O
RIGINAL
A
RTICLE
Noggin Elicits Retinal Fate in
Xenopus Animal Cap Embryonic
Stem Cells
AQ1
LEILAN,aANTONIOVITOBELLO,aMICHELEBERTACCHI,aFEDERICOCREMISI,a ROBERTVIGNALI,a MASSIMILIANO
ANDREAZZOLI,a GIANCARLODEMONTIS,bGIUSEPPINABARSACCHI,a SIMONACASAROSA
AQ27 a,c
aDipartimento di Biologia, Universita` degli Studi di Pisa, Pisa, Italy;bDipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` degli Studi di Pisa, Pisa, Italy;cCentre for Integrative Biology (CIBIO), Universita` di Trento, Mattarello, Italy
Key Words. Cell differentiation•Retina•Noggin•Stem cells
ABSTRACT
Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the fac-tors necessary to drive multipotent stem cells toward reti-nal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a sin-gle secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells.
AQ2 Reverse-transcription polymerase
chain reaction,
AQ3 immunohistochemistry, and in situ hybrid-ization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro.
Fol-lowing in vivo transplantation, ACES cells expressing AQ4 high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology andc-fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells. STEM
CELLS2009; 000: 000–000 Disclosure of potential conflicts of interest is found at the end of this article.
I
NTRODUCTIONDriving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Cells of the animal cap of the blastula stage Xenopus embryo may be similar to mammalian embryonic stem cells. They are multipotent, and can be diverged from their default ectodermal state by the action of inducing tissues or growth factors, giving rise to neuroectoderm, mesoderm, or endoderm [1]. Animal caps can generate anterior brain structures, including eye cups, under the action of specific inducing signals [2]. The expres-sion of the neural inducer
AQ5 noggin in animal cap embryonic
stem (ACES) cells activates neural markers [3] and eye field transcription factors [4] (EFTFs). Additionally, EFTF over-expression in early Xenopus embryos induces ectopic eyes outside the nervous system [4]. Besides its ability to activate EFTF expression in ACES cells, little is known about the
capability of Noggin to induce retinal fates, either in vitro or in vivo. In whole embryos, reducing bone morphogenic pro-tein (BMP) signaling by expression of a dominant-negative BMP receptor or by noggin overexpression allows blasto-meres that normally do not contribute to the retina to do so [5]. Thus, the initial step in the retinal lineage appears to be regulated by position within the BMP/Noggin field of epider-mal versus neural induction. For what concerns in vitro stud-ies, its function during mammalian embryonic stem (ES) cell AQ6 differentiation is not completely clear. It surely enhances neural differentiation in many different protocols [6], but what is its role in retinal differentiation is more controversial [7, 8]. Thus, the capability of Noggin to bring to completion the developmental pathway leading to retinal differentiation has not been fully understood yet. We therefore decided to investigate the retina-forming potential of Xenopus ACES cells expressing different doses of noggin, both in vitro and in in vivo transplantations.
Author contributions: L.L. and M.B.: Collection and assembly of data, data analysis and interpretation; A.V.: Conception and design, collection and assembly of data, data analysis and interpretation; F.C.: Data analysis and interpretation, manuscript writing; R.V.: Data analysis and interpretation, financial support; M.A.: Contribution of reagents, data analysis and interpretation, manuscript writing; G.B.: Data analysis and interpretation, financial support, manuscript writing; G.C.D.: Collection and assembly of data, data analysis and interpretation, manuscript writing; S.C.: Conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing.
Correspondence: Simona Casarosa,
AQ26 Ph.D., Centre for Integrative Biology CIBIO, Via delle Regole 101, 38123 Mattarello (TN), Italy. Telephone: +39-0461-882745; Fax: +39-0461-883937; e-mail: [email protected]; Website: http://portale.unitn.it/cibio Received December 8, 2008; accepted for publication June 26, 2009; first published online inSTEMCELLSEXPRESSMonth 00, 2009.VC AlphaMed
Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.167
S
TEMC
ELLS2009;000:000–000 www.StemCells.com
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M
ATERIALS ANDM
ETHODS EmbryoAQ22
preparation and staging, microinjections, in situ hybrid-ization,
AQ23
and immunohistochemistry were performed according to Casarosa et al. [9]. Thenoggin construct used for microinjections is pCS2-Xnoggin (kind gift of R. Harland [10]). Embryos were comicroinjected with in vitro transcribed noggin and GFP mRNAs (mMessage Machine;
AQ7 Ambion, Austin, TX, http:// www.ambion.com;
AQ8 Applied BioSystems, Foster City, CA, http:// www.appliedbiosystems.com), in both blastomeres of two-cell stage embryos. Thehermes [11], vsx1 [12], c-fos [13], Rx1 [9], Xotch [14], Nkx2.4 [15], and Emx1 [16] probes were used for in situ hybridization. The following antibodies were used for immu-nohistochemistry: anti-Islet-1 (1:50; Developmental Studies Hy-bridoma Bank), anti-otx2 [17] (1:200), anti-opsin (1:500; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and rhoda-mine-conjugated anti-mouse and anti-rabbit antibodies (1:1000; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). For histological observations, paraffin sections of stage-43 trans-planted embryos were stained with hematoxylin and eosin (H&E).
Animal caps were prepared and cultured according to Zuber et al. [4]. To visualize and count proliferating cells, animal caps were cultured in 1 MBS (10 MBS: NaCl 0.88 M, KCl 0.01 M, MgSO48 mM, HEPES 0.1 M, NaHCO30.024 M; gentamicin
50 mg/ml) to which 30lMEdU was added from stage 36. Ani- AQ9 mal caps were then collected and analyzed either 2 or 10 hours later. Detection of EdU was performed according to the manufac-turer’s instructions (Click-iT EdU imaging kits; Molecular AQ10 Probes, Invitrogen). For transplantations, animal caps were cul-tured until stage 15, then cut in half and grafted in the appropri-ate location of the isochronic host embryo. Host embryos always came from the same fertilization as the animal caps. Prior to the graft, either one of the bilateral eye fields (eye field transplants) or a portion of the posterior one fourth of the neural plate (poste-rior transplants) was removed. Transplanted embryos were cul-tured on agar-coated plates in 1 MBS (7 ml/ltof CaCl20.1 M AQ11 was added prior to use) for 24 hours at 18C, then transferred to 0.1 MMR (10 MMR: NaCl 1 M, KCl 0.02 M, MgSO4 10 AQ12 mM, HEPES 0.05 M, EDTA 1 mM; gentamicin 50lg/ml) until stage 43. To analyze responses to light, embryos were grown on a 12-hour light/12-hour dark cycle (LD 12:12) starting immedi-ately after transplantation, then analyzed at stage 43 forc-fos in
C O L O R Figure 1. In
AQ21 vitro analysis ofnoggin-injected animal cap embryonic stem (ACES) cells by reverse-transcription polymerase chain reaction (RT-PCR) (A, B), EdU incorporation, immunohistochemistry, and in situ hybridization (C–J). (A): Multiplex RT-PCR of terminal differentiation reti-nal markers. Onlynoggin doses higher than 15 pg elicit expression of markers of specific retinal cell types in ACES cells. Expression of the gen-eral neuronal markerSox2 is activated by all doses of noggin tested, showing that noggin correctly neutralizes ACES cells. (B): Multiplex RT-PCR of retinal progenitor markers. All doses ofnoggin tested induce the expression of retinal progenitor proliferation and differentiation markers. Pax2, vax2, and zic2 were tested as molecules implicated in retinal dorsoventral patterning [21]. (C, D): EdU incorporation (in red; GFP in green); (E, F) opsin immunohistochemistry (in red; GFP in green); (G, H) hermes in situ hybridization (in red; GFP in green); (I, J) vsx1 in situ hybridization (in blue). (C, E, G, I): 2.5-pg noggin ACES cells; (D, F, H, J) 20-pg noggin ACES cells. Abbreviations: EdU, ___; GFP, green fluorescent protein; P, pigment;þRT, with RT-PCR; RT, without RT-PCR; uninj.: noninjected embryos; we: whole embryos.
situ hybridization and at stage 46 for electrophysiology, as described. Care of the animals was in accordance with institu-tional guidelines. Images were obtained using epifluorescence mi-croscopy and stereomimi-croscopy (Nikon Eclipse E600 and Nikon SMZ1500 [Tokyo, http://www.nikon.com]) connected with a digi-tal camera (Photometrix CoolSnap; Roper Scientific, Trenton, NJ, http://www.roperscientific.com).
For multiplex reverse-transcription polymerase chain reaction (RT-PCR), animal caps were cultured in 1 MMR until stage 39, then snap frozen. Total RNAs were extracted with Trizol (Invitro-gen), treated with RQ1 DNAse (Promega, Madison, WI, http:// www.promega.com), and reverse-transcribed by Superscript III Reverse Transcriptase (Invitrogen) in the presence of poly-T pri-mers, in a total volume of 20 ll. Multiplex RT-PCR was then performed using HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany, http://www1.qiagen.com) following the protocol of Klink et al. [18], with minor modifications. A list of the primers used can be found in the supplemental online data.
For electrophysiology, animals were dark-adapted overnight, and eyes dissected under dim red light. Retinae were finely chopped and stored for 90 minutes in darkness in Ringer solution (NaCl 113 mM, KCl 2.5 mM, CaCl2 1 mM, MgCl2 1.6 mM,
EDTA 0.01 mM, glucose 10 mM, Hepes 3 mM, pH 7.7, with NaOH) with 30 U/ml DNAse (Sigma-Aldrich) and 200 nM 9-cis retinal (Sigma-Aldrich) to promote rhodopsin generation from free opsin. Retinal fragments were then transferred to the recording chamber and responses to light acquired using the suction pipette technique [19]. Recording pipettes were drawn from borosilicate glass using a two-stage air-cooled puller (
AQ13 BB-CH; Mecanex,
Ge-neva, Switzerland). Pipettes were filled with Ringer and connected to a
AQ14 LIST EPC-7 amplifier through a silver chloride-coated silver wire. Currents were filtered at 50 Hz before sampling at 1 kHz by a Digidata 1200 (Axon Instruments/Molecular Devices Corp.,
Union City, CA, http://www.moleculardevices.com). Light stimuli were generated by a green light-emitting diode (LED; Optodiode AQ15 520 L) driven by the digital to analog output of the Digidata. The LED was mounted on the second camera port of the microscope and the emitted light focused on the preparation by the60 objec-tive as a 1.2-mm-diameter circular spot.
R
ESULTS ANDD
ISCUSSION AQ16 Xenopus laevis embryos at two-cell stage were microinjected in both blastomeres with noggin and GFP mRNAs. Animal caps from embryos overexpressing different doses of noggin were explanted at blastula stages (stage 9) and in vitro cul-tured until stage 39 (a stage in which embryonic retinogenesis is almost complete). RT-PCR for retinal differentiation markers showed that all noggin doses tested (2.5 pg to 20 pg mRNA) were able to activate in ACES cells general neuronal markers such as Sox2 [20] (Fig.1A) and genes expressed in F1 proliferating, undifferentiated retinal precursors such as CycD1, Xotch, Xash1, and NeuroD [9] (Fig. 1B). However, only dosages of 15 pg or more ofnoggin mRNA were able to elicit the expression of terminal differentiation markers of specific retinal cell types, such asXbh1 and Brn3d (ganglion cells [22]), Prox1 (horizontal cells [23]), and opsin and NRL (photoreceptors [24]) (Fig. 1A).Immunohistochemistry and in AQ17 situ hybridization experiments showed similar results, as only ACES cells injected with highnoggin doses expressed opsin protein as well ashermes and vsx1 mRNAs (markers of pho-toreceptors, ganglion cells [11], and bipolar cells [12]: FiguresTable 1. The percentage of 20-pg noggin-injected ACES cells expressing the different markers
Marker Cells labeled No. of positive explants Total no. of cells No. of positive cells % of positive cells
N-tubulin Differentiated neurons 9/9 5707 2782 48.75 hermes Ganglion cells 12/12 2625 512 19.50
Opsin Photoreceptors 18/18 5211 947 18.17
vsx1 Progenitors and bipolar cells 9/9 4775 2034 42.60 Xotch Proliferating progenitors 18/18 5008 1622 32.39 EdU Proliferating cells 7/7 4464 1293 28.97 The total number of cells in each explant was estimated by Hoechst nuclear staining.
C O L O R
Figure 2. In vivo analysis ofnoggin-injected animal cap embryonic stem (ACES) cells. (A--C): Eye field transplants; (D--F) posterior trans-plants. (A, D): GFP ACES cells. Insets show sections at the level of the transplant, showing incorporation of GFP-expressing ACES cells in the epidermis; (B, E) 2.5-pg noggin ACES cells; (C, F) 20-pg noggin ACES cells. Abbreviation: GFP, green fluorescent protein.
1E, 1F; Figure 1G, 1H; and Figure 1I, 1J, respectively). Table T1 1 shows frequencies of marker staining and sample sizes. A percentage of ACES cells expressing high noggin doses (Table 1) was also found to proliferate, as seen by incorpora-tion of EdU (Fig. 1C, 1D) and by the expression ofRx1 and Xotch, retinal progenitor markers [9, 14] (data not shown and supplemental online Fig. 1A). Notably, ACES cells expressing high noggin doses do not express markers of other types of forebrain neurons, such asNkx2.4 [15] and Emx1 [16] (0/30 explants in both cases; supplemental online Fig. 1B, 1C). We can thus propose that all the cells that compose the 20-pg noggin-expressing explants are committed to the retinal
line-age, and are at different levels of differentiation. We perform our analysis at stage 39 (it is difficult to culture these explants for longer periods) so theXotch-positive cells we find can be considered (at least in part) as Mu¨ller glia progenitors, since this cell type is not yet differentiated at this stage [25]. Although the expression pattern of the analyzed markers is variable, a trend is apparent, with opsin expressed predomi-nantly in the external layer of the caps;hermes, in an internal concentric site; and vsx1, with a more diffuse pattern that includes also the area between opsin and hermes (Fig. 1F, 1H, 1J). This cell distribution is reminiscent of retina lamina-tion and suggests that noggin-injected caps may display a
Table 2. Percentages of embryos with incomplete and complete (% eye with lens) eyes and of embryos where no effect was obtained, following the transplantation of noggin-expressing ACES cells
Noggin dose/site of transplant No. of embryos % incomplete eye % eye with lens % no effect
2.5 pg/eye field 94 11.70 6.38 81.92
20 pg/eye field 189 13.76 73.54a 12.70
2.5 pg/posterior 88 17.05 0 82.95
20 pg/posterior 135 93.33a 0 6.67
The dose of noggin mRNA used to inject ACES cells and the site of transplant are indicated.
a p< .001, Z test (vs. no effect). C O L O R
Figure 3. In vivo analysis ofnoggin-injected animal cap embryonic stem cells by immunohistochemistry and in situ hybridization. (A, B, I, J): Opsin immunohistochemistry marking photoreceptors (in red; GFP in green); (C, D, K, L) Otx2 immunohistochemistry marking bipolar cells (in red; GFP in green); (E, F, M, N) Islet1 immunohistochemistry (in red; GFP in green); (G, H, O, P) hermes in situ hybridization marking gan-glion cells. (A, C, E): 2.5-pg noggin eye field transplants; (B, D, F) 20-pg noggin eye field transplants; (G, I, K) 2.5-pg noggin posterior trans-plants; (H, J, L) 20-pg noggin posterior transplants. Abbreviation: GFP, green fluorescent protein.
basic retinal patterning. In any case, these data strongly sug-gest that treatment with high doses of noggin is able to start a developmental program exclusively leading to retinal differentiation.
We then tested the retina-forming potential of noggin-expressing ACES cells in vivo. To this purpose, one of the presumptive eye fields was removed from a neurula stage embryo (stage 15) and replaced with isochronic ACES cells expressing either GFP alone or differentnoggin dosesþ GFP. Removal of one of the presumptive eye fields at this develop-mental stage prevents eye formation (as seen in Fig.
F2 2A).
Ec-topic posterior transplantations were also performed, replacing a portion of the posterior neural plate with ACES cells. GFP-expressing ACES cells integrated in the host embryos. Indeed, eye field transplants integrated in the head epidermis, showing a localized fluorescence, whereas posteriorly transplanted ACES cells distributed extensively along the tail epidermis (Fig. 2A, 2D; sections are shown in the insets). In no cases (0/146) were GFP-expressing ACES cells detected in the neu-ral tissue, and they never expressed neuneu-ral markers (data not shown). Transplants obtained from low noggin-expressing ACES cells were
AQ18 neutralized, as they expressed the neural markerb-tubulin [26] (in situ hybridization; data not shown), but eye field transplants did not rescue eye formation in the vast majority of cases (81.92%). Only a low percentage of embryos showed a poorly differentiated eye-like structure (6.38%) or pigment condensation (11.70%) (Fig. 2B and Table
T2 2). Eye-like structures were never formed in posterior transplants (Fig. 2E and Table 2). Conversely, transplantations made with high noggin-expressing ACES cells formed eyes. Eye field transplants showed complete eyes with a lens (73.54%; Fig. 2C, Table 2, and
AQ19 Fig. 4A, 4B). The formation
of an almost complete eye was also frequent in posterior transplants (93.33%; Fig. 2F, Table 2, and Fig. 4C, 4D), even if in this ectopic location a lens was never formed (no
expres-sion ofb-crystallin [27] as seen by in situ hybridization; data not shown). The expression of retinal terminal differentiation markers was consistent with the morphological features of the different phenotypes. The eyes formed by high noggin-expressing ACES cells always expressed such markers both in the eye field and in the posterior transplants (Fig.3B, 3D, 3F, F3 3H, 3J, 3L, 3N, 3P). On the contrary, the low noggin-express-ing ACES cells never expressed retinal differentiation markers in the posterior transplants, and at a low percentage in the eye field transplants, this percentage corresponds to those trans-plants showing the formation of an eye-like structure (Fig. 3A, 3C, 3E, 3G, 3I, 3K, 3M, 3O and supplemental online Fig. 2). This possibly means that differentiation, at least after a certain level, goes in parallel with organization of structure and morphogenesis, as is the case in other tissues [28]. H&E staining of paraffin sections of the eyes formed by high nog-gin-expressing ACES cells shows a considerable level of or-ganization and structure, not only in the eye field transplants (Fig. 4A, 4B) but also in the posterior transplants (Fig. 4C, F4 4D), where fiber layers and morphologically differentiated photoreceptors (arrows and asterisks in Fig. 4D) are present.
Finally, we analyzed the functionality of the eyes originat-ing from the eye field transplants of high noggin-expressing ACES cells. Suction pipette recordings from single-rod photo-receptors show similar dark current amplitudes (i.e., current influx through cGMP-gated channels), kinetics, and sensitivity to light in wild-type (Fig.5A, 5C) and 20-pgnoggin-express- F5 ing eye field transplants (Fig. 5B, 5D). To demonstrate the an-atomical and functional integrity of the different layers of the retina, we performed c-fos in situ hybridization experiments on retinae of wild-type (Fig. 5E, 5F) and 20-pg noggin-expressing eye field transplants (Fig. 5G, 5H) fixed during the light (Fig. 5E, 5G) or the dark (Fig. 5F, 5H) period of a 12-hour light/12-12-hour dark cycle (LD 12:12). It is known that, in the retina of animals grown on a LD 12:12 cycle, the
C O L O R
Figure 4. Histological analysis (H&E staining) of the 20-pgnoggin transplants. (A, B): Eye field transplants. The resulting eyes show morpho-logically differentiated cell types, a laminated retinal sheet, and fiber layers between the nuclear layers. (C, D): Posterior transplants. Even if overall organization is not complete, cells are organized in nuclear layers divided by fiber layers (arrows in [D]) and morphological differentiation of photoreceptors is evident (asterisks in [D]). Abbreviations:
AQ24 CMZ, _____; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexi-form layer; ONL, outer nuclear layer; OPL, outer plexiplexi-form layer.
immediate early gene c-fos is transiently expressed in the inner cell layer (INL) and in the ganglion cell layer (GCL) starting from 30 minutes after the onset of light, whereas it is completely absent in these layers during the dark period [29]. This activation can be seen as an indication of the transduc-tion of the light stimulus from the photoreceptors to second-order neurons in the retina. Our data show that the retinae originating from the transplants behave identically to wild-type retinae, that is, they activatec-fos mRNA expression af-ter the onset of light in the INL and GCL, when grown on a LD 12:12 cycle. Taken together, our data show that the eyes originating from the 20-pgnoggin-expressing eye field
trans-plants are able to respond to light, and in this respect are thus functionally indistinguishable from normal eyes.
C
ONCLUSIONSOur data bring a novel contribution to the field of retinal differ-entiation, as we show here for the first time that Noggin is able to start a process of terminal differentiation specifically into retinal neurons, and not into other types of forebrain neurons. The significance of our data is twofold. First, noggin-induced ACES cells can activate markers of terminal differentiation of
C O L O R Figure 5. Functional properties of 20-pg noggin-expressing eye field transplants. (A, B): A rod photore-ceptor outer segment projecting from a retinal fragment is shown inside the recording pipette for either a stage-46 wild-type (wt)Xenopus (A) or a time-matched 20-pg noggin-expressing eye field transplant (B). Images were acquired under infrared (k>850 nm) light using a cooled charge-coupled device camera (Leica DFC-350FX; Heerbrugg, Switzer-land, http://www.leica.com) mounted on an inverted microscope (Leica DMI 4000B).(C, D): Sweeps plot the time course of the inward dark cur-rent suppression by light flashes of increasing intensities for either a stage-46 wt Xenopus (C) or for a time-matched 20-pg noggin-express-ing eye field transplant (D). The smooth lines above the noisy records plot the time course of the 10-ms-long light stimulus. Stimuli inten-sities were 1, 1.5, 22, and 170 pho-tonslm2flash1. Traces in (C) and (D) are the average of 9 or 4 sweeps, respectively. Dark current amplitudes (i.e., number of cGMP-gated chan-nels open in the dark) and light sensi-tivities were similar for wt and transplanted rods. Qualitatively simi-lar results were obtained in addi-tional seven wt and eight transplanted rods. (E–H): c-fos in situ hybridization on wild-type (E, F) and 20-pg noggin-expressing eye field transplants (G, H) collected in light (E, G) or dark (F, H). Both wild-type and transplanted retinae show c-fos expression in the inner nuclear layer and ganglion cell layer only during the light period. Abbre-viations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer;pA, ___; s, seconds. AQ25
retinal cell types and support the formation of functionally nor-mal eyes, capable of responding to light. Thus, in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without addi-tional cues. Second, this makesXenopus ACES cells a model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells.
A
CKNOWLEDGMENTSWe thank Y. Bozzi for comments, and M. Fabbri, G. De Mat-ienzo, and S. Di Maria for technical help. This work was
sup-ported by Telethon-Italy (Grant GGP07275). This work was also supported by Ministero Affari Esteri Cooperazione Italia-Cina RP (NFNS 30370453) and Convenzione di Cooperazione Interuniversitaria Internazionale Pisa-Pechino to G.B., and Cofinanziamento PRIN-Universita` di Pisa to G.B. and R.V. L.L. and A.V. contributed equally to this work. A.V. is cur-rently affiliated with Friedrich Miescher Institute for Biomedi-cal Research, Basel, Switzerland.
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ISCLOSURE OFP
OTENTIALC
ONFLICTS OFI
NTERESTThe authors indicate no potentialconflicts of interest. AQ20
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