriptional changes in sciatic nerve cells following constitutive MAPK signaling. Download figure Open in new tab Figure 5. Comparative transcriptomics show downregulation of myelin sheath component transcripts in Braf -mutant sciatic nerves. a) RNA-seq analysis was performed in control and Mpz- Braf mutant sciatic nerves (n = 4 per genotype). Volcano plots depict the DEGs in Braf -mutant sciatic nerves relative to controls at P5 (left panel) and P21 (right panel). An adjusted p -value < 0.5 and Log2FC 1 (red) were used as thresholds to define DEGs. The top 15 DEGs are labeled on each graph according to Manhattan distance. b) Volcano plots illustrate the differentially enriched pathways provided by GSEA through WebGestalt https://www.webgestalt.org/ 117 using the mouse Wikipathway database. Pathways are significantly enriched when false discovery rate (FDR) < 0.05 and normalized enrichment score (NES) 1 (red). Circle diameter is proportional to gene ratio (count of core enrichment genes / count of pathway genes). c) Enrichment plot for “cholesterol biosynthesis”, the most significant gene set downregulated in mutant sciatic nerves (p-value and FDR < 0.001, NES = -2.3). d) Dot plot displaying log2 fold-change (Log2FC) of myelin-associated genes ( Mpz , Pmp22 , and Mbp in bold, representing transcripts of the primary myelin sheath proteins). The myelin sheath comprises 70% to 85% lipids and 15 to 30% proteins 46 . Notably, the term “spinal cord injury” was enriched in DEGs at both P5 and P21 ( Fig 5b ). By P21, cholesterol biosynthesis and metabolism pathways were significantly downregulated in mutant nerves ( Fig 5b, c ). Strongly downregulated DEGs at P21 included Hmgcr (11.5-fold reduction), encoding the rate-limiting enzyme in cholesterol biosynthesis 47 , and Fa2h (7.9-fold reduction), encoding fatty acid 2-hydroxylase, a key enzyme in myelin glycosphingolipid synthesis 48 ( Supplemental Table S2 ). While Pmp2 (Fabp8, a fatty acid-binding protein) and Omg (oligodendrocyte myelin glycoprotein) were significantly upregulated at P5 in mutants, their expression normalized to control levels by P21 ( Fig 5d ). Transcripts for other myelin proteins (e.g., Mpz , Pmp22 [peripheral myelin protein 22], Mbp [myelin basic protein]) showed no significant changes at P5, but other myelin-associated transcripts, including myelin and lymphocyte protein ( Mal ), periaxin ( Prx ), the myelin-associated 2’,3’-cyclic nucleotide 3’ phosphodiesterase ( Cnp ), myelin-associated glycoprotein ( Mag ), and plasmolipin ( Pllp ) were already modestly but significantly reduced ( Fig 5d ; Supplemental Table S1 ). By P21, when control sciatic myelination is nearly complete ( Fig 2 ), Mpz , Mbp and Pmp22 transcripts were also significantly downregulated in mutants ( Fig 5d ). Thus, sustained MAPK signaling drives transcriptional downregulation of myelin components, contributing to the observed demyelination and axonal degeneration phenotype. Constitutive MAPK signaling disrupts transcriptional control of myelination To investigate how constitutively active MAPK signaling modulates myelination-associated transcription factors (TFs), we analyzed co-variance patterns in Mpz- Braf and control sciatic nerves. At P5, key myelination activators Egr2 (Krox20; early growth factor response 2 49 ), and Sox10 50 , remained unchanged. However, by P21, mutant SCs exhibited a significant decrease in Egr2 and the melanocyte determinant Mitf , with concomitant ectopic increases in neural crest or multipotency maintenance TFs Sox2, Foxd3 , Tfap2a and Tfap2b, with no change in Sox10 ( Fig 6a ; Supplemental Table S2 ). Concurrently, myelination transcriptional repressors 51 were significantly upregulated in mutant nerves at P21, including Jun , Egr1 , Egr3, and Zeb2 ( Fig 6a ). Increased Jun translated to higher Jun protein levels in mutants beyond P21, whereas Egr2 protein loss became detectable at P42 ( Fig 6b ). Download figure Open in new tab Figure 6. Egr2 and its target genes decrease in Mpz- Braf nerves with concomitant increase in expression of negative regulators of myelination a) Dot plot displaying log2 fold-change (Log2FC) of known positive ( Sox10 , Egr2 ) or negative ( Jun , Sox2 ) transcriptional regulators of myelination in RNAseq of mutant vs. control sciatic nerves at P5 and P21. b) Western blot of Jun and Egr2 in sciatic nerve lysates from P21 and P42 Mpz- Braf and control mice. Gapdh was detected as a loading control. N=13 individuals of each genotype. Statistical significance was assessed by an unpaired Mann-Whitney test; ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001. c) Enrichment plot for up- or down-regulated genes (top and bottom panels, respectively) showing that most genes are commonly deregulated in sciatic nerves from either the Mpz-Braf or homozygous Egr2 hypomorphic allele mice 132 . d-e) Spearman’s rho to assess significance of correlation was calculated from bulk RNAseq of Mpz- Braf sciatic nerves at P5 (top panels) or P21 (bottom panels), with analogous datasets at P8 and P30 from Egr2- Cre -driven ( d ) overexpression of constitutively active Mek1 (Egr2 -Cre ; Mek1 DD) or ( e ) knockout of Shp2 (Egr2 -Cre ; Shp2 f/f ). To corroborate these findings, we compared our model to a hypomyelinating neuropathy model with germline expression of two hypomorphic Egr2 alleles (Le et al., 2005). Gene Set Enrichment Analysis (GSEA) analysis revealed significant co-variance of 90% (99/110) upregulated and 91.5% (98/107) downregulated DEGs ( Fig 6c ; p < 0.001). Sciatic nerve hypomyelination is therefore linked to increased Jun expression, followed by reduced Egr2 -driven transcription. During remyelination after sciatic nerve injury, axonal Nrg1 type III stimulates SC Pmp2 expression, facilitating fatty acid uptake and mitochondrial ATP production 52 . In Mpz- Braf mice, RNAseq revealed significant Pmp2 and Nrg1 overexpression ( Supplemental Table S2 ). Notably, the major source of Nrg1 after P5 is the SC rather than the axon itself 53 . This reactive overexpression to induced injury or potentially physiological wear, associated with “onion bulb” formation around neuropathic axons, contributes to an ultimately detrimental chronic repair response 54 . Nrg1-III normally binds SC-specific ErbB3 to activate MAPK signaling. We hypothesized that Mpz- Braf sciatic nerves (P5/P21) should mirror the transcriptional response of an Egr2 -driven gain-of-function Mek1 DD allele (P8/P30), which bypasses the natural Erbb3 decline in maturing SC and hypermyelinates 30 . Since Mek1 functions downstream of Braf , we observed robust positive correlation ( p < 0.0001) at both myelination stages as well as significant Erbb3 upregulation between P5 and P21 ( Fig 6d ; Supplemental Table S2) . Conversely, Egr2 -Cre-driven Shp2 depletion (encoded by Ptpn11 , a Noonan syndrome RASopathy gene 55 ) causes hypomyelinating neuropathy 30 . Comparing Mpz- Braf and Egr2 Cre ;Shp2 f/f sciatic transcriptomes revealed significant anti-correlation of DEG at initial myelinization (P5 vs. P8) but positive correlation post-weaning (P21 vs. P30; Fig 6e ). Finally, to test whether oncogenic Braf effects could be recapitulated in Egr2 -expressing (rather than Mpz -expressing) SCs in vivo , we crossed heterozygous LSL- Braf V600E/+ mice with the same Egr2 -Cre drivers. The Egr2 -Cre allele is expressed by SCs from mid-gestation on (Topilko et al., 1994; Supplemental Figure S5A ). However, no mutant neonates survived, deviating significantly ( p < 10 -6 ) from expected Mendelian ratios. This fully penetrant embryonic lethality, likely due to broader expression of Egr2 beyond SCs 56 , 57 , prevented further postnatal myelination comparisons ( Supplemental Figure S5B ). Uninjured sciatic nerves initiate and sustain reprogramming to an early repair program Transcripts significantly overexpressed at P5 and sustained through P21 may reflect direct rather than indirect effects of MAPK signaling. We therefore examined 175 genes that were significantly upregulated by ≥2-fold in both P5 and P21 mutant sciatic nerves. Pathway enrichment analysis (Wikipathways, KEGG, REACTOME ( Supplemental Figure S6a ) and GSEA ( Supplemental Figure S6b ; Fig. 7a ) revealed that “spinal cord injury” (WP2431) remained one of the most significantly enriched terms (cf. Fig 5b ). Notably, cell adhesion molecule L1 ( Chl1 ), a highly specific repair-type SC marker identified in single-cell RNAseq 23 , was significantly enriched. Comparing the Mpz- Braf sciatic nerve transcriptome to a 6- to 8-week mouse axotomy model at seven days post-injury 58 showed significant positive correlation at P5 ( Supplemental Figure S6c ), which strengthened at P21 ( Fig 7b ). Following physical injury (e.g., crush or axotomy), SCs re-express immaturity genes and transcribe novel repair-associated genes. Jun, a repair SC-specific TF that represses myelination by opposing Egr2 59 , was significantly more transcribed and translated at P21 in the Mpz- Braf sciatic nerve than controls ( Fig 6a-b ). Thus, juvenile Mpz- Braf mice misactivate a molecular repair response during physiological myelination. Download figure Open in new tab Figure 7. Activation of injury-related genes in Mpz- Braf sciatic nerves. a ) GSEA analysis of upregulated genes at P21 shows highly significant correlation with the Wikipathway annotation "Spinal cord injury" (WP2431). b ) These are also significantly and positively correlated with genes upregulated in regenerating nerves of young adult mice 7 days after axotomy. c ) Transcription of Shh , Gdnf and Nrg1 are greatly increased at both P5 and P21, while Gdnf-family member Artn and Nrg1 receptor ErbB3 are only significantly upregulated at P21. Teased fibers from sciatic nerves of P21 control and Mpz- Braf mice immunostained against ( d ) S100b (yellow) and Shh (magenta), with DAPI nuclear counterstain (gray), or ( f ) Pmp2 (orange) and Erbb3 (blue). Scale bars: 50 µm ( d ) or 10 µm (f ). To determine whether Jun-dependent repair ligand effectors were also upregulated, we examined Shh (Sonic hedgehog) and the neurotrophic factor Gdnf (glial cell line-derived neurotrophic factor) 60 . Both were significantly upregulated as early as P5 ( Fig 7c ). Cleaved Shh, critical for multipotent NC maintenance 61 and reactivated in repair SCs 62 was confirmed by immunolabeling of fiber bundles ( Fig 7d ) and Western blot ( Fig. 7e ; Supplemental Figure S7e ). S100b, a calcium-binding intermediate filament in SCs and their multipotent progenitors 63 , was also co-localized and significantly overexpressed by P21 in mutants. Additionally, Nrg1-III and the Gdnf family member Artemin (Artn), secreted by Jun-positive repair SCs to promote axon outgrowth 53 , 64 , were upregulated at P5 ( Nrg1 ) and P21 ( Nrg1 , Artn ) ( Fig. 7c ). The Nrg1 receptor ErbB3 , a nerve damage hallmark 65 , was transcriptionally and translationally upregulated over time ( Fig 7c, f-g ; Supplemental Figure S7b ), with increased Erbb3 immunofluorescence in mutants. This distribution was complementary to Pmp2-positive myelin in individual cells ( Fig. 7f ; Supplemental Figure S7d ). Vimentin ( Vim ), an intermediate filament upregulated in injured axons 66 , also increased over time ( Fig. S7c; Supplemental Table S2 ). Despite repair program activation, progressive axonal degeneration in mutants implies that sustained MAPK signaling is counterproductive for SC-mediated trophic activity 67 in vivo . Schwann cell differentiation of CFC patient-derived stem cells is impaired While the Mpz- Braf model demonstrated phenotypic consequences of MAPK overactivation, germline BRAF V600E is embryonic lethal. To study a RASopathy-associated variant 68 , we differentiated human induced pluripotent stem cells (hiPSCs) from two unrelated CFC patients with heterozygous BRAF Q257R variants 68 , 69 into SCs in vitro 70 and compared to two unrelated control lines. Control hiPSCs followed expected differentiation 70 , but one mutant line (RMK0138C) required more frequent passaging to maintain similar density and failed to develop Schwann cell progenitor (SCP)-like morphology (elongated, refringent soma and long projections) even after 90 days ( Fig 8a ). Download figure Open in new tab Figure 8. Patient-derived immature Schwann cells express inappropriate gene signatures in vitro . a) Summary of hiPSC-directed differentiation into SC, derived from two unrelated WT (HFF15 shown here and AG08H) and from unrelated adolescent girls, both carrying the BRAF p.Q257R variant (CFC patients RMK0056C, RMK0138C) over time. Representative bright-field images shown at D13, D19, a day after passage on D32, and at D95-100. Scale bar = 100 µm for all frames. b ) Euler diagram showing the number of common/specific dysregulated genes ( p -value 1 or < -1) in RMK0056C and RMK0138C mutant cells compared to control cells (HFF15) at D21 of differentiation. (C) Bubble plot of enrichment analysis of the common 380 dysregulated genes in RMK0056C and RMK0138C. Gene set names from the REACTOME database are indicated on the y-axis. Gene sets are ranked according to significance level (-log adjusted p-value). The size of the dots represents the number of queried genes present in the corresponding pathway, and the color corresponds to the gene ratio (number of queried genes present in the gene set / gene set size). (D) Enrichment plot for Egr2- and Sox10-mediated initiation of SC myelination using Reactome database, corresponding to the significant gene set downregulated in RMK0056C and RMK0138C cell lines after 30 days of differentiation. (E) Enrichr-KG analysis of the most significantly enriched terms across three additional databases (Descartes Cell Types and Tissue 2021, Jensen DISEASES and CCLE Proteomics 2020) highlight the close lineage relationship between Schwann cell progenitors and either melanocytes (“melanoma” and “skin cancer”, the A2058 cell line) or neuroendocrine cells (“visceral neurons in lung/heart”, the SKNAS and NCIH446 cell lines). RNAseq at (d)18, a committed SCP-like stage, and d31, analogous to immature SC ( Fig 8a ) reveealed 380 DEGs in both mutant lines ( Supplemental Table S3 ). Reactome 71 pathway analysis highlighted their unexpected downregulation of “epidermal cornification” (REAC:R-HSA-6809371), including eight keratin genes ( Fig 8c ; Supplemental Table S3 and suppression of “EGR2 and SOX10-mediated initiation of SC myelination” (REAC:R-HSA-9619665). Downregulated transcripts included EGR2 itself , MBP , PRX and LAMA2 , encoding the alpha-2 chain of laminin-211, a component of SC basal lamina necessary to restrain small-caliber fiber hypermyelination 72 ( Fig 8c-d ) . In contrast, POU3F2 , a TF transiently required in neonatal murine SC 73 , was significantly upregulated in both CFC patient-derived cell lines ( Supplemental Table S3 ). DEGs co-expressed with POU3F2 evoked multipotency of neural crest-derived SCPs. Upregulated annotations included “melanoma” and the proteome of amelanotic, BRAF V600E -mutated melanoma cells (A2058); proteomes of P53 -mutated lung neuroendocrine carcinoma (NCI-H446) and NRAS Q61L -mutated neuroblastoma (SK-N-AS) cells, and transcripts common to “visceral” (parasympathetic) autonomic neurons ( Fig 8e ). These findings suggest that prolonged postnatal MAPK signaling in immature SC, both in mice and humans, sustains transcriptional programs typical of other neural crest lineages 28 , 74 . Thus, persistent MAPK pathway activation in cycling SC prevents their terminal differentiation in vitro and in vivo . Discussion Hyperplastic neuropathy as a shared RASopathy feature Our findings demonstrate that constitutive activation of BRAF-mediated signaling in a subpopulation of NC-derived cells recapitulates systemic effects found across multiple germline RASopathy syndromes. A key phenotype was rapid onset of hyperplastic neuropathy, reminiscent of “hypertrophic neuropathy” described in case reports 18 , 20 , 75 – 78 . This phenotype had not been reported in prior systemic RASopathy animal models 79 , suggesting that neuropathy should be systematically assessed in RASopathy patients, given the mechanistic convergence on sustained ERK1/2 activation in Schwann cells. Growth retardation and endocrine dysregulation Like other RASopathy models (e.g. germline mutations in Kras 80 , Braf 81 – 84 , Raf1 85 , Ptpn11 86 or Sos1 87 ), the Mpz- Braf mouse presents early postnatal failure to thrive. Severe feeding difficulties are common in BRAF -associated germline RASopathies 18 , 88 – 90 . However, Mpz- Braf mice exhibited normal feeding behavior despite motility issues, while growth delay due to Igf1 deficiency preceded weaning. No megacolon was observed at any age as a sign of gut dysmotility, but transcripts co-expressed specifically in adult enteric glia, such as Plp1 and Gfap 91 , were upregulated over time in Mpz- Braf SC ( Supplemental Table S3 ). This suggests the repair phenotype reverts SCPs to a commitment state preceding glial specialization. By restricting strong MAPK activation to Mpz -expressing cells, including SCs and SCPs 92 , we unintentionally also targeted a subset of Gh-producing pituitary cells (somatotrophs) that can express Mpz under conditions of systemic inflammation ( https://cells.ucsc.edu/?ds=mouse-pituitary-inflam&gene=Mpz ) 93 . Postnatal Igf1 -null mice, which survive at low rates, exhibit severe growth retardation 94 , 95 , while liver-specific Igf1 rescue restores both body size and fertility 96 . The fully penetrant cryptorchidism in male Mpz- Braf but not Igf1 -null mice, despite no Mpz expression in germ cells, suggests a neurogenic mechanism. Cryptorchidism is common in RASopathies 4 , 97 , and genitofemoral nerve damage in rats causes testicular ascent 98 . This supports a role for peripheral nerve myelination in maintaining testicular descent and fertility 99 , 100 . Uncoupling of Gh-Igf1 by sustained MAPK signaling mirrors the effects of blocking differentiation of both SC ( via Nrg1-ErbB3) and pituitary progenitors (eg. Prop1 -driven Braf models) 2 , implicating constitutive MAPK activation in additional human disorders. Future directions in congenital hypomyelinating neuropathy and lineage plasticity Severe, early-onset CMTs, caused by mutations in MPZ , EGR2 , PMP22 or PRX 22 , share features with our model: demyelination, profuse SC proliferation, distal sensory loss, and the enlarged, “gelatinous” peripheral nerves first reported in 1893 101 , which principally affect the legs. Mpz- Braf mice exhibited hindlimb tremor and locomotor deficits, extending to other body areas by P42 but with generalized nerve enlargement by P21. Our findings confirm that SCs initiate these neuromuscular outcomes. The absence of apparent suffering (e.g., aversion, unkempt fur, flattened ears), makes the weanling model tractable for behavioral or electrophysiological studies of RASopathy-associated neuropathies 102 . Furthermore, patient-derived, BRAF Q257R -expressing NC cells are stochastically disadvantaged in adopting a differentiated SC phenotype in vitro , as compared to BRAF -wildtype cells. Prospective nerve biopsies in unresolved cases of either RASopathies or early-onset CMT could confirm these findings, linking transcriptional dysregulation to clinical phenotypes and improving molecular diagnoses while opening new interventional avenues. Methods Ethical statement This study complies with all relevant ethical regulations. Animal care and experiments were in accordance with French and European legislation and approved by the French ACUC (C2EA-14; project numbers 37-08102012 and 9522-2017040517496865v5). Human iPSC studies adhered to the Declaration of Helsinki 103 and French bioethics law (2021-1017) 104 consistent with our institutional review board protocols. Mouse breeding and genotyping Transgenic P0-Cre +/° ; Braf V600E/+ and Krox20-Cre +/° ; Braf V600E/+ mice were generated by crossing LSL-Braf V600E/+ mice 11 (RRID:IMSR_JAX:017928) with P0(Mpz)- Cre (RRID:IMSR_JAX:017927) 36 , 105 or Krox20- Cre (RRID:IMSR_JAX:025744) 106 drivers. Genotyping was performed by PCR (see Supplemental Methods ). Mice were housed under standard conditions (12-hour light/dark cycles, ad libitum access to food/water). Western Blots Sciatic nerve lysates were prepared in RIPA buffer with protease inhibitors, separated by 12% SDS-PAGE, and probed with antibodies against phospho-Erk1/2 (RRID:AB_331646), Erk1/2 (RRID:AB_330744), c-Jun (RRID:AB_2130165) and Gapdh (RRID:AB_2107445). Alternately, primary antibodies to Shh (RRID:AB_528466) 107 , Erbb3 (RRID:AB_358277), Egr2 (RRID:AB_10862073), alpha-tubulin (RRID:AB_2241126), vimentin (RRID:AB_2881439) or vinculin (RRID:AB_10603627) were used. Signals were detected using relevant secondary antibodies (RRID:AB_330924 and RRID:AB_2099233) followed by chemiluminescence detection and quantified with ImageJ v.1.54f. Culture and differentiation of human pluripotent stem cells (hiPSC) Two female patients with cardio-facio-cutaneous (CFC) syndrome bearing a heterozygous germline BRAF Q257R variant, or their legal guardians, provided written consent as approved by the UCSF Human Research Protection Program (CHR #10-02794), to produce the human induced pluripotent stem cell (hiPSC) lines, RMK0056C and RMK0138C, as described 69 . The AG08H and HFF15 clones of healthy donor male hiPSC were derived from commercial cell lines consented for research, AG08498 (RRID:CVCL_1Y51) and HFF-1 SCRC-1041 (RRID:CVCL_3285) as described 108 . Clones were initially grown in mTeSR1 (Stemcell) on human ES cell-qualified Matrigel™ before cryopreservation. Thawed hiPSC were cultured on Synthemax II-SC (Corning) at 0.025 mg/mL and maintained with daily changes of StemMACS iPS-Brew XF (Miltenyi Biotec). At 60-80% confluence, they were passed with ReleSR (StemCell Technologies), with 10 μM Y-27632 for the first day. Differentiation of hiPSCs into SC 70 was initiated at 60% confluence and passed every 6 days until day 31. Thereafter, upon 90% confluence, they were split 1:2. RNA sequencing and analyses Sciatic nerves and pituitary glands from WT or Braf V600E/+ animals, and pellets of WT or BRAF Q257R/+ hiPSC-derived SCP and immature Schwann cells (iSC) were stored at -80°C. Tissues were lysed in the FastPrep-24 homogenizer (MP Biomedicals) and total RNA extracted with the RNeasy Lipid Tissue Mini (Qiagen) or, for cell pellets, NucleoSpin RNA Plus S (Macherey-Nagel) kits. RNA concentrations were measured on a NanoDrop TM 1000 Spectrophotometer (ThermoFisher). Libraries were prepared with the KAPA mRNA HyperPrep kit (Roche) and sequenced on a NovaSeq 6000 (Illumina). Quality was assessed using fastQC (v0.11.5). Raw sequencing reads were aligned to GRCm39/GRCh38 using STAR (v2.7.2b). Bam files were indexed and sorted with Samtools (v1.7). After mapping, reads per feature (Ensembl or GENCODE v34 annotations for mouse or human data, respectively) were determined using StringTie v2.1.6, and differential expression analyzed with a Wald test in DESeq2 (v1.34.0; FDR < 0.05). Data visualization made use of R packages Pheatmap and EnhancedVolcano 109 . Expression-dependent enrichment analyses were conducted with Gene Set Enrichment Analysis (GSEA) v4.3.2 110 using mouse Wikipathway and human Reactome databases, otherwise with g:Profiler 111 or Enrichr-KG 112 , interrogating quantitative proteomics of the Cancer Cell Line Encyclopedia 113 KEGG, Reactome, Gene Ontology datasets. These were significantly enriched by Enrichr-KG 112 , using databases of annotations related to human disease 114 , and a large single-cell atlas of developmental human gene expression 115 . Nerve and fiber immunostaining Sciatic nerves were fixed in 4% buffered paraformaldehyde and stained with primary antibodies to Mbp (RRID:AB_94975); Ki-67 (RRID:AB_10854564); Caspr (RRID:AB_869934); Sox10 (RRID:AB_2941085); Pmp2 (RRID:AB_2166978); Erbb3 (RRID:AB_358277); S100b (RRID:AB_1856538); or Shh (RRID:AB_528466) 107 , followed by secondary antibodies and DAPI counterstaining. Further details are in the Supplemental Methods . Image stacks of teased fibers were acquired with a spinning disk confocal microscope (Nikon SoRa) and NIS-Elements software and nerve images on a Zeiss LSM800 with Airyscan and Zen software v2.3. Transmission electron microscopy (TEM) and morphometry Sciatic nerves were fixed in 2% PFA + 2.5% glutaraldehyde, post-fixed with 1% OsO 4 , contrasted in 1% uranyl acetate before Epon resin embedding. Semi-thin sections at 1 µm were counterstained with toluidine blue. Ultra-thin sections were imaged on an FEI Morgagni 120 kv microscope. Myelin morphological parameters were analyzed with MyelTracer 40 . Axon density was assessed with ImageJ using the semi-thin sections. Statistics Statistical analyses were performed with Prism v10.2.0 (GraphPad), using unpaired Mann-Whitney or two-way ANOVA non-parametric tests. For multiple comparisons, two-way ANOVA followed by Šidák’s post-hoc tests, or Student t-tests followed by Benjamini-Hochberg adjustment, were used. Mendelian segregation was assessed with a chi-square contingency test. Data are presented as mean ± SEM; significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data availability Raw and processed RNA-sequencing data from this study have been deposited to the Gene Expression Omnibus (GEO) and assigned the accession identifiers GSE262047 and GSE262048. Websites "Catalogue Of Somatic Mutations In Cancer" (COSMIC), https://cancer.sanger.ac.uk/cosmic 1 . Rare disease ORPHA codes are maintained at https://www.orpha.net and available on https://www.orphadata.com/ 116 . fastQC (v0.11.5) is available at https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ and EnhancedVolcano at https://bioconductor.org/packages/EnhancedVolcano . NIH Fiji/ImageJ software ( https://imagej.net/ij/ ) is available from https://imagej.net/ij/ . Author contributions E.M. wrote the first draft, performed and analyzed experiments; D.A. provided analyses, performed experiments and revised the article; P.Q., G.M. and M.M. performed experiments; N.B.M. and V.D. provided protocols and guidance; C.E.Y. and N.B. provided control hiPSC lines and guidance; L.A.W. provided patient hiPSC lines; A.B. provided constructive criticism and funding; H.C.E. supervised the experiments, provided funding, wrote and revised the article. Competing interests The authors declare no competing interests. Funding The authors declare that they have no conflicts of interest. This work was made possible by the Association Française contre les Myopathies (AFM-Téléthon) grant ‘MoThARD’ to VD and HCE, the National Institutes of Health New Innovator award (1DP2OD007449) to LAW, and Horizon Europe HORIZON-MISS-2021-CANCER-02-03 MELCAYA (101096667) to HCE. EM was supported by the AFM-Téléthon, the Asociación Española de Nevus Gigante Congénito (AsoNevus, Spain), Naevus 2000 France-Europe (France) and the Marseille Rare Diseases Institute (MarMaRa) of Aix-Marseille University. Acknowledgements We are grateful to C. Humbert, C. Castro, N. Trévisiol and C. Aubert of the MMG Genomics and Bioinformatics facility (GBiM) for assistance with RNAseq and analyses; Dr. F. Magdinier and O. Hadadeh of the Marseille Stem Cell (MaSC) core facility for control hiPSC cell lines and their quality control; Dr. T.-T. Mac, Adeline Querdray and Dr. T. Fauquier for their technical assistance and advice; the PiCSL-France BioImaging core facility, supported by ANR-10-INBS-04, and Dr. C. Leterrier and Dr. L. Fourel of the Institute of NeuroPhysiopathology, CNRS-Aix Marseille University, for their assistance with spinning-disk confocal microscopy. Funder Information Declared Association Francaise contre les Myopathies, https://ror.org/0162y2387 , MoThARD National Institutes of Health , 1DP2OD007449 European Commission , HORIZON-MISS-2021-CANCER-02-03 MELCAYA (101096667) Asociación Española de Nevus Gigante Congénito, Spain Naevus 2000 France-Europe, France Marseille Rare Diseases Institute, Aix-Marseille Univ, Marseille, France Footnotes Reduced word count, retained references that will be removed in published version but are in the supplemental information, small copy edits, title revision. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262047 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262048 References 1. ↵ Sondka , Z. et al. COSMIC: a curated database of somatic variants and clinical data for cancer . Nucleic Acids Res . 52 , D1210 – D1217 ( 2024 ). OpenUrl CrossRef PubMed 2. ↵ Gualtieri , A. et al. Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans . Nat. Commun . 12 , 2028 ( 2021 ). OpenUrl CrossRef PubMed 3. Jaeger , Z. J. et al. RASopathies. Part II: Cutaneous and extracutaneous manifestations . J. Am. Acad. Dermatol . S0190–9622(25)02340–0 ( 2025 ). 4. ↵ Rauen , K. A. The RASopathies . Annu. Rev. Genomics Hum. Genet. 14 , 355 – 369 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 5. ↵ Siano , M. A. et al. Endocrine system involvement in patients with RASopathies: A case series . Front. Endocrinol . 13 , ( 2022 ). 6. ↵ Tartaglia , M. , Aoki , Y. & Gelb , B. D . The molecular genetics of RASopathies: An update on novel disease genes and new disorders . Am. J. Med. Genet. C Semin. Med. Genet . 190 , 425 – 439 ( 2022 ). OpenUrl PubMed 7. ↵ Carli , D. , Resta , N. , Ferrero , G. B. , Ruggieri , M. & Mussa , A . Mosaic RASopathies: A review of disorders caused by somatic pathogenic variants in the genes of the RAS/MAPK pathway . Am. J. Med. Genet. C Semin. Med. Genet . 190 , 520 – 529 ( 2022 ). OpenUrl PubMed 8. ↵ Kratz , C. P. , Rapisuwon , S. , Reed , H. , Hasle , H. & Rosenberg , P. S. Cancer in Noonan, Costello, Cardiofaciocutaneous and LEOPARD Syndromes . Am. J. Med. Genet. Part C 157 , 83 – 89 ( 2011 ). OpenUrl 9. ↵ Peltonen , S. et al. Pediatric malignancies in neurofibromatosis type 1: A population-based cohort study . Int. J. Cancer 145 , 2926 – 2932 ( 2019 ). OpenUrl PubMed 10. ↵ Davies , H. et al. Mutations of the BRAF gene in human cancer . Nature 417 , 949 – 954 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 11. ↵ Mercer , K. et al. Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts . Cancer Res . 65 , 11493 – 11500 ( 2005 ). OpenUrl Abstract / FREE Full Text 12. ↵ Haling , J. R. et al. Structure of the BRAF-MEK Complex Reveals a Kinase Activity Independent Role for BRAF in MAPK Signaling . Cancer Cell 26 , 402 – 413 ( 2014 ). OpenUrl CrossRef PubMed 13. ↵ Yao , Z. et al. BRAF Mutants Evade ERK-Dependent Feedback by Different Mechanisms that Determine Their Sensitivity to Pharmacologic Inhibition . Cancer Cell 28 , 370 – 383 ( 2015 ). OpenUrl CrossRef PubMed 14. ↵ Gripp , K. W. et al. Further delineation of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate cardio-facio-cutaneous syndrome from Costello syndrome . Am. J. Med. Genet. A . 143A , 1472 – 1480 ( 2007 ). OpenUrl CrossRef PubMed 15. ↵ Koudova , M. , Seemanova , E. & Zenker , M . Novel BRAF mutation in a patient with LEOPARD syndrome and normal intelligence . Eur. J. Med. Genet . 52 , 337 – 340 ( 2009 ). OpenUrl CrossRef PubMed 16. Nava , C. et al. Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype-phenotype relationships and overlap with Costello syndrome . J. Med. Genet . 44 , 763 – 71 ( 2007 ). OpenUrl Abstract / FREE Full Text 17. ↵ Nyström , A.-M. et al. Noonan and cardio-facio-cutaneous syndromes: two clinically and genetically overlapping disorders . J. Med. Genet . 45 , 500 – 506 ( 2008 ). OpenUrl Abstract / FREE Full Text 18. ↵ Manci , E. A. et al. Cardiofaciocutaneous syndrome (CFC) with congenital peripheral neuropathy and nonorganic malnutrition: an autopsy study . Am. J. Med. Genet. A . 137 , 1 – 8 ( 2005 ). OpenUrl CrossRef PubMed 19. De Ridder , W. , van Engelen , B. & van Alfen , N . Neurological features of Noonan syndrome and related RASopathies: Pain and nerve enlargement characterized by nerve ultrasound . Am. J. Med. Genet. A . 188 , 1801 – 1807 ( 2022 ). OpenUrl CrossRef PubMed 20. ↵ Draaisma , F. et al. Hypertrophic neuropathy: a possible cause of pain in children with Noonan syndrome and related disorders . Eur. J. Pediatr . 182 , 3789 – 3793 ( 2023 ). OpenUrl CrossRef PubMed 21. ↵ Callegari , I. et al. Mutation update for myelin protein zero-related neuropathies and the increasing role of variants causing a late-onset phenotype . J. Neurol . 266 , 2629 – 2645 ( 2019 ). OpenUrl CrossRef PubMed 22. ↵ Pearce , J. M. S . Dejerine-Sottas Disease (Progressive Hypertrophic Polyneuropathy) . Eur. Neurol . 55 , 115 – 117 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 23. ↵ Eid , S. A. et al. Single-cell RNA-seq uncovers novel metabolic functions of Schwann cells beyond myelination . J. Neurochem . 166 , 367 – 388 ( 2023 ). OpenUrl CrossRef PubMed 24. Geren , B. B . The formation from the schwann cell surfaceof myelin in the peripheral nerves of chick embryos . Exp. Cell Res . 7 , 558 – 562 ( 1954 ). OpenUrl CrossRef PubMed Web of Science 25. ↵ Le Douarin , N. , Dulac , C. , Dupin , E. & Cameron-Curry , P . Glial cell lineages in the neural crest . Glia 4 , 175 – 84 ( 1991 ). OpenUrl CrossRef PubMed 26. Etchevers , H. C. , Dupin , E. & Le Douarin , N. M . The diverse neural crest: from embryology to human pathology . Development 146 , ( 2019 ). 27. Hockman , D. et al. Striking parallels between carotid body glomus cell and adrenal chromaffin cell development . Dev. Biol . ( 2018 ) doi: 10.1016/j.ydbio.2018.05.016 . OpenUrl CrossRef PubMed 28. ↵ Furlan , A. et al. Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla . Science 357 , eaal3753 ( 2017 ). OpenUrl Abstract / FREE Full Text 29. ↵ Ishii , A. , Furusho , M. & Bansal , R . Sustained activation of ERK1/2 MAPK in oligodendrocytes and schwann cells enhances myelin growth and stimulates oligodendrocyte progenitor expansion . J. Neurosci. Off. J. Soc. Neurosci . 33 , 175 – 186 ( 2013 ). OpenUrl Abstract / FREE Full Text 30. ↵ Sheean , M. E. et al. Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination . Genes Dev . 28 , 290 – 303 ( 2014 ). OpenUrl Abstract / FREE Full Text 31. Belin , S. et al. Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypomyelinating neuropathy . Hum. Mol. Genet . 28 , 1260 – 1273 ( 2019 ). OpenUrl CrossRef PubMed 32. ↵ Ishii , A. , Furusho , M. , Dupree , J. L. & Bansal , R . Strength of ERK1/2 MAPK Activation Determines Its Effect on Myelin and Axonal Integrity in the Adult CNS . J. Neurosci. Off. J. Soc. Neurosci . 36 , 6471 – 6487 ( 2016 ). OpenUrl Abstract / FREE Full Text 33. ↵ Pipis , M. , Rossor , A. M. , Laura , M. & Reilly , M. M . Next-generation sequencing in Charcot–Marie–Tooth disease: opportunities and challenges . Nat. Rev. Neurol . 15 , 644 – 656 ( 2019 ). OpenUrl CrossRef PubMed 34. ↵ Feltri , M. L. et al. P0-Cre transgenic mice for inactivation of adhesion molecules in Schwann cells . Ann. N. Y. Acad. Sci . 883 , 116 – 123 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 35. ↵ Kelnar , C. J. H . Noonan syndrome: the hypothalamo-adrenal and hypothalamo-gonadal axes . Horm. Res . 72 Suppl 2 , 24 – 30 ( 2009 ). OpenUrl CrossRef PubMed 36. ↵ Feltri , M. L. et al. A novel P 0 glycoprotein transgene activates expression of lac Z in myelin-forming Schwann cells . Eur. J. Neurosci . 11 , 1577 – 1586 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 37. ↵ Hagedorn , L. , Suter , U. & Sommer , L . P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors . Development 126 , 3781 – 3794 ( 1999 ). OpenUrl Abstract 38. ↵ Lundberg , E. , Kriström , B. , Jonsson , B. , Albertsson-Wikland , K. , & study group . Growth hormone (GH) dose-dependent IGF-I response relates to pubertal height gain . BMC Endocr. Disord . 15 , 84 ( 2015 ). OpenUrl PubMed 39. ↵ Alvey , L. M. , Jones , J. F. X. , Tobin-O’Brien , C. & Pickering , M . Bands of Fontana are caused exclusively by the sinusoidal path of axons in peripheral nerves and predict axon path; evidence from rodent nerves and physical models . J. Anat . 234 , 165 – 178 ( 2019 ). OpenUrl PubMed 40. ↵ Kaiser , T. et al. MyelTracer: A Semi-Automated Software for Myelin g-Ratio Quantification . eNeuro 8 , ENEURO.0558-20.2021 ( 2021 ). OpenUrl 41. ↵ Kozlowski , M. M. et al. Pmp2+ Schwann Cells Maintain the Survival of Large-Caliber Motor Axons . J. Neurosci . 45 , e1362242025 ( 2025 ). OpenUrl Abstract / FREE Full Text 42. ↵ Woodhoo , A. & Sommer , L . Development of the Schwann cell lineage: from the neural crest to the myelinated nerve . Glia 56 , 1481 – 1490 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 43. ↵ Gerdes , J. et al. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67 . J. Immunol. Baltim. Md 1950 133 , 1710 – 1715 ( 1984 ). OpenUrl 44. ↵ Miller , I. et al. Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence . Cell Rep . 24 , 1105 – 1112 .e5 ( 2018 ). OpenUrl CrossRef PubMed 45. ↵ Kuhlbrodt , K. , Herbarth , B. , Sock , E. , Hermans-Borgmeyer , I. & Wegner , M . Sox10, a novel transcriptional modulator in glial cells . J. Neurosci . 18 , 237 – 250 ( 1998 ). OpenUrl Abstract / FREE Full Text 46. ↵ Poitelon , Y. , Kopec , A. M. & Belin , S . Myelin Fat Facts: An Overview of Lipids and Fatty Acid Metabolism . Cells 9 , 812 ( 2020 ). OpenUrl CrossRef 47. ↵ Ohashi , K. et al. Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene . J. Biol. Chem . 278 , 42936 – 42941 ( 2003 ). OpenUrl Abstract / FREE Full Text 48. ↵ Maldonado , E. N. , Alderson , N. L. , Monje , P. V. , Wood , P. M. & Hama , H . FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin . J. Lipid Res . 49 , 153 – 161 ( 2008 ). OpenUrl Abstract / FREE Full Text 49. ↵ Topilko , P. et al. Krox-20 controls myelination in the peripheral nervous system . Nature 371 , 796 – 799 ( 1994 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Peirano , R. I. , Goerich , D. E. , Riethmacher , D. & Wegner , M . Protein zero gene expression is regulated by the glial transcription factor Sox10 . Mol. Cell. Biol . 20 , 3198 – 3209 ( 2000 ). OpenUrl Abstract / FREE Full Text 51. ↵ Jessen , K. R. & Mirsky , R . Negative regulation of myelination: relevance for development, injury, and demyelinating disease . Glia 56 , 1552 – 1565 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 52. ↵ Hong , J. et al. PMP2 regulates myelin thickening and ATP production during remyelination . Glia 72 , 885 – 898 ( 2024 ). OpenUrl CrossRef PubMed 53. ↵ Stassart , R. M. et al. A role for Schwann cell-derived neuregulin-1 in remyelination . Nat. Neurosci . 16 , 48 – 54 ( 2013 ). OpenUrl CrossRef PubMed 54. ↵ Heming , M. et al. Multi-omic identification of perineurial hyperplasia and lipid-associated nerve macrophages in human polyneuropathies . Nat. Commun . 16 , 7872 ( 2025 ). OpenUrl CrossRef PubMed 55. ↵ Serra-Nédélec , A. D. R. et al. Noonan syndrome-causing SHP2 mutants inhibit insulin-like growth factor 1 release via growth hormone-induced ERK hyperactivation, which contributes to short stature . Proc. Natl. Acad. Sci . 109 , 4257 – 4262 ( 2012 ). OpenUrl Abstract / FREE Full Text 56. ↵ Gerschenfeld , G. et al. Neural tube-associated boundary caps are a major source of mural cells in the skin . eLife 12 , e69413 ( 2023 ). OpenUrl CrossRef PubMed 57. ↵ Bouchoucha , Y. X. , Charnay , P. & Gilardi-Hebenstreit , P . Ablation of Egr2-Positive Cells in Male Mouse Anterior Pituitary Leads to Atypical Isolated GH Deficiency . Endocrinology 154 , 270 – 282 ( 2013 ). OpenUrl CrossRef PubMed 58. ↵ Arthur-Farraj , P. J. et al. Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury . Cell Rep . 20 , 2719 – 2734 ( 2017 ). OpenUrl CrossRef PubMed 59. ↵ Parkinson , D. B. et al. c-Jun is a negative regulator of myelination . J. Cell Biol . 181 , 625 – 637 ( 2008 ). OpenUrl Abstract / FREE Full Text 60. ↵ Piirsoo , M. , Kaljas , A. , Tamm , K. & Timmusk , T . Expression of NGF and GDNF family members and their receptors during peripheral nerve development and differentiation of Schwann cells in vitro . Neurosci. Lett . 469 , 135 – 140 ( 2010 ). OpenUrl CrossRef PubMed 61. ↵ da Costa , M. C. , Trentin , A. G. & Calloni , G. W . FGF8 and Shh promote the survival and maintenance of multipotent neural crest progenitors . Mech. Dev . 154 , 251 – 258 ( 2018 ). OpenUrl CrossRef PubMed 62. ↵ Hashimoto , M. et al. Neuroprotective effect of sonic hedgehog up-regulated in Schwann cells following sciatic nerve injury . J. Neurochem . 107 , 918 – 927 ( 2008 ). OpenUrl CrossRef PubMed 63. ↵ Takahashi , K. et al. Immunohistochemical study on the distribution of alpha and beta subunits of S-100 protein in human neoplasm and normal tissues . Virchows Arch. B Cell Pathol. Incl. Mol. Pathol . 45 , 385 – 396 ( 1984 ). OpenUrl PubMed Web of Science 64. ↵ Huang , L. et al. c-Jun gene-modified Schwann cells: upregulating multiple neurotrophic factors and promoting neurite outgrowth . Tissue Eng. Part A 21 , 1409 – 1421 ( 2015 ). OpenUrl CrossRef PubMed 65. ↵ Carroll , S. L. , Miller , M. L. , Frohnert , P. W. , Kim , S. S. & Corbett , J. A . Expression of neuregulins and their putative receptors, ErbB2 and ErbB3, is induced during Wallerian degeneration . J. Neurosci. Off. J. Soc. Neurosci. 17 , 1642 – 1659 ( 1997 ). OpenUrl Abstract / FREE Full Text 66. ↵ Perlson , E. et al. Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve . Neuron 45 , 715 – 726 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 67. ↵ Nave , K.-A . Myelination and the trophic support of long axons . Nat. Rev. Neurosci . 11 , 275 – 283 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 68. ↵ Josowitz , R. et al. Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes . Stem Cell Rep . 7 , 355 – 369 ( 2016 ). OpenUrl 69. ↵ Yeh , E. et al. Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment . Mol. Psychiatry 23 , 1687 – 1698 ( 2018 ). OpenUrl CrossRef PubMed 70. ↵ Hörner , S. J. et al. hiPSC-Derived Schwann Cells Influence Myogenic Differentiation in Neuromuscular Cocultures . Cells 10 , 3292 ( 2021 ). OpenUrl CrossRef 71. ↵ Milacic , M. et al. The Reactome Pathway Knowledgebase 2024 . Nucleic Acids Res . 52 , D672 – D678 ( 2024 ). OpenUrl CrossRef PubMed 72. ↵ Ghidinelli , M. et al. Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination . PLoS Biol . 15 , e2001408 ( 2017 ). OpenUrl CrossRef PubMed 73. ↵ Jaegle , M. et al. The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development . Genes Dev . 17 , 1380 – 1391 ( 2003 ). OpenUrl Abstract / FREE Full Text 74. ↵ Adameyko , I. et al. Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin . Cell 139 , 366 – 379 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 75. ↵ Ando , Y. et al. A novel KRAS gene mutation of Noonan syndrome with severe peripheral nerve hypertrophy . J. Neurol. Sci . 333 , e605 ( 2013 ). OpenUrl 76. DeRoos , S. T. , Ryan , M. M. & Ouvrier , R. A . Peripheral neuropathy in cardiofaciocutaneous syndrome . Pediatr. Neurol . 36 , 250 – 252 ( 2007 ). OpenUrl CrossRef PubMed 77. Maridet , C. , Sole , G. , Morice-Picard , F. & Taieb , A . Hypertrophic neuropathy in Noonan syndrome with multiple lentigines . Am. J. Med. Genet. A . 170 , 1570 – 1572 ( 2016 ). OpenUrl CrossRef PubMed 78. ↵ Vizcaino , M. A. et al. Localized Hypertrophic Neuropathy as a Neoplastic Manifestation of KRAS-Mediated RASopathy . J. Neuropathol. Exp. Neurol . 79 , 647 – 651 ( 2020 ). OpenUrl CrossRef PubMed 79. ↵ Hernández-Porras , I. & Guerra , C . Modeling RASopathies with Genetically Modified Mouse Models . Methods Mol. Biol. Clifton NJ 1487 , 379 – 408 ( 2017 ). OpenUrl 80. ↵ Hernández-Porras , I. et al. K-RasV14I recapitulates Noonan syndrome in mice . Proc. Natl. Acad. Sci. U. S. A . 111 , 16395 – 16400 ( 2014 ). OpenUrl Abstract / FREE Full Text 81. ↵ Urosevic , J. et al. Constitutive activation of B-Raf in the mouse germ line provides a model for human cardio-facio-cutaneous syndrome . Proc. Natl. Acad. Sci. U. S. A . 108 , 5015 – 5020 ( 2011 ). OpenUrl Abstract / FREE Full Text 82. Andreadi , C. et al. The intermediate-activity L597VBRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway . Genes Dev . 26 , 1945 – 1958 ( 2012 ). OpenUrl Abstract / FREE Full Text 83. Inoue , S.-I. et al. New BRAF knockin mice provide a pathogenetic mechanism of developmental defects and a therapeutic approach in cardio-facio-cutaneous syndrome . Hum. Mol. Genet . 23 , 6553 – 6566 ( 2014 ). OpenUrl CrossRef PubMed 84. ↵ Moriya , M. et al. Adult mice expressing a Braf Q241R mutation on an ICR/CD-1 background exhibit a cardio-facio-cutaneous syndrome phenotype . Hum. Mol. Genet . 24 , 7349 – 7360 ( 2015 ). OpenUrl CrossRef PubMed 85. ↵ Wu , X. et al. MEK-ERK pathway modulation ameliorates disease phenotypes in a mouse model of Noonan syndrome associated with the Raf1(L613V) mutation . J. Clin. Invest . 121 , 1009 – 1025 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 86. ↵ Araki , T. et al. Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation . Nat. Med . 10 , 849 – 857 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 87. ↵ Chen , P.-C. et al. Activation of multiple signaling pathways causes developmental defects in mice with a Noonan syndrome–associated Sos1 mutation . J. Clin. Invest . 120 , 4353 – 4365 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 88. ↵ Allanson , J. E. et al. Cardio-facio-cutaneous syndrome: Does genotype predict phenotype? Am. J. Med. Genet. C Semin. Med. Genet . 157 , 129 – 135 ( 2011 ). OpenUrl CrossRef PubMed 89. Feng , B. et al. Molecular and phenotypic spectrum of cardio-facio-cutaneous syndrome in Chinese patients . Orphanet J. Rare Dis . 18 , 284 ( 2023 ). OpenUrl CrossRef PubMed 90. ↵ Lee , Y. et al. Clinical and molecular spectra of BRAF-associated RASopathy . J. Hum. Genet . 66 , 389 – 399 ( 2021 ). OpenUrl PubMed 91. ↵ Rao , M. et al. Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system . Glia 63 , 2040 – 2057 ( 2015 ). OpenUrl CrossRef PubMed 92. ↵ Lee , M. et al. P0 is constitutively expressed in the rat neural crest and embryonic nerves and is negatively and positively regulated by axons to generate non-myelin-forming and myelin-forming Schwann cells, respectively . Mol. Cell. Neurosci . 8 , 336 – 350 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 93. ↵ Yan , T. , Wang , R. , Yao , J. & Luo , M . Single-cell transcriptomic analysis reveals rich pituitary–Immune interactions under systemic inflammation . PLOS Biol . 21 , e3002403 ( 2023 ). OpenUrl PubMed 94. ↵ Baker , J. , Liu , J.-P. , Robertson , E. J. & Efstratiadis , A . Role of insulin-like growth factors in embryonic and postnatal growth . Cell 75 , 73 – 82 ( 1993 ). OpenUrl CrossRef PubMed Web of Science 95. ↵ Powell-Braxton , L. et al. IGF-I is required for normal embryonic growth in mice . Genes Dev . 7 , 2609 – 2617 ( 1993 ). OpenUrl Abstract / FREE Full Text 96. ↵ Stratikopoulos , E. , Szabolcs , M. , Dragatsis , I. , Klinakis , A. & Efstratiadis , A . The hormonal action of IGF1 in postnatal mouse growth . Proc. Natl. Acad. Sci . 105 , 19378 – 19383 ( 2008 ). OpenUrl Abstract / FREE Full Text 97. ↵ Champion , K. J. et al. Germline mutation in BRAF codon 600 is compatible with human development: De novo p.V600G mutation identified in a patient with CFC syndrome . Clin. Genet. 79 , 468 – 474 ( 2011 ). OpenUrl CrossRef PubMed 98. ↵ Gutiérrez-Pérez , O. , Rojas-Castañeda , J. C. , Chavez-Saldaña , M. , Reyes , G. & Vigueras-Villaseñor , R. M . Infertility in rats subjected to genitofemoral nerve section is not associated with testicular damage . Andrologia 46 , 151 – 157 ( 2014 ). OpenUrl PubMed 99. ↵ Prince , F. P . Ultrastructural evidence of indirect and direct autonomic innervation of human Leydig cells: comparison of neonatal, childhood and pubertal ages . Cell Tissue Res . 269 , 383 – 390 ( 1992 ). OpenUrl CrossRef PubMed Web of Science 100. ↵ Tanyel , F. C . Commentary: The fate of germ cells in cryptorchid testis . Front. Endocrinol . 15 , 1393040 ( 2024 ). OpenUrl 101. ↵ Déjerine , J. & Sottas , J . Sur la névrite interstitielle hypertrophique et progressive de l’enfance . Comp Rend Soc Biol Paris 45 , 63 – 96 ( 1893 ). OpenUrl 102. ↵ Duman , M. , Jaggi , S. , Enz , L. S. , Jacob , C. & Schaeren-Wiemers , N . Theophylline Induces Remyelination and Functional Recovery in a Mouse Model of Peripheral Neuropathy . Biomedicines 10 , 1418 ( 2022 ). OpenUrl PubMed 103. ↵ The World Medical Association, (WMA) . WMA Declaration of Helsinki . Ethical Principles for Medical Research Involving Human Subjects https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/ ( 2022 ). 104. ↵ Journal Officiel, F. LOI n° 2021-1017 du 2 août 2021 relative à la bioéthique . ( 2021 ). 105. ↵ Feltri , M. L. et al. P0-Cre transgenic mice for inactivation of adhesion molecules in Schwann cells . Ann. N. Y. Acad. Sci . 883 , 116 – 123 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 106. ↵ Voiculescu , O. , Charnay , P. & Schneider-Maunoury , S . Expression pattern of a Krox-20/Cre knock-in allele in the developing hindbrain, bones, and peripheral nervous system . Genes. N. Y. N 2000 26 , 123 – 126 ( 2000 ). OpenUrl CrossRef 107. ↵ Ericson , J. , Morton , S. , Kawakami , A. , Roelink , H. & Jessell , T. M . Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity . Cell 87 , 661 – 673 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 108. ↵ Dion , C. et al. SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite . Nucleic Acids Res . 47 , 2822 – 2839 ( 2019 ). OpenUrl CrossRef PubMed 109. ↵ Blighe , K. , Rana , S. & Lewis , M. Blighe K , Rana S , Lewis M (2023). EnhancedVolcano: Publication-ready volcano plots with enhanced coloring and labeling . R package version 1.20.0. ( 2023 ). 110. ↵ Subramanian , A. et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles . Proc. Natl. Acad. Sci . 102 , 15545 – 15550 ( 2005 ). OpenUrl Abstract / FREE Full Text 111. ↵ Kolberg , L. et al. g:Profiler—interoperable web service for functional enrichment analysis and gene identifier mapping (2023 update) . Nucleic Acids Res . 51 , W207 – W212 ( 2023 ). OpenUrl CrossRef PubMed 112. ↵ Evangelista , J. E. et al. Enrichr-KG: bridging enrichment analysis across multiple libraries . Nucleic Acids Res . 51 , W168 – W179 ( 2023 ). OpenUrl CrossRef PubMed 113. ↵ Nusinow , D. P. et al. Quantitative Proteomics of the Cancer Cell Line Encyclopedia . Cell 180 , 387 – 402 .e16 ( 2020 ). OpenUrl CrossRef PubMed 114. ↵ Grissa , D. , Junge , A. , Oprea , T. I. & Jensen , L. J . Diseases 2.0: a weekly updated database of disease–gene associations from text mining and data integration . Database 2022 , baac019 ( 2022 ). OpenUrl CrossRef PubMed 115. ↵ Cao , J. et al. A human cell atlas of fetal gene expression . Science 370 , eaba7721 ( 2020 ). OpenUrl Abstract / FREE Full Text 116. ↵ Aymé , S. , Bellet , B. & Rath , A . Rare diseases in ICD11: making rare diseases visible in health information systems through appropriate coding . Orphanet J. Rare Dis . 10 , 35 ( 2015 ). OpenUrl CrossRef PubMed 117. ↵ Liao , Y. , Wang , J. , Jaehnig , E. J. , Shi , Z. & Zhang , B. WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs . Nucleic Acids Res . 47 , W199 – W205 ( 2019 ). OpenUrl CrossRef PubMed 118. Le , N. et al. Analysis of congenital hypomyelinating Egr2Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination . Proc. Natl. Acad. Sci. U. S. A . 102 , 2596 – 2601 ( 2005 ). OpenUrl Abstract / FREE Full Text Back to top Previous Next Posted January 10, 2026. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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