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 .
 summaryprecise control of the thyroid hormone ( t3)-dependent transcriptional program is required by multiple cell systems , including muscle stem cells . deciphering how this is achieved and how the t3 signal is controlled in stem cell niches is essentially unknown . 
 we report that in response to proliferative stimuli such as acute skeletal muscle injury , type 3 deiodinase ( d3 ) , the thyroid hormone - inactivating enzyme , is induced in satellite cells where it reduces intracellular thyroid signaling . satellite cell - specific genetic ablation of dio3 severely impairs skeletal muscle regeneration . 
 this impairment is due to massive satellite cell apoptosis caused by exposure of activated satellite cells to the circulating th . 
 the execution of  this proapoptotic program requires an intact foxo3/myod axis , both genes positively regulated by intracellular th . 
 thus , d3 is dynamically exploited in  vivo to chronically attenuate th signaling under basal conditions while also being available to acutely increase gene programs required for satellite cell lineage progression . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions