muscle regeneration is a multistep process that includes myofiber degradation , regeneration , and remodeling ( ten broek et  al . , the repair process is characterized by the activation  of a primary myogenic stem cell population referred to as  satellite cells ,  which give rise to activated proliferating myoblasts or myoblast precursor cells ( mpcs ) , followed by cell differentiation and fusion into regenerated myofibers . satellite cells , that are normally quiescent , can be activated to proliferate and generate committed progeny in response to a variety of stimuli , including degenerative muscle diseases ( brack and rando , 2012 ; dhawan and rando , 2005 ; rudnicki et  al . , 2008 ) . the active thyroid hormone ( th ) , t3 , derives in large part from the monodeiodination of the prohormone thyroxine ( t4 ) by one of two iodothyronine selenodeiodinases ( d1 or d2 ) . conversely , th signaling terminates consequent to inactivation of t3 and t4 induced by removal of a tyrosyl ring iodine by type 3 deiodinase ( d3 ) . d3 converts the active hormone t3 to inactive metabolites thereby terminating th action within cells . this provides a mechanism by which th action can be terminated in a tissue - specific chronologically programmed fashion ( bianco et  al . , 2002 ) . the high expression of d3 in fetal compartments and the growth retardation and partial neonatal mortality of d3-null mice ( hernandez et  al . , 2006 ) normal th levels are required for efficient muscle homeostasis , function , and regeneration ( mcintosh et  al . , 1994 ; simonides and van hardeveld , 2008 ) . indeed , a broad set of genes are positively or negatively regulated at the transcriptional level by th ( salvatore et  al . , 2014 ; simonides and van hardeveld , 2008 ) . one of the genes transcriptionally stimulated by t3 is myod ( muscat et  al . , 1995 ) , which is a master regulator of the myogenic developmental and regeneration program . while it is well known that muscle function is altered in patients with thyrotoxicosis or hypothyroidism , it has also been shown that th excess impairs the regeneration process in the mdx mouse ( anderson et  al . , 1994 ) . there are two sources of t3 in muscle tissue ; one is the fraction that enters the cells directly from the plasma , the second is locally produced from t4-to - t3 conversion via d2 action ( dentice et  al . similarly , little is known about how the balance between the t3-activating and -inactivating deiodinases in muscle and in muscle progenitor cells is determined . clarification of these issues would be a significant advance in the understanding of the cellular pathways governing the progression of muscle stem cell lineage . the aim of our study was to dissect the role of the intracellular th metabolism and signaling in muscle progenitor cells . we identified d3 in satellite cells and mpcs , and found that it is induced upon stem cell activation early after muscle injury . this event was associated with the expansion of the satellite cell population that occurs after muscle injury . despite normal plasma t3 concentrations , selective depletion of d3 in the satellite cell compartment resulted in severe cell apoptosis thereby disrupting the normal pattern of tissue response to acute injury and causing a marked delay in muscle regeneration . thus , we demonstrate that d3 and modulation of local th metabolism represent a survival mechanism during the progression of the muscle stem cell lineage . to assess whether d3 is expressed in satellite cells , we measured its expression in facs - sorted cells from tg : pax7-ngfp mice ( rocheteau et  al . , 2012 ) and found elevated d3 levels , which declined as differentiation proceeded ( figures 1a and 1b ) , a pattern observed also in c2c12 as well as in primary cultures of skeletal muscle enriched in satellite cells ( pp6 cells ; qu et  al . , 1998 ) d3 levels increased rapidly during muscle development , and peaked in the second postnatal week , after which they progressively declined to reach very low levels in the adult ( figure  s1b ) . d3 expression , detectable in isolated pax7-positive cells , increased in dividing myod1-positive cells of muscle fibers ( figures 1c and s1c ) . interestingly , d3 activity in limb muscles was significantly higher in the  dystrophic mdx mouse than in controls ( figure  1d ) , which is consistent with the increased proliferation of satellite cells in the dystrophic context . we examined d3 expression during regeneration after cardiotoxin ( ctx ) injection ( charg and rudnicki , 2004 ) . d3 mrna , protein , and enzymatic activity were significantly increased at early stages after ctx injection ( 48  days ) in the tibialis anterior ( ta ) muscle . then d3 declined , reaching nearly normal adult levels by day 15 ( figures 1e and 1f ) . coimmunofluorescence analysis showed d3 staining in the pax7-positive cells as well as in the newly regenerating fibers and in f4/80 macrophages infiltrating the injury site ( figures 2a and 2b ) . d3 showed a dynamic cell - type specific expression , i.e. , a robust expression at day 7 in the satellite cell compartment and declined thereafter ( figures 2a and 2b ) . to determine whether d3 might serve as a novel marker of a specific subset of pax7-positive cells , we isolated satellite cells and fractionated them according to ngfp intensity by flow cytometry . we analyzed facs - isolated pax7-ngfp ( top 20% ) , pax7-ngfp ( intermediate 60% ) , and pax7-ngfp ( bottom 20% ) from ctx - injured or control - uninjured muscles . interestingly , within the pax7-ngfp - expressing cells , d3 expression was higher in pax7-ngfp cells , which have the highest commitment to progress toward a myogenic lineage ( figure  2c ) ( mourikis et  al . , 2012 ) . within the d3-positive pax7-ngfp fraction , this is illustrated by the expression profile of well - characterized positively ( serca2 , foxo3 , pgc-1 , troponin 2 ) and negatively ( pgc-1 and troponin 1 ) ( figures 2d and s2 ) regulated th target genes , which points to a relatively  hypothyroid state  in the pax7-ngfp / d3high - expressing cells ( figure  2d ) . the pattern of d3 expression and the expression profile of the th - responsive gene in gfp - sorted cells provide functional evidence that intracellular th concentrations are tightly regulated in satellite cells during lineage progression . to this aim , we generated a satellite cell - specific conditional dio3 knockout mouse ( see experimental procedures ) . the homozygous dio3 mice were viable and fertile , with no alterations in d3 protein , body mass , fertility , or serum th concentrations ( figures s1 and s3 ; data not shown ) . adeno - cre infection of primary myoblasts resulted in effective deletion of the selenocysteine insertion sequence ( secis ) element and inactivation of d3 ( see experimental procedures ) , without affecting d3 mrna levels ( figure  s1d ) in agreement with the genetic strategy used . we crossed the dio3 floxed mouse with a tg : pax7-cre mouse ( mourikis et  al . , 2012 ) in order to specifically delete d3 in the satellite cells . we subjected 2-month - old dio3 ; tg : pax7-cre mice ( cd3ko ) to tamoxifen ( tam ) treatment followed by ctx injury ( figure  3a ) . after tam treatment , d3 in satellite cells was markedly and specifically reduced as demonstrated by immunofluorescence staining ( figures 3b and s3 ) . as expected , d3 protein levels were significantly reduced , but not abolished , in muscle by tam treatment , in agreement with the expression of d3 in pax7-negative cells , e.g. , in infiltrating macrophages ( figures 2b and 3b ) . after ctx injection ( 4  days ) , the pattern of regeneration in the ta was similar in the control and cd3ko mice . however , following d3 depletion at day 7 post - ctx , we observed a significant reduction in the number of pax7-positive cells ( figures 3a3c ) . this reduction was due to massive apoptosis , as measured by tunel assay and parp cleavage in regenerating muscles ( figures 3c3f , s3d , and s3e ) . examination of muscle sections at later times showed a striking difference between the cd3ko and wild - type ( wt ) animals , a difference particularly evident at day 14 postinjury and thereafter ( figure  4a ) . indeed , while regeneration was active at this time in control muscle , with most of the myonuclei in the central position of myofibers and a sharp increase in the myogenic markers myod , myogenin , and mhc , these markers were greatly decreased in cd3ko muscles ( figures 4b and 4c ) . there was also a dramatic reduction in the number of regenerating fibers in the mutant animals ( figure  4d ) , which is consistent with a block in the initial wave of muscle regeneration . at day 40 , when regeneration was almost complete and the number of centrally located nuclei was similar in the cd3ko and wt mice ( figure  4d ) , regeneration was persistently aberrant and incomplete in the mutant tissues ( figure  4a ) . in addition , the cross - sectional area was significantly reduced in cd3ko mice versus control mice ( figure  4e ) . systemic hypothyroidism significantly rescued apoptosis in cd3ko mice ( figures 3d3f ) , indicating that apoptosis was due to an excessive intracellular th concentration . overall , these results indicate that d3 depletion in satellite cells and the consequent cell apoptosis significantly impair the regeneration process . in dio3 myoblasts , apoptosis followed d3 depletion or th treatment ( figures 5a , 5b , s4a , and s4b ) . this did not occur in adeno - cre - infected cells from wt mice or in the fibroblast - like primary cell population from dio3 mice ( figures s4c  s4f ) . since d3 serves only to inactivate th , we speculated that apoptosis would not occur in the absence of th in the serum , which was in fact the case ( figure  5a ) . furthermore , apoptosis was dependent on , and related to , cell proliferation . when differentiated dio3 cells were infected with the adeno - cre virus or treated with supraphysiological th concentrations , no apoptosis was detected , nor did apoptosis occur in cells arrested in s phase before th treatment ( figures s4 g and s4h ) . these results indicate that proliferating , but not cell cycle - arrested or differentiated , mpcs , undergo apoptosis following exposure to elevated th concentrations . accordingly , in cultured fibers , th treatment significantly induced apoptosis in proliferating myoblasts ( figures s4i and s4l ) . finally , we tested whether apoptosis occurred also in  vivo after th treatment . in ctx - injured th - treated mice , impairment of the regeneration process ( figures s5a  s5c ) was associated with a significant reduction in pax7-positive cells and a corresponding increase in apoptosis ( figures 5c and 5d ) . interestingly , after th treatment , the expression levels of th - regulated myogenic markers were normally increased in uninjured muscle , but not in ctx muscle ( figure  s5d ) . this finding is in agreement with the concept that systemic thyrotoxicosis causes massive apoptosis of activated myogenic precursor cells . these results demonstrate that d3 expression protects activated satellite cells from a physiological , but spatiotemporally excessive , intracellular th concentration . to identify the molecular mechanisms underlying d3-dependent apoptosis , we evaluated the effects of d3 depletion on the expression of various transcription and myogenic factors . d3 depletion was associated with a spike in  th - dependent signaling ( figure  s5e ) and a transient induced transcription of differentiation markers ( figures 6a and 6b ) , which is in agreement with the marked similarity between the apoptotic cascade and muscle differentiation ( fernando et  al . , 2002 ) . interestingly , foxo3 was significantly induced upon d3 depletion in  vitro and confirmed in  vivo ( figures 6a , s5f , and s5 g ) . the upregulation of foxo3 preceded the peak of myod , a well - established th - responsive gene , thereby confirming data showing that foxo3 induces myod in muscle cells ( hu et  al . , 2008 ) ( figures 6a and 6b ) . to verify that th induces foxo3 levels , we measured the response of foxo3 to th treatment in myogenic cells . foxo3 was induced by th at mrna and protein levels ( figure  6c ) . this effect was abrogated when a dominant - negative th receptor ( tr ) was transfected ( figures 6d and s4b ) , which indicates that it requires a functional tr . th also significantly increased foxo3 transcriptional activity , as demonstrated by the significant increase in the activity of a foxo3-responsive artificial promoter upon treatment with th , as well as its reduction by intracellular t3 depletion obtained by reverse t3-mediated ( dentice et  al . , importantly , the dependence of foxo3 on th levels was confirmed in  vivo by the appropriate foxo3 levels in both hypo- and hyperthyroid muscles ( figure  6f ) . interestingly , foxo3 was reduced in d2ko cells and muscles ( figures 6 g and 6h ) , in which intracellular t3 concentrations are reduced , despite normal circulating t3 ( marsili et  al . , 2011 ) , indicating a direct correlation between intracellular th and foxo3 levels . in summary , this set of experiments provides compelling evidence that th is a master regulator of foxo3 and demonstrates that foxo3 responds to the modulation of th signaling in muscle . to assess the functional relevance of the foxo3-myod axis in th - dependent apoptosis , we analyzed separately the roles of foxo3 and myod in th - treated cells . interestingly , th failed to induce apoptosis in cells expressing a dominant - negative foxo3 protein ( figures 7a and s4b ) and in myoblasts genetically depleted of foxo3 ( figures 7b and 7c ) , which indicates that foxo3 plays a crucial role in th - induced apoptosis . we also assessed the effects of th in myoblasts in which myod expression was efficiently blocked by rnai ( figures s6a and s6b ) . this result indicates that an active myod is also required for th - induced apoptosis , and is consistent with a linear cascade in which th induces foxo3 , which lies upstream of myod . consistent with this model , both foxo3 and myod deficiency were rescued by transfecting a constitutively active pakt - resistant foxo3 ( figure  7c ) as well as by myod overexpression ( figure  s6b ) , thereby restoring in both cases the capacity of th to induce apoptosis . these gain- and loss - of - function analyses clearly indicate that foxo3 is a critical mediator of the apoptosis driven by th . finally , we asked whether th - mediated muscle differentiation also requires an intact foxo3 . the expression of several well - known t3-dependent myogenic markers was not responsive to th in foxo3-depleted cells ( figure  7f ) , and this was not due to altered expression of th receptors ( figure  s6c ) . these results indicate that foxo3 is a critical mediator of th action in skeletal muscle and that , notwithstanding exposure to excessive th , foxo3-depleted myoblasts do not undergo apoptosis upon th treatment and are phenotypically hypothyroid at nuclear level . we show that a time - dependent and cell - autonomous degradation of th signaling via d3 expression is required for correct satellite cell amplification and muscle repair . in the absence of d3 , proliferating myoblasts undergo apoptosis , and this is functionally critical in regenerating muscle , but less evident under homeostatic conditions ( figure  s7 ) . to our knowledge , this study is the first to describe how a ubiquitous endocrine signal  th  is integrated and adapted in the stem cell niche to enable cell lineage progression . in muscle stem cells , only activated proliferating cells apoptose , not those that are quiescent or arrested in s phase ( figure  5 ) . therefore , differentiating cells express d2 in order to locally produce extra t3 to allow proper differentiation . vice versa , proliferating myoblasts require lower than circulating th levels , and to this aim activate d3-mediated t3 inactivation . levels of t3 present in the plasma . therefore , satellite cells are able to customize their intracellular t3 concentrations based on their functional state and dynamic needs . it is not yet clear why this is the case , but we speculate that temporally excessive th creates a metabolic or mitotic catastrophe , which can not be overcome by the proliferating cells . in  vitro , d3 is potently induced by growth factors ( egf , fgf , and pdgf ) and cytokines ( dentice et  al . , 2009 ) . we demonstrated in neoplastic contexts that the wnt and shh pathways are potent inducers of dio3 ( dentice et  al . , we found that -catenin , sonic hedgehog , c - met , cmyc , and pax7 induced d3 in myoblasts in  vitro ( figure  s6d ) . it is conceivable that d3 is induced by a set of integrated signals from growth factors , cytokines , morphogens , and other signaling pathways that are active at the injury site . t3 is a circulating hormone whose plasma concentrations are relatively stable in the healthy state . in plasma , the hypothalamic - pituitary - thyroid axis prevents potentially adverse oscillations of t4 and t3 via a potent homeostatic mechanism that controls hormone secretion by the thyroid gland ( bianco and kim , 2006 ) . a major question in this context is how muscle stem cells customize their th signature and adapt it to their physiology . the rapid increase in d3 in proliferating muscle stem cells and the consequent attenuation of t3 nuclear receptor saturation is an ideal solution to this problem . the local metabolism of th is a highly regulated process during embryonic and fetal life , as well as during muscle regeneration . in general , d3 is reciprocally regulated with d2 to maintain local control of the t3 concentration . these interlocking pathways facilitate specific chronotropic and tissue - specific changes in t3 concentrations during periods when developmental or regenerative programs call for a transient increase or decrease in specific cells ( gereben et  al . , 2008 ) . the rapid increase of d3 in proliferating myoblasts clearly indicates that the ambient plasma and cellular concentrations of th are too high to sustain their proliferation . d3 is also expressed in activated macrophages at the injury site ( boelen et  al . , 2011 ) , although its role in this cell context is presently unknown . this mechanism also occurs in the mdx mouse model , where d3 is expressed at significantly higher levels compared with healthy muscle ( figure  1d ) . skeletal muscle stem cells are a heterogeneous population of cells that span from stem - like cells to cells more committed toward myogenic lineage progression and differentiation . there are at least two populations of pax7-positive satellite cells resident in skeletal muscle . one population rapidly contributes to muscle repair ; the second is more stem cell - like and remains longer in a quiescent state in the recipient muscle . these functional differences are reflected by the different levels of pax7 ( rocheteau et  al . , 2012 ) . in this context , pax7 represents a reversible dormant stem cell state during homeostasis ( mourikis et  al . , 2012 ) . here we show that t3 availability is differentially regulated in these two subpopulations of myogenic cells via d3 action . during muscle homeostasis , d3 is expressed in the satellite cells that are poised for myogenic commitment ( pax7 ) , wherein d3 expression does not simply reflect a secondary homeostatic regulation of this enzyme , but causes an intracellular attenuation of the th signal compared to the pax7 cells . this is reflected in the divergent expression of oppositely regulated th - responsive genes , which change depending on the reduced th signaling mediated by d3 ( figures 2e and 2f ) . such a regulation could correlate with the metabolic status of the cells , e.g. , pax7 quiescent cells are more metabolically active than the corresponding pax7 ( rocheteau et  al . , 2012 ) . here we show that the control of th signaling in the muscle stem compartment occurs , and that it is necessary for muscle cell lineage progression . this is in agreement with the critical role of th action in neurons , where th signaling represses sox2 levels and regulates neurogenesis ( lpez - jurez et  al . , 2012 ) . hyperthyroidism in humans may cause a severe muscle myopathy associated with a failure in muscle regeneration ( anderson et  al . , 1994 ; simonides and van hardeveld , 2008 ) . while it was thought that the effects of hyperthyroidism occurred at the level of myofibers ( simonides and van hardeveld , 2008 ) , the reduced expression of pax7 and increased apoptosis in hyperthyroid muscle in mice characterized in this study point to a failure at stem cell level , which may well occur also in humans . here importantly , the identification of foxo3 as a t3 target links two potent systems controlling multiple aspects of muscle physiology . in silico examination of the mouse foxo3 locus showed multiple potential candidate sequences containing tr - binding elements , although none was evolutionally conserved , and chip assays failed to identify functional relevance under our experimental conditions . nevertheless , the accumulating evidence indicating crosstalk between t3 and the akt pathway ( figure  6 ) points to a complex network involving foxo3 regulation by t3 . intriguingly , foxo3 is a potent inducer of d2the t3-producing enzyme  thus suggesting that a positive autoregulatory loop might be one mechanism by which foxo3 is a target of t3 , and also indirectly sustains t3 intracellular concentration by inducing d2 ( figure  7 g ) . asakura and colleagues suggested that myod is a proapoptotic transcription factor in muscle stem cells ( asakura et  al . , 2007 ) . here we show that myod , a target of both t3 and foxo3 ( dentice et  al . , 2010 ; hu et  al . , 2008 ) , is required for the proapoptotic cascade triggered by t3 . finally , we show that not only does foxo3 act as a th target , but also that its action is required to sensitize muscle stem cells to th levels . in summary , the absence of d3 in activated muscle stem cells causes a time - dependent and irreversible stem cell death due to excessive intracellular nuclear t3 . d3 depletion renders these cells incapable of overcoming the t3 excess , thus promoting the apoptotic pathway . the finding that d3 plays a crucial role in controlling nuclear t3 in muscle stem cells and in the triggering of a death pathway via the foxo3-myod axis has broad implications and opens a future research scenario for the development of translational therapeutic strategies based on the local control of potent endocrine signals by modulating their metabolic pathways . primary muscle cultures ( pp6 ) were isolated as described ( qu et  al . , 1998 ) from the indicated mouse lines . foxo3-depleted ( foxo - ko ) pp6 cells were prepared from foxo3 mice as previously described ( dentice et  al . , 2010 ) . transient transfections were performed using lipofectamine 2000 ( life technologies ) according to the manufacturer s instructions . single myofibers were prepared from the extensor digitorumlongus and gastrocnemius muscles of 6- to 12-week - old mice as previously described ( rosenblatt et  al . , 1995 ) . pax7 isolation by facs has been described elsewhere ( rocheteau et  al . , 2012 ) . anti - myod ( sc-304 ) , myogenin ( sc-12732 ) , tubulin ( sc-8035 ) , and anti - foxo3 antibodies were purchased from santa cruz biotechnology . polyclonal anti - mhc antibody ( mf-20a ) and anti - pax7 antibody were from developmental studies hybridoma bank . anti - d3 antibody is described elsewhere ( huang et  al . , 2003 ) . anti - total akt was from upstate , and anti - pakt and anti - parp were from cell signaling technology . cells isolated by facs were prepared from tg : pax7-ngfp mice as previously described ( rocheteau et  al . , 2012 ) . in brief , muscle dissection was done with  a  scalpel by removing the tissue from the bone in dmem . the collagenase / trypsin solution was added to continue the digestion until the process was completely terminated and muscle totally digested . satellite cells have been cultured in 1:1 dmem : mcdb containing 20% serum fbs and its ( 1   , insulin - transferrin - selenium ; gibco ) . cells were plated on matrigel ( bd biosciences ; catalog # 354234 ) or collected in a lysis buffer ( rneasy micro kit , quiagen ) for rna extraction . foxo3 , foxodn , and dbe plasmids are described elsewhere ( dentice et  al . , 2010 ) . tr plasmid was kindly provided by dr . s. cheng . to knock down myod , two different stealth sirna targeting two different regions of the coding sequence ( myod-7 , gacgacuucuaugaugacccguguu ; and myod-8 , cacuacaguggcgacucagaugcau ) were designed with the block - it technology ( invitrogen ) and purchased from invitrogen corporation . effective myod knockdown was assessed by measuring endogenous myod levels in satellite cells following 48  hr stealth sirna transfection ( figure  s6a ) . all three deiodinases contain a critical selenocysteine residue in the catalytic domain , which is encoded by a uga codon and requires a specific secondary mrna loop ( secis element ) in the 3 utr of the mrna that overrides the stop function and inserts selenocysteine ( berry et  al . , 1991 ) . given the absolute requirement of a secis element for correct d3 protein translation , we selectively ablated the secis region in the dio3 gene , thus preserving the dio3as transcript encoded in the antisense orientation in the dio3 locus , the function of which is not known ( figure  s2 ) . we generated a plasmid harboring floxed sites in the dio3 locus , and specifically flanking the secis mrna structure located at nt 1,001 and nt 1,706 from the atg in the dio3 mrna ( figure  s2 ) . in the absence of the secis , the tga codon , within the d3 catalytic domain , is recognized as stop codon , and protein translation terminated . once we obtained the homozygous dio3 mice , we crossed these mice with heterozygous tg : pax7-cre mice , expressing the cre - ert2 fusion protein . the cre - ert2 protein consists of cre recombinase fused to a triple - mutant form of the human estrogen receptor and catalyzes the lox sites recombination tam inducible . the expected recombination event occurred in the progeny , as documented in the pcr in figure  s1d . dio3 not expressing recombinase , but tam treated , were used as negative controls . for immunofluorescent staining , cells were fixed with 4% formaldehyde and permeabilized in 0.1% triton x-100 , then blocked with 0.5% goat serum and incubated with primary antibody . dissected muscle was snap frozen in liquid nitrogen , sectioned ( 7  m thick ) , and stained with hematoxylin and eosin stain ( h&e ) or immunofluorescence using standard protocols . western blots were run in triplicate , and bands were quantitated in one representative gel . ctx was injected as described previously ( yan et  al . , 2003 ) . briefly , 1020  l of 10  m ctx ( naja mossambica mossambica , sigma - aldrich ) was injected into the right ta and gastrocnemius muscles of anesthetized 12-week - old c57bl/6 male mice ( the jackson laboratory ) ( wt ) , and age- and gender - matched cd3ko mice . the uninjected left ta muscles served as controls . for ctx experiments in cd3ko mice , animals were treated daily with 75  g / g tam for 5  days , and ctx was injected on day 4 . mice were killed 4 , 14 , or 40  days after ctx injection . for ctx experiments in wt mice , animals were treated daily with th ( 2.5  g / mouse t3  + 10  g / mouse t4 ) 4  days before ctx injection ; mice were killed 4 and 14  days after ctx injection . hypothyroidism was obtained by supplying the drinking water with 0.1% mmi and 1% kclo4 for 6  weeks . animal experimental protocols were approved by the animal research committee of the university of naples federico ii . differences between samples were assessed by the student s two - tailed t test for independent samples . in all experiments , asterisks indicate significance at p  < 0.05 , p  < 0.01 , and p  < 0.001 throughout . , s.b . , and l.d.v . provided observations and scientific interpretations ; p.r.l . and s.t . provided essential reagents and scientific insight ; m.d . performed most experiments and analysis , prepared figures , and wrote the paper ; d.s . designed the overall study , supervised the experiments , analyzed the results , and wrote the paper ; and all authors discussed the results and provided input on the manuscript .