Thyroid hormone triggers the developmental loss of receptor α1 … · 2012. 11. 14. · Thyroid...

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Thyroid hormone triggers the developmental loss of axonal regenerative capacity via thyroid hormone receptor α1 and krüppel-like factor 9 in Purkinje cells Hasan X. Avci a,b , Clement Lebrun a,b , Rosine Wehrlé a,b , Mohamed Doulazmi a,b , Fabrice Chatonnet c , Marie-Pierre Morel a,b , Masatsugu Ema d , Guilan Vodjdani e , Constantino Sotelo f , Frédéric Flamant c , and Isabelle Dusart a,b,1 a Université Pierre et Marie Curie (UPMC) Paris 06, Unité Mixte de Recherche 7102, 75005 Paris, France; b Centre National de la Recherche Scientique, Unité Mixte de Recherche 7102, 75005 Paris, France; c Centre National de la Recherche Scientique, Institut National de la Recherche Agronomique, École Normale, Supérieure de Lyon, Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, 69007 Lyon, France; d Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki 305-8575, Japan; e Centre de Recherche de lInstitut du Cerveau et de la Moelle Epinière, Unité Mixte de Recherche 7225-Centre National de la Recherche Scientique/Unité Mixte de Recherche S975-Institut National de la Santé et de la Recherche Médicale, Université Pierre et Marie Curie Paris 06, 75013 Paris, France; and f Cátedra de Neurobiología del Desarrollo Remedios Caro Almela,Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Cienticas, 03550 San Juan de Alicante, Spain Edited by Thomas M. Jessell, Columbia University College of Physicians and Surgeons, New York, NY, and approved July 6, 2012 (received for review December 5, 2011) Neurons in the CNS of higher vertebrates lose their ability to regenerate their axons at a stage of development that coincides with peak circulating thyroid hormone (T 3 ) levels. Here, we exam- ined whether this peak in T 3 is involved in the loss of axonal re- generative capacity in Purkinje cells (PCs). This event occurs at the end of the rst postnatal week in mice. Using organotypic culture, we found that the loss of axon regenerative capacity was trig- gered prematurely by early exposure of mouse PCs to T 3 , whereas it was delayed in the absence of T 3 . Analysis of mutant mice showed that this effect was mainly mediated by the T 3 receptor α1. Using gain- and loss-of-function approaches, we also showed that Krüppel-like factor 9 was a key mediator of this effect of T 3 . These results indicate that the sudden physiological increase in T 3 during development is involved in the onset of the loss of axon regenerative capacity in PCs. This loss of regenerative capacity might be part of the general program triggered by T 3 throughout the body, which adapts the animal to its postnatal environment. axon regeneration | cerebellum | neural development | lentiviral vectors I n higher vertebrates, there is a period of development during which CNS neurons can regenerate their axons after injury (1). The molecular mechanisms underlying the subsequent loss of this ability are not fully understood, but the onset of an inhibitory en- vironment in the developing CNS is known to prevent axon re- generation (13), together with intrinsic neuronal maturation (46). In mice, this event takes place during the rst postnatal week for neurons of the entorhinal cortex and motor cortex, as well as retinal ganglion cells and Purkinje cells (PCs) (711). Interestingly, this loss of regenerative capacity coincides with a strong increase in circulating thyroid hormone (T 3 ) levels. T 3 levels in serum are low during mouse prenatal development but increase sharply (more than 1,000-fold) during the rst week after birth before decreasing to adult levels during the second week (1214). This hormonal burst leads to profound changes in gene expression that are required to adapt the physiology of various organs to postnatal life (15). We suspected that the loss of axonal regenerative ability might be one of these critical physiological changes. We therefore in- vestigated the involvement of high T 3 levels in the onset of the loss of regenerative capacity of postnatal PCs. Because Krüppel- like factor 9 (Klf9) is a transcriptional target of T 3 in some neuronal cell types (16, 17) and is involved in the developmental loss of regenerative ability of retinal ganglion cells (5), we also examined whether T 3 acts on PCs by regulating Klf9 expression. Results Early Exposure to T 3 Accelerates the Loss of Ability to Regenerate PC Axons. To investigate the effect of early T 3 exposure on axon regeneration ability, we used a coculture assay to quantify the regenerative capacity of PCs (18, 19). PCs from newborn mice [postnatal day (P) 0] were grown in organotypic culture for 7 d in vitro (div) and were then axotomized and placed in front of the ventral half of a cerebellar slice taken from age-matched (P0 + 7 div) calbindin-decient mice (Calb1 -/- ) for another 7 div to allow for regeneration (Fig. 1A). Hence, all Calb1-immunoreactive axons in the ventral half of the slice corresponded to regenerative axons. Two parameters were measured to determine regeneration ca- pacity: the area covered by Calb1 + regenerating axons and the mean length of the three longest regenerating axons in the Calb1 -/- slice (Fig. S1). Hereafter, we refer to the area of regeneration as an indirect index for the number of PCs able to regenerate, whereas the mean length of the three longest axons serves as an indirect index for the growth rate of regenerating axons. When 30 nM T 3 was added to the culture medium throughout the cell culture period, the regenerative capacity of PC axons was strongly inhibited (Fig. 1 B and C). The differences from untreated cultures were statistically signicant both for the number of re- generating PCs and for the axon growth rate (Fig. 1 F and G). However, PC survival was very low in this experiment (Fig. 1 B and C and Fig. S2). PC death induced by axotomy can be prevented by inhibiting PKC with Gö6976 (18). When similar experiments were carried out in the presence of 1 μM Gö6976 (Fig. 1 D and E and Fig. S2), the two parameters reecting regenerative capacity were enhanced in both the presence and absence of T 3 (Fig. 1 DG). In the presence of T 3 , no considerable regeneration was observed in slices with very high PC survival (Fig. 1E), whereas slices with lower PC survival showed marked regeneration in he absence of T 3 (Fig. 1D). Thus, independent of the survival effect of Gö6976, T 3 sig- nicantly reduced the capacity of axons to regenerate (Fig. 1 F and G). Remarkably, mean axonal length in slices grown in the pres- ence of T 3 was only 28% of that observed in the absence of T 3 (Fig. 1G). Thus, T 3 not only reduced the number of PCs capable of promoting axon regeneration but limited the axon growth rate of those that were able to regenerate. We then investigated the time dependency of this effect by performing axotomy in the presence of Gö6976 at various time points (0, 3, 5, 7, and 14 div) and analyzing PC regeneration 7 d later. In the absence of T 3 , the capacity of PCs to regenerate axons Author contributions: H.X.A., C.S., and I.D. designed research; H.X.A., C.L., R.W., F.C., M.-P.M., G.V., and I.D. performed research; M.E., G.V., and F.F. contributed new re- agents/analytic tools; H.X.A., R.W., M.D., F.C., M.-P.M., and I.D. analyzed data; and H.X.A., G.V., C.S., F.F., and I.D. wrote the paper. The authors declare no conict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1119853109/-/DCSupplemental. 1420614211 | PNAS | August 28, 2012 | vol. 109 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1119853109 Downloaded by guest on February 28, 2021

Transcript of Thyroid hormone triggers the developmental loss of receptor α1 … · 2012. 11. 14. · Thyroid...

Page 1: Thyroid hormone triggers the developmental loss of receptor α1 … · 2012. 11. 14. · Thyroid hormone triggers the developmental loss of axonal regenerative capacity via thyroid

Thyroid hormone triggers the developmental loss ofaxonal regenerative capacity via thyroid hormonereceptor α1 and krüppel-like factor 9 in Purkinje cellsHasan X. Avcia,b, Clement Lebruna,b, Rosine Wehrléa,b, Mohamed Doulazmia,b, Fabrice Chatonnetc,Marie-Pierre Morela,b, Masatsugu Emad, Guilan Vodjdanie, Constantino Sotelof, Frédéric Flamantc,and Isabelle Dusarta,b,1

aUniversité Pierre et Marie Curie (UPMC) Paris 06, Unité Mixte de Recherche 7102, 75005 Paris, France; bCentre National de la Recherche Scientifique, UnitéMixte de Recherche 7102, 75005 Paris, France; cCentre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, École Normale,Supérieure de Lyon, Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, 69007 Lyon, France; dGraduate School of Comprehensive HumanSciences, University of Tsukuba, Ibaraki 305-8575, Japan; eCentre de Recherche de l’Institut du Cerveau et de la Moelle Epinière, Unité Mixte de Recherche7225-Centre National de la Recherche Scientifique/Unité Mixte de Recherche S975-Institut National de la Santé et de la Recherche Médicale, Université Pierreet Marie Curie Paris 06, 75013 Paris, France; and fCátedra de Neurobiología del Desarrollo “Remedios Caro Almela,” Instituto de Neurociencias, UniversidadMiguel Hernández-Consejo Superior de Investigaciones Cientificas, 03550 San Juan de Alicante, Spain

Edited by ThomasM. Jessell, Columbia University College of Physicians and Surgeons, New York, NY, and approved July 6, 2012 (received for review December 5, 2011)

Neurons in the CNS of higher vertebrates lose their ability toregenerate their axons at a stage of development that coincideswith peak circulating thyroid hormone (T3) levels. Here, we exam-ined whether this peak in T3 is involved in the loss of axonal re-generative capacity in Purkinje cells (PCs). This event occurs at theend of the first postnatal week in mice. Using organotypic culture,we found that the loss of axon regenerative capacity was trig-gered prematurely by early exposure of mouse PCs to T3, whereasit was delayed in the absence of T3. Analysis of mutant miceshowed that this effect was mainly mediated by the T3 receptorα1. Using gain- and loss-of-function approaches, we also showedthat Krüppel-like factor 9 was a key mediator of this effect of T3.These results indicate that the sudden physiological increase in T3during development is involved in the onset of the loss of axonregenerative capacity in PCs. This loss of regenerative capacitymight be part of the general program triggered by T3 throughoutthe body, which adapts the animal to its postnatal environment.

axon regeneration | cerebellum | neural development | lentiviral vectors

In higher vertebrates, there is a period of development duringwhich CNS neurons can regenerate their axons after injury (1).

The molecular mechanisms underlying the subsequent loss of thisability are not fully understood, but the onset of an inhibitory en-vironment in the developing CNS is known to prevent axon re-generation (1–3), together with intrinsic neuronal maturation (4–6). In mice, this event takes place during the first postnatal week forneurons of the entorhinal cortex andmotor cortex, as well as retinalganglion cells and Purkinje cells (PCs) (7–11). Interestingly, thisloss of regenerative capacity coincides with a strong increase incirculating thyroid hormone (T3) levels. T3 levels in serum are lowduring mouse prenatal development but increase sharply (morethan 1,000-fold) during the first week after birth before decreasingto adult levels during the second week (12–14). This hormonal burstleads to profound changes in gene expression that are required toadapt the physiology of various organs to postnatal life (15).We suspected that the loss of axonal regenerative ability might

be one of these critical physiological changes. We therefore in-vestigated the involvement of high T3 levels in the onset of theloss of regenerative capacity of postnatal PCs. Because Krüppel-like factor 9 (Klf9) is a transcriptional target of T3 in someneuronal cell types (16, 17) and is involved in the developmentalloss of regenerative ability of retinal ganglion cells (5), we alsoexamined whether T3 acts on PCs by regulating Klf9 expression.

ResultsEarly Exposure to T3 Accelerates the Loss of Ability to Regenerate PCAxons. To investigate the effect of early T3 exposure on axonregeneration ability, we used a coculture assay to quantify the

regenerative capacity of PCs (18, 19). PCs from newborn mice[postnatal day (P) 0] were grown in organotypic culture for 7 din vitro (div) and were then axotomized and placed in front of theventral half of a cerebellar slice taken from age-matched (P0 +7 div) calbindin-deficient mice (Calb1−/−) for another 7 div to allowfor regeneration (Fig. 1A). Hence, all Calb1-immunoreactive axonsin the ventral half of the slice corresponded to regenerative axons.Two parameters were measured to determine regeneration ca-pacity: the area covered by Calb1+ regenerating axons and themean length of the three longest regenerating axons in the Calb1−/−

slice (Fig. S1). Hereafter, we refer to the area of regeneration as anindirect index for the number of PCs able to regenerate, whereasthe mean length of the three longest axons serves as an indirectindex for the growth rate of regenerating axons.When 30 nM T3 was added to the culture medium throughout

the cell culture period, the regenerative capacity of PC axons wasstrongly inhibited (Fig. 1 B and C). The differences from untreatedcultures were statistically significant both for the number of re-generating PCs and for the axon growth rate (Fig. 1 F and G).However, PC survival was very low in this experiment (Fig. 1 B andC and Fig. S2). PC death induced by axotomy can be prevented byinhibiting PKC with Gö6976 (18). When similar experiments werecarried out in the presence of 1 μM Gö6976 (Fig. 1 D and E andFig. S2), the two parameters reflecting regenerative capacity wereenhanced in both the presence and absence of T3 (Fig. 1 D–G). Inthe presence of T3, no considerable regeneration was observed inslices with very high PC survival (Fig. 1E), whereas slices with lowerPC survival showed marked regeneration in he absence of T3 (Fig.1D). Thus, independent of the survival effect of Gö6976, T3 sig-nificantly reduced the capacity of axons to regenerate (Fig. 1 F andG). Remarkably, mean axonal length in slices grown in the pres-ence of T3 was only 28% of that observed in the absence of T3 (Fig.1G). Thus, T3 not only reduced the number of PCs capable ofpromoting axon regeneration but limited the axon growth rate ofthose that were able to regenerate.We then investigated the time dependency of this effect by

performing axotomy in the presence of Gö6976 at various timepoints (0, 3, 5, 7, and 14 div) and analyzing PC regeneration 7 dlater. In the absence of T3, the capacity of PCs to regenerate axons

Author contributions: H.X.A., C.S., and I.D. designed research; H.X.A., C.L., R.W., F.C.,M.-P.M., G.V., and I.D. performed research; M.E., G.V., and F.F. contributed new re-agents/analytic tools; H.X.A., R.W., M.D., F.C., M.-P.M., and I.D. analyzed data; and H.X.A.,G.V., C.S., F.F., and I.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1119853109/-/DCSupplemental.

14206–14211 | PNAS | August 28, 2012 | vol. 109 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1119853109

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decreased with time, as shown by the gradual decrease in the areaof regeneration from 0 to 14 d (Fig. 1H) and the decrease in theaverage length of the three longest regenerative axons between 5and 7 d (Fig. 1I). Addition of T3 throughout the cell culture periodreduced the number of PCs able to regenerate (area) and the axongrowth rate (longest axons) when axotomy was performed after

3 div (Fig. 1H and I). Thus, early exposure to T3 accelerates the lossof PC regenerative capacity in our in vitro conditions.

Absence of T3 in Vivo Delays Developmental Loss of PC Axon Regen-erative Ability in Organotypic Culture.We then investigated whetherthe absence of T3 in vivo delayed the loss of regenerative capacity

Fig. 1. T3 accelerates the developmental loss of PCaxon regenerative capacity. (A) Slice culture re-generation assay. Cerebellar slices from newbornmice (P0) were cultured, thus preserving PCs andtheir targets (deep cerebellar nuclei neurons) in thesame slice. Axotomy (axt) was performed in vitro.After 7 div, the ventral halves containing the deepcerebellar nuclei neurons were amputated andreplaced by the ventral half of a cerebellar slicetaken from age-matched (P0 + 7 div) Calb1−/− mice.Hence, all Calb1-immunoreactive axons in the ven-tral half of the slice were regenerative axons. Theseslice cocultures were kept for another 7 div to al-low regeneration to proceed. Photomicrographsof cocultured slices immunostained with Calb1 anti-bodies are shown: untreated coculture (B), T3-treatedcocultures (C and E), and Gö6976-treated cocul-tures (D and E). The dotted lines indicate the sitesof axotomy, and arrowheads show regeneratingaxons. Note that the axons are thicker in T3-treatedcultures than in untreated cultures. (Scale bar: 475μm.) (F and G) Quantitative analysis of regenerationin the presence and absence of T3 and Gö6976.Axotomy was always performed after 7 div. (H andI) Quantitative analysis of regeneration to deter-mine the time dependency of the T3 effect. Axot-omy was performed at various time points (0, 3, 5, 7,and 14 div). All cultures were grown in the presenceof Gö6976, with (black bars) or without (white bars)T3. The area covered by Calb1+ regenerating axons(F and H) and mean length of the three longestregenerating axons in the Calb1−/− slice (G and I)are shown. Values are means ± SEM [***P < 0.001,nonparametric Kruskal–Wallis one-way ANOVAwith the Mann–Whitney post hoc test (F and G) andtwo-way ANOVA (T3 effect and age) with post hocprotected least significant difference (PLSD) of theFisher exact test (H and I)]. In F and G, n = 27, 27, 34,and 46 for Ctrl, T3, Gö6976, and Gö6976 + T3, re-spectively. In H and I, n = 24, 32, 24, 22, and 23 in theabsence of T3 and n = 23, 36, 20, 21, and 22 in thepresence of T3 for axotomy at 0, 3, 5, 7, and 14 div,respectively.

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beyond P10, the age at which it is normally completed (9, 18).Cerebellar explants were prepared from three groups of P10 lit-termates (euthyroid, hypothyroid, and hyperthyroid pups; Fig.2A). The blood level of T3 was measured in each group of animalsto validate their thyroid status (Table S1). In coculture assays (Fig.2 B and C) performed in the absence of T3, the area covered byregenerating GFP+ PC axons was 12-fold higher in hypothyroidthan in euthyroid cerebella (Fig. 2 D–G and J) and the regeneratedaxons were 77% longer (Fig. 2 D–G and K). Hyperthyroidism hadno significant additional influence (Fig. 2 H–K). Addition of T3 tothe culture medium strongly reduced axon regeneration by PCsfrom hypothyroid animals, although it did not affect PCs fromeuthyroid or hyperthyroid animals (Fig. 2 J and K). Thus, theabsence of T3 in vivo prolongs the period during which PCs areable to regenerate in organotypic culture assays, whereas anexcess of T3 does not seem to have any influence on this ability.

T3 Receptor α1 Is Involved in the T3-Induced Loss of PC Axon Regen-erative Capacity in Organotypic Cultures. T3 acts directly on genetranscription by binding to the T3 receptor (TR) α1 or TRβ1/2[thyroid hormone receptor alpha1 (THRA1) and thyroid hor-mone receptor beta1/2 (THRB1/2) according to the MGI no-menclature] nuclear receptors, encoded by the genes TRα andTRβ [Thra and Thrb (20)]. Both receptors are expressed by PCs(21–23). We used a mouse model (TRαAMI line) in which CRE/loxP recombination is used to trigger the expression of a domi-nant-negative mutant receptor, TRα1L400R (24). Cerebellar slices

from newborn TRαAMI mice were transduced with a lentiviralvector (Lv) expressing Cre recombinase driven by the CMVpromoter (Fig. S3 A and B) and were grown in the presence ofT3. Gö6976 was added at the time of axotomy for 48 h to in-crease survival (Fig. S3C). It is important to note that the re-generative axons elongated in a WT TRα1 environment (Calb1−/−slices). T3 exposure failed to inhibit the regenerative ability ofPCs expressing mutated TRα1L400R (Fig. 3 A and B), whoseaxons covered an area fourfold larger than those of control PCs,and whose axons were more than twice as long (Fig. 3 C and D).To determine whether this effect was cell-autonomous, we

compared the behavior of Cre-transduced PCs vs. nontransducedPCs on slices from WT and TRαAMI animals (Fig. S4). Themajority of PCs expressing TRα1L400R were able to regenerate,whereas the majority of those expressing TRα1 did not re-generate (Fig. 3E). Although our results do not exclude the in-volvement of TRβ1/2, they clearly show that T3 acts on TRα1 andinhibits PC axonal regeneration in a cell-autonomous manner inorganotypic culture.

Krüppel-Like Factor 9 Mediates the Inhibitory Effect of T3 on PC AxonRegeneration. Klf9 is a T3 target gene in several neuronal celltypes (16, 17). It encodes a transcription factor identified as aninhibitor of axonal regeneration in murine retinal ganglion cells(5). To determine whether Klf9 is under the control of TRα1 inPCs, we crossed the TRαAMI mouse line with the ROSA26-lox-STOP-lox-EYFP floxed and Ptf1a Cre deleter line (Fig. S5A).

Fig. 2. T3 depletion prolongs the period of de-velopmental plasticity of PC axons. (A) To study PCregenerative capacity in P10 animals in T3-depletedconditions, we generated euthyroid (Eu-TH), hypo-thyroid (Hypo-TH), and hyperthyroid (Hyper-TH)pups of the L7-GFP-BAC line. Thyroid status can bedetected visually: Euthyroid pups are bigger thanhypothyroid pups but smaller than hyperthyroidpups. (B and C) Only half of the litter expressed GFPin all PCs when Swiss females were crossed withtransgenic L7-GFP-BAC heterozygous males. Thedorsal half of cerebellar slices from L7-GFP-BACmice were cultured and apposed to the ventral halffrom their GFP-negative littermates. Thus, all theGFP-immunoreactive axons present in the ventralhalf were regenerative PC axons. The ventral halfwas visualized by immunostaining with anti-Calb1antibodies (C). Photomicrographs of euthyroid (Dand E), hypothyroid (F and G), and hyperthyroid (Hand I) cocultures. E, G, and I are magnified views ofD, F, and H, respectively. The arrowheads indicateregenerating axons, and the dotted lines indicatethe sites of axotomy. Quantitative analysis of thearea of axon regeneration (J) and the longestregenerative axons (K) is shown. The cocultureswere grown in the presence (black bars) or absence(white bars) of T3. Values are means ± SEM (***P <0.001, Kruskal–Wallis test with Mann–Whitney posthoc test). (Scale bar: B and C, 450 μm; D, F, and H,240 μm; E, G, and I, 80 μm.) n = 17, 18, and 17 for Eu-TH, Hypo-TH, and Hyper-TH, and n = 15, 18, and 17in the presence of T3 for Eu-TH, Hypo-TH, and Hy-per-TH, respectively.

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After dissociating the cerebella of P7 animals, YFP-expressingcells (PCs and interneurons) were sorted out by FACS to mea-sure the expression of Klf9 mRNA by quantitative RT-PCR.Among the cells expressing KLF9, only PCs express TRα1L400Rand YFP (Fig. S5B). They showed a considerable reduction ofKlf9 mRNA by 75% (Fig. 4A).To address the possibility that KLF9 might mediate the in-

hibitory effect of T3 on PC axon regeneration, we then comparedKlf9 expression between hypothyroid and euthyroid animals by insitu hybridization at P10. Klf9 mRNA was detected in both PCsand inner granule cells (Fig. 4B), and its level was markedlydecreased by T3 depletion in both neuron populations (Fig. 4C).Similar results were obtained when the expression of β-gal wasquantified in the Klf9 KO mice. In this mouse line, Klf9 wasreplaced by the reporter gene lacZ, and it is therefore driven bythe Klf9 promoter (Fig. S6).To determine whether T3 acts on PCs through KLF9 expres-

sion, we compared the effect of T3 on regeneration of PCs fromKlf9−/− and WTmice on Calb1−/− slices in the presence of Gö6976(Fig. 4 D–G). Klf9−/− PCs displayed a smaller T3-induced decreasein regenerative capacity than WT PCs: The area and length ofregenerative PC axons elongating in a WT environment for KLF9were, respectively, twofold and 1.45-fold higher (Fig. 4 F and G).These moderate effects contrasted with the dramatic effect of T3on WT PCs (Fig. S7).We finally investigated whether KLF9 overexpression itself

inhibited PC axon regeneration in a WT KLF9 environmentin vitro. When cerebellar slices of newborn mice were transducedwith an Lv encoding Klf9 and grown in the absence of T3, thecapacity of PCs to regenerate axons was markedly lower thanthat of PCs expressing GFP (Fig. 4 H–K, in the presence ofGö6976). To determine whether this effect was cell-autonomous,we compared PCs transduced with either Lv-KLF9 or control Lv-GFP, as well as nontransduced PCs of the same slices. Almost90% of the Lv-KLF9–transduced PCs were unable to regenerate

even in the absence of T3, whereas in all three control PCpopulations, the majority of PCs regenerated (Fig. S8).Together, our findings demonstrate that the T3-induced de-

velopmental loss of PC axon regenerative capacity is largelymediated by KLF9.

DiscussionThis study shows that T3 is a key factor to determine the time atwhich PCs lose their ability to regenerate their axons in orga-notypic cultures. This function of T3 is mainly mediated by TRα1and involves its downstream target Klf9. This effect of T3 ondeveloping mammalian CNS neurons is a unique finding, be-cause T3 had previously been shown to promote axon regener-ation in sectioned adult mammal nerves in the peripheral nervoussystem (25).

Role of T3, TRα1, and KLF9 in the Developmental Loss of PC AxonRegenerative Ability. We have previously shown that newbornmouse PCs lose the ability to regenerate their axons by the end ofthe first week in organotypic culture (18, 19). In these experi-ments, T3 was present in the culture medium through the additionof 25% (vol/vol) horse serum. Here, in the absence of T3 (usingserum-free medium), PCs lose their ability to regenerate later.Thus, T3 determines the onset of the loss of PC regenerative ca-pacity, an event that coincides with peak circulating levels of T3.The respective functions of the T3 nuclear receptors TRα1 and

TRβ1 in PCs are controversial. In vivo PC dendritogenesis isreduced in mice expressing dominant-negative TRα1L400R orTRα1R384C (26, 27), as well as in ex vivo in TRα KO mice (28).However, the TRβ1 dominant-negative mutation also affects PCdifferentiation (29). Here, we found that TRα1L400R expressionby PCs was sufficient to protect them from the inhibitory effectof T3 on axon regeneration. This effect was cell-autonomousbecause the behavior of PCs was dependent on which form ofTRα1 was expressed; TRα1L400R-expresssing PCs were able to

Fig. 3. TRα1 mediates the T3-induced loss of PCregenerative capacity. Photomicrographs of new-born mouse (P0) cocultured slices transduced withLv encoding the Cre recombinase (LvCre) and im-munostained with anti-Calb1 antibodies: slices fromWT (A) and TRαAMI (B) mouse lines. The dotted linesindicate the site of axotomy, and the arrowheadspoint to regenerating axons. (Scale bar: 240 μm.)Quantitative analysis of PC axon regeneration: areaof axon regeneration (C) and longest regenerativeaxons (D). All cultures were grown in the presenceof Gö6976 at the time of axotomy to increase PCsurvival. Values from WT and TRαAMI animals areplotted as the mean ± SEM (***P < 0.001, Mann–Whitney test). n = 14 WT and 66 TRαAMI. (E) Distri-bution of crossing and noncrossing PC axons (Fig. S4C and I). The histogram illustrates percentages ofPCs transduced or nontransduced with Lv-Cre onWT or TRαAMI slices that either cross or do not crossthe site of axotomy. The frequency distribution ofsections in the four groups was compared betweenthe various experimental conditions using the Fisherexact test (***P < 0.001). WT: n = 32 nontransduced,n = 15 transduced; TRαAMI: n = 66 nontransduced,n = 77 transduced.

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regenerate, whereas WT TRα1-expressing PCs were not. Thus,our work demonstrates the involvement of TRα1 but does notrule out an eventual implication of TRβ1.Several lines of evidence suggest that Klf9 is a direct tran-

scriptional target of T3 (17). Here, we show that this is also likelyto be the case in PCs. The Klf9 gene encodes a transcriptionfactor that, together with Klf4, regulates the developmental lossof the regenerative capacity of retinal ganglion cell axons (5). Weused gain- and loss-of-function approaches to establish thatKLF9 largely mediates the T3-induced loss of PC regenerativeability during development. Altogether, our results show theinvolvement of the T3/TRα1/KLF9 pathway in the loss of PCaxon regenerative ability during development.

T3 Orchestrates a General Change in Vertebrate Brain Properties,Reducing Plasticity. The role of T3 in PC dendritic developmentand synaptogenesis has been clearly demonstrated in vivo (30,31) and in vitro (21, 32). T3 also acts on other cell types (30, 31).In particular, it promotes the differentiation of oligodendrocytes(33), which express molecules that inhibit axon growth (2, 3).Furthermore, the loss of axonal regenerative capacity coincideswith myelination (1, 34). Thus, T3 could indirectly abrogate PC

regenerative capacity by promoting oligodendrocyte differentia-tion and myelin formation. However, we have previously shownthat PCs lose their ability to regenerate their axons in the pres-ence of T3, even in the absence of oligodendrocytes (19). In ourcoculture essay, the environment in which axotomized axonelongate is independent of other conditions (i.e., with slices fromCalb1−/− being WT for TRα1 or KLF9 expression), we show thatthe action of T3 via TRα1 and KLF9 on PC regenerative capacityis cell-autonomous. The transition from the ability to the in-ability to regenerate axons is likely due to profound modifi-cations of both neuron differentiation and the environment.Interestingly, our results also reveal that this transition is per-manent, because once the P10 animals have been exposed to T3,the regenerative ability cannot be reversed by omitting T3 in theslice culture medium. Thus, T3 controls at least two importantprocesses that reduce brain plasticity. We found recently that thepromotion of myelin formation by T3 is not a cell-autonomousprocess but is secondary to some events taking place in neurons(35). T3 would thus orchestrate a complex network of cellularinteractions, which all contribute to determine the correct timingfor PCs to lose their regenerative capacity, suggesting that theloss of axonal regenerative ability could be part of a general

Fig. 4. KLF9 reduces the capacity of PCs to regen-erate their axons. (A) Graphic representation of Klf9mRNA expression in the cerebellum at P7. Shownhere is the ratio of Klf9 expression in TRαAMI/+ andTRα WT (TRα WT) mice, both in a ROSA26-lox-STOP-lox-EYFP (R26YFP)/+; Ptf1aCre/+ background. The leftbar represents YFP− cells (granule cells and othercell types), and the right bar represents YFP+ cells(PCs and interneurons; a description of the experi-ment is provided in Fig. S5). When TRα1L400R isexpressed in PCs, the Klf9 mRNA level is reduced byfourfold compared with WT (*P < 0.05, Mann–Whitney test). Photomicrographs of in situ hybrid-ization of euthyroid (Eu-TH; B) and hypothyroid(Hypo-TH; C) sections from P10 mice with a Klf9probe; the arrows point to the PC layers. Photo-micrographs of newborn WT (D) and Klf9−/− mouse(E) dorsal slices apposed to Calb1−/− ventral slices,cultured in the presence of T3, and immunostainedwith anti-Calb1 antibodies are shown. Quantitativeanalysis of the area of axon regeneration (F) andthe longest regenerative axons (G) is shown. Pho-tomicrographs of dorsal cerebellar slices transducedwith GFP-expressing Lv (LV-GFP; H) or KLF9-expres-sing Lv (LV-KLF9; I), apposed to Calb1−/− ventral slicescultured in the absence of T3, and immunostainedwith anti-Calb1 antibodies are shown. Quantitativeanalysis of the area of axon regeneration (J) and thelongest regenerative axons (K) is shown. The dottedlines indicate the site of axotomy, and the arrow-heads point to regenerating axons. Black bars rep-resent cultures grown in the presence of T3, andwhite bars represent those grown in the absenceof T3. All cultures were grown in the presence ofGö6976 at the time of axotomy to increase PC sur-vival. (Scale bars: B and C, 60 μm; D, E, H, and I,240 μm.) Values are means ± SEM (**P < 0.01, ***P <0.001 Mann–Whitney test). n = 32 WT, 41 KO, 14 Lv-GFP, and 13 Lv-KLF9.

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Page 6: Thyroid hormone triggers the developmental loss of receptor α1 … · 2012. 11. 14. · Thyroid hormone triggers the developmental loss of axonal regenerative capacity via thyroid

process of brain maturation. It remains to be shown whether thismaturation also involves other neuronal cell types.The role of T3 in terminating axon regeneration during de-

velopment seems to be conserved across many vertebrate spe-cies. Xenopus laevis, for example, displays robust regeneration ofneural structures as larvae but partially or totally loses this ca-pability after T3-induced metamorphosis (36, 37). In chicks,circulating T3 levels increase before hatching (38), and thiscoincides with the loss of axon regenerative capacity (39). Birdsare classified as precocial or altricial according to their degree ofmaturation and physiological capabilities at hatching. In pre-cocial birds, thyroid function is already well developed during thelater part of incubation and hatchings have relatively maturesensory and locomotor capabilities, whereas thyroid maturationoccurs mainly after hatching in altricial birds, as is also the casewith their sensory and motor functions (38). Interestingly, insheep, which are mature at birth, the increase in circulating T3levels occurs before birth (40) and axon regenerative ability islost in the late embryonic period (41). This implies that humanslikely lose their axon regenerative ability before birth, becausethe T3 peak occurs prenatally (40).Thus, our results indicate that the physiological T3 burst in

mammals is involved in the developmental loss of PC axon

regenerative capacity. This effect may be part of a more generalprocess of T3-dependent maturation that readies the animal forits postnatal environment.

Materials and MethodsA complete description of the material and methods used in this study isprovided in SI Materials and Methods.

Animals. All animal procedures were approved by the Ile de France EthicsCommittee (p3/2009/020).

Slice Culture. Cerebellar organotypic cultures were prepared from newborn(P0) and P10 Swiss mice as previously described (19, 32). Each experiment wasperformed three times with at least four animals each time. For each ex-periment, the number of slices is given in the figure legend, together withthe statistic tests.

ACKNOWLEDGMENTS. We thank Teddy Fauquier for providing variousmouse lines and Isabelle Caillé, Ekrem Dere, Fatiha Nothias, and Alain Trem-bleau for helpful discussions. This work was supported financially by theCentre National de la Recherche Scientifique, Université Pierre et Marie Cu-rie (Grant ANR-07-NEURO-043-01) and International Foundation for Re-search in Paraplegia.

1. Schwab ME, Bartholdi D (1996) Degeneration and regeneration of axons in the le-sioned spinal cord. Physiol Rev 76:319–370.

2. Schwab ME (2004) Nogo and axon regeneration. Curr Opin Neurobiol 14(1):118–124.3. He Z, Koprivica V (2004) The Nogo signaling pathway for regeneration block. Annu

Rev Neurosci 27:341–368.4. Cai D, et al. (2001) Neuronal cyclic AMP controls the developmental loss in ability of

axons to regenerate. J Neurosci 21:4731–4739.5. Moore DL, et al. (2009) KLF family members regulate intrinsic axon regeneration

ability. Science 326:298–301.6. Liu K, Tedeschi A, Park KK, He Z (2011) Neuronal intrinsic mechanisms of axon re-

generation. Annu Rev Neurosci 34:131–152.7. Li D, Field PM, Raisman G (1995) Failure of axon regeneration in postnatal rat en-

torhinohippocampal slice coculture is due to maturation of the axon, not that of thepathway or target. Eur J Neurosci 7:1164–1171.

8. Chen DF, Jhaveri S, Schneider GE (1995) Intrinsic changes in developing retinal neuronsresult in regenerative failure of their axons. Proc Natl Acad Sci USA 92:7287–7291.

9. Dusart I, Airaksinen MS, Sotelo C (1997) Purkinje cell survival and axonal regenerationare age dependent: an in vitro study. J Neurosci 17:3710–3726.

10. Gianola S, Rossi F (2001) Evolution of the Purkinje cell response to injury and re-generative potential during postnatal development of the rat cerebellum. J CompNeurol 430(1):101–117.

11. Oishi Y, Baratta J, Robertson RT, Steward O (2004) Assessment of factors regulatingaxon growth between the cortex and spinal cord in organotypic co-cultures: Effects ofage and neurotrophic factors. J Neurotrauma 21:339–356.

12. Morreale de Escobar G, Calvo R, Escobar del Rey F, Obregón MJ (1994) Thyroid hor-mones in tissues from fetal and adult rats. Endocrinology 134:2410–2415.

13. Gauthier K, et al. (1999) Different functions for the thyroid hormone receptorsTRalpha and TRbeta in the control of thyroid hormone production and post-nataldevelopment. EMBO J 18:623–631.

14. Hadj-Sahraoui N, Seugnet I, Ghorbel MT, Demeneix B (2000) Hypothyroidism prolongsmitotic activity in the post-natal mouse brain. Neurosci Lett 280(2):79–82.

15. Kress E, Samarut J, Plateroti M (2009) Thyroid hormones and the control of cellproliferation or cell differentiation: Paradox or duality? Mol Cell Endocrinol 313(1-2):36–49.

16. Martel J, Cayrou C, Puymirat J (2002) Identification of new thyroid hormone-regu-lated genes in rat brain neuronal cultures. Neuroreport 13:1849–1851.

17. Denver RJ, Williamson KE (2009) Identification of a thyroid hormone response ele-ment in the mouse Kruppel-like factor 9 gene to explain its postnatal expression inthe brain. Endocrinology 150:3935–3943.

18. Ghoumari AM, Wehrlé R, De Zeeuw CI, Sotelo C, Dusart I (2002) Inhibition of proteinkinase C prevents Purkinje cell death but does not affect axonal regeneration. JNeurosci 22:3531–3542.

19. Bouslama-Oueghlani L, Wehrlé R, Sotelo C, Dusart I (2003) The developmental loss ofthe ability of Purkinje cells to regenerate their axons occurs in the absence of myelin:An in vitro model to prevent myelination. J Neurosci 23:8318–8329.

20. Yen PM (2001) Physiological and molecular basis of thyroid hormone action. PhysiolRev 81:1097–1142.

21. Heuer H, Mason CA (2003) Thyroid hormone induces cerebellar Purkinje cell dendriticdevelopment via the thyroid hormone receptor alpha1. J Neurosci 23:10604–10612.

22. Wallis K, et al. (2010) The thyroid hormone receptor alpha1 protein is expressed inembryonic postmitotic neurons and persists in most adult neurons.Mol Endocrinol 24:1904–1916.

23. Bradley DJ, Young, WS, 3rd, Weinberger C (1989) Differential expression of alphaand beta thyroid hormone receptor genes in rat brain and pituitary. Proc Natl AcadSci USA 86:7250–7254.

24. Quignodon L, Vincent S, Winter H, Samarut J, Flamant F (2007) A point mutation inthe activation function 2 domain of thyroid hormone receptor alpha1 expressed afterCRE-mediated recombination partially recapitulates hypothyroidism. Mol Endocrinol21:2350–2360.

25. Panaite P-A, Barakat-Walter I (2010) Thyroid hormone enhances transected axonalregeneration and muscle reinnervation following rat sciatic nerve injury. J NeurosciRes 88:1751–1763.

26. Venero C, et al. (2005) Anxiety, memory impairment, and locomotor dysfunctioncaused by a mutant thyroid hormone receptor alpha1 can be ameliorated by T3treatment. Genes Dev 19:2152–2163.

27. Fauquier T, et al. (2011) Severe impairment of cerebellum development in mice ex-pressing a dominant-negative mutation inactivating thyroid hormone receptor al-pha1 isoform. Dev Biol 356:350–358.

28. Hashimoto K, et al. (2001) An unliganded thyroid hormone receptor causes severeneurological dysfunction. Proc Natl Acad Sci USA 98:3998–4003.

29. Portella AC, et al. (2010) Thyroid hormone receptor β mutation causes severe im-pairment of cerebellar development. Mol Cell Neurosci 44(1):68–77.

30. Oppenheimer JH, Schwartz HL (1997) Molecular basis of thyroid hormone-dependentbrain development. Endocr Rev 18:462–475.

31. Koibuchi N (2008) The role of thyroid hormone on cerebellar development. Cere-bellum 7:530–533.

32. Boukhtouche F, et al. (2010) Induction of early Purkinje cell dendritic differentiationby thyroid hormone requires RORα. Neural Dev 5:18.

33. Barres BA, Lazar MA, Raff MC (1994) A novel role for thyroid hormone, glucocorti-coids and retinoic acid in timing oligodendrocyte development. Development 120:1097–1108.

34. Filbin MT (2003) Myelin-associated inhibitors of axonal regeneration in the adultmammalian CNS. Nat Rev Neurosci 4:703–713.

35. Picou F, Fauquier T, Chatonnet F, Flamant F (2012) A bimodal influence of thyroidhormone on cerebellum oligodendrocyte differentiation.Mol Endocrinol 26:608–618.

36. Beattie MS, Bresnahan JC, Lopate G (1990) Metamorphosis alters the response tospinal cord transection in Xenopus laevis frogs. J Neurobiol 21:1108–1122.

37. Gibbs KM, Chittur SV, Szaro BG (2011) Metamorphosis and the regenerative capacityof spinal cord axons in Xenopus laevis. Eur J Neurosci 33:9–25.

38. McNabb FMA (2006) Avian thyroid development and adaptive plasticity. Gen CompEndocrinol 147(7):93–101.

39. Hasan SJ, Keirstead HS, Muir GD, Steeves JD (1993) Axonal regeneration contributesto repair of injured brainstem-spinal neurons in embryonic chick. J Neurosci 13:492–507.

40. Fisher DA, Polk DH, Wu SY (1994) Fetal thyroid metabolism: A pluralistic system.Thyroid 4:367–371.

41. Meuli-Simmen C, et al. (1996) The fetal spinal cord does not regenerate after in uterotransection in a large mammalian model. Neurosurgery 39:555–560, discussion 560–561.

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