Neural crest regulates myogenesis through the transient ... - Nature

LETTER

doi:10.1038/nature09970

Neural crest regulates myogenesis through the transient activation of NOTCH Anne C. Rios1, Olivier Serralbo1*, David Salgado1* & Christophe Marcelle1

human histone H2B (H2B)–RFP reporter gene driven by an ubiquitous promoter to evaluate the normal distribution of electroporated cells. After 24 h, 11% of H2B–RFP-positive cells were HES1–d2EGFPpositive (Fig. 1c, d and Supplementary Fig. 2l). The H2B–RFP-positive cells were distributed among PAX7- (60%), MYF5- (46%) and MyHCpositive (10%) populations. In contrast, nearly all (92%) HES1-positive cells were MYF5-positive (distributed in the DML and the transition zone), whereas only 24% and 2% expressed PAX7 and MyHC, respectively (Fig. 1e–i and Supplementary Fig. 3a, b). We followed the morphogenic movements of NOTCH-activating cells using live video microscopy. Epithelial cells that activated the NOTCH reporter in the DML rapidly translocated in the transition zone (Fig. 1j–l and

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How dynamic signalling and extensive tissue rearrangements interact to generate complex patterns and shapes during embryogenesis is poorly understood1–3. Here we characterize the signalling events taking place during early morphogenesis of chick skeletal muscles. We show that muscle progenitors present in somites require the transient activation of NOTCH signalling to undergo terminal differentiation. The NOTCH ligand Delta1 is expressed in a mosaic pattern in neural crest cells that migrate past the somites. Gain and loss of Delta1 function in neural crest modifies NOTCH signalling in somites, which results in delayed or premature myogenesis. Our results indicate that the neural crest regulates early muscle formation by a unique mechanism that relies on the migration of Delta1-expressing neural crest cells to trigger the transient activation of NOTCH signalling in selected muscle progenitors. This dynamic signalling guarantees a balanced and progressive differentiation of the muscle progenitor pool. Early skeletal muscle (the primary myotome, which is composed of mononucleated post-mitotic muscle fibres, or myocytes) is formed from the generation of muscle cells at the four borders of the dermomyotome, the dorsal-most epithelial compartment of somites4–10. Most of the dermomyotome undergoes an epithelial to mesenchymal transition that leads to the emergence of a population of resident muscle progenitors that massively contributes to the growth of all trunk muscles11–14. The medial border of the dermomyotome, the dorsomedial lip (DML), remains epithelial for a considerable period of time, during which it generates muscle cells that contribute to the growth of the primary myotome. DML stem/progenitor cells can adopt two fates during the first days of embryonic muscle development4–6: to self-renew and remain in the epithelial border of the dermomyotome or to translocate in the myotome and undergo terminal myogenic differentiation. How this balance is regulated is unknown. In the chick embryo, the epithelial DML population comprises a majority (77%) of PAX7-positive cells interspersed by (23%) PAX7/ MYF5-positive cells (Supplementary Figs 1a–e, 2a–e). In the transition zone, cells shut-off the expression of PAX7, but maintain MYF5 expression. Fully elongated myocytes express skeletal muscle myosin heavy chain (MyHC; also known as MYC). NOTCH family members are expressed in the DML, the transition zone and the myotome during the first phase of myogenesis (Fig. 1a)15. The NOTCH target genes HES1/cHairy2 and lunatic fringe (LFNG) are expressed in a salt-andpepper pattern within the DML. Many transition zone cells express HES1, whereas LFNG expression is low in this region. Their expression is low in the myotome (Fig. 1a, b and Supplementary Fig. 2f). Both genes act as bona fide NOTCH targets in somites, as their messenger RNA expression is upregulated after electroporation of a constitutive form of NOTCH (NOTCH intracellular domain (NICD); Supplementary Fig. 2g–i, m–o). To quantify the distribution of NOTCH activity, we electroporated a NOTCH reporter construct consisting of the mouse Hes1 promoter region upstream of a destabilized GFP (d2EGFP; half life, 2 h), that efficiently responds to NOTCH activation and inhibition (Supplementary Fig. 2j–l). We co-electroporated a

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Figure 1 | Notch is active during early myogenesis. a, Scheme showing the expression of NOTCH signalling family members in the DML, transition zone (TZ) and myotome. Serrate2 is also known as JAGGED2. b, Expression of chick HES1/chick Hairy2 in the DML and the TZ. ISH, in situ hybridization. c–h, Confocal stacks showing the expression (in green) of a HES1–d2EGFP reporter construct and (in red) RFP (c, d), PAX7 (e, f) and MYF5 (g, h) in dorsal (d, f, h) and transverse (c, e, g) views of somites 24 h after electroporation. i, Quantification of c–h. Error bars show standard deviation (s.d.). ***P , 0.0001. j–l, Time-lapse confocal analysis (see Supplementary Movie 1) showing the translocation of two NOTCH-activating DML cells (blue and white arrowheads) into the transition zone. T0, start of the movie; T1, 4 h 50 min after the start; T2, 10 h after the start. My, myotome; NT, neural tube. Scale bars, 50 mm.

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EMBL Australia; Australian Regenerative Medicine Institute (ARMI), Monash University, Building 75, Clayton, Victoria 3800, Australia. *These authors contributed equally to this work.

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©2011 Macmillan Publishers Limited. All rights reserved

LETTER RESEARCH Supplementary Movie 1). We followed their fate as they further differentiated, using a construct that contains the Hes1 promoter region upstream of a stable GFP (EGFP half life of over 24 h). After 24 and 48 h, the proportion of MyHC-positive myocytes was more than twice (24 h: 34%; 48 h: 60%) that of control RFP-electroporated embryos (24 h: 13%; 48 h: 28%; Supplementary Fig. 3c–g), further indicating that activation of NOTCH signalling is associated with myogenesis. Altogether, this indicates that NOTCH signalling is activated in DML cells that engage in the myogenic program before they translocate into the transition zone. NOTCH signalling remains active in the transition zone and is extinguished before cells undergo terminal myogenic differentiation and elongate into myocytes. We inhibited NOTCH activity in somites using a truncated, dominant-negative form of the NOTCH transcriptional co-activator mastermind (DN MAML1)16,17 and small interfering RNAs (siRNAs) against NOTCH1 (ref. 18). DN MAML1 and the NOTCH1 siRNA gave similar results one day later, that is, a drastic reduction of myogenic differentiation (Fig. 2). This was characterized by a sharp reduction of MYF5-positive cells (7% DN MAML1 ; 3% siRNA NOTCH), compared to controls (49% CAGGS–IRES–EGFP ; 53% siRNA luciferase), and by a halt of terminal differentiation (0% MyHC-positive cells for DN MAML1 and siRNA NOTCH; controls: 12% CAGGS–IRES–EGFP; 8% siRNA luciferase; Fig. 2s, t), with no change in dermomyotome cell proliferation (Supplementary Fig. 4a–d). Virtually all cells in which a

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Figure 2 | NOTCH signalling is necessary for myogenesis. a–r, Confocal stacks of somites in dorsal (b, d, f, h, j, l, n, p, r) and transverse (a, c, e, g, i, k, m, o, q) view, 24 h after electroporation of (in green) CAGGS– EGFP as controls (a–f), DN MAML1 (g–l) and siRNA against chick NOTCH1 (m–r), and stained (in red) for PAX7 (a, b, g, h, m, n), MYF5 (c, d, i, j, o, p) and MyHC (e, f, k, l, q, r). s, Quantification of a–l. t, Quantification of m–r, siRNA against luciferase as controls are not shown. Error bars show s.d. ***P , 0.0001. Scale bars, 50 mm.

NOTCH signalling was inhibited remained epithelial in the dermomyotome (98% DN MAML1; 97% siRNA NOTCH; controls: 56% CAGGS–IRES–EGFP; 57% siRNA luciferase). Altogether, this strongly indicates that NOTCH signalling is necessary for the initial phases of myogenesis of DML cells. We induced a gain of NOTCH function by electroporating NICD in newly formed somites. One day later, most electroporated cells had translocated in the transition zone (Fig. 3a–f); however, most (83%) expressed the dermomyotomal marker PAX7 (Fig. 3a, b, g), and only a few (3%) were MYF5 positive (Fig. 3c, d, g). Although few electroporated cells entered the myotome region, they did not elongate and never (0%) initiated MyHC expression (Fig. 3e–g). This result is coherent with studies that showed that NOTCH signalling inhibits muscle differentiation in various contexts19–21. However, characterizing the electroporated cells 6 h after electroporation of NICD, we observed a robust increase in the proportion of electroporated cells expressing MYF5 (80%; controls, 17%; Fig. 3h–k and Supplementary Fig. 5n). Strong MYF5 activation was maintained 12 h after electroporation (89%; controls, 20%; Fig. 3l–o and Supplementary Fig. 5n). After 6 h, all MYF5-positive electroporated cells were positioned in the epithelial DML (Fig. 3j), at 12 h, most electroporated cells had translocated in the transition zone (Fig. 3n). The same observations were made with MYOD (Supplementary Fig. 5a–m). Altogether, this indicates that the first steps of myogenesis (the activation of MYF5 and MYOD) are promoted by a short activation of NOTCH signalling. However, a sustained activation of NOTCH reverses the myogenic program, resulting in a downregulation of MYF5 and MYOD expression and a return to a PAX7-positive state. To prove this, we used a doxycyclin-inducible system to drive NICD expression. In the continuous presence of doxycyclin, NICD expression was maintained in electroporated cells and, consistent with our previous observation (Fig. 3a–f), most of them translocated in the transition zone but did not maintain MYF5 expression (6%, Fig. 3r, s, v; controls, 42% MYF5-positive, Fig. 3p, q, v). When doxycyclin was removed, NICD was strongly expressed 6 h later, but was almost undetectable after overnight incubation (Supplementary Fig. 6c, d, f). Remarkably, after this transient activation of NOTCH signalling, most electroporated cells had translocated in the transition zone and the myotome and nearly all (97%) expressed MYF5 (Fig. 3t–v). In addition, electroporated cells that were positioned in the myotome had elongated into myocytes, indicating that they initiated terminal differentiation. The lack of electroporated cells in the DML (arrowheads in Fig. 3t) indicates a depletion of the DML progenitor cell population and suggests that the pulse of NOTCH signalling massively disrupted the balance between maintenance and differentiation of this cell population. This shows that NOTCH signalling displays a complex behaviour on myogenesis, acting as a potent stimulator of the myogenic program for DML cells, but only during a limited time window. In search for a signal controlling the mosaic activation of NOTCH we observed in the DML, we noted that neural crest cells that migrate in close proximity to the DML express the NOTCH ligand Delta1 (DLL1) in a salt-and-pepper pattern (Fig. 4a and Supplementary Fig. 8a, b). A provocative hypothesis was thus that migrating DLL1-expressing neural crest cells may activate NOTCH signalling in selected progenitors within the DML. We eliminated the neural crest cell population by electroporating into the neural tube a diphtheria toxin fragment A complementary DNA (DTA) under the control of a neural-crestspecific promoter (Supplementary Fig. 7a–f). This led to a considerable decrease in the expression of MYF5 on the electroporated side (Fig. 4b, arrowheads, and Supplementary Fig. 8c; n 5 13/15). The inhibition of non-canonical, planar cell polarity (PCP) WNT signalling in Xenopus affects neural crest migration22 without affecting its induction. In the dorsal neural tube, we electroporated a mutant form of the WNT intracellular effector Dishevelled that specifically inhibits the WNT/ PCP pathway23–25. This led to a considerable reduction in MYF5 expression compared to the control side (Fig. 4c and Supplementary 2 6 M AY 2 0 1 1 | VO L 4 7 3 | N AT U R E | 5 3 3


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Figure 3 | Myogenesis requires the transient activation of NOTCH. a–f, Confocal stacks of somites in dorsal (b, d, f) and transverse (a, c, e) view 24 h after electroporation of NICD (in green), and stained (in red) for PAX7 (a, b), MYF5 (c, d) and MyHC (e, f). g, Quantification of a–f. Error bars show s.d. ***P , 0.0001. h–o, Time-course analysis of MYF5 expression (in green) in dorsal (i, k, m, o) and transverse (h, j, l, n) view, 6 h (h–k) and 12 h (l–o) after electroporation of CAGGS–H2B–RFP as control (h, i, l, m) or HA–NICD a U2-DTA+CAAGS–EGFP

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(j, k, n, o). In red, staining for RFP (h, i, l, m) or HA (j, k, n, o). p–u, Confocal stacks of somites in dorsal (q, s, u) and transverse (p, r, t) view electroporated with a doxycyclin-expression-inducible system. Embryos were electroporated with an empty vector as controls (p, q) or HA–NICD (r, u), treated for 1 h (t, u) or overnight (p, s) with doxycyclin and stained for GFP (in green) and MYF5 (in red). v, Quantification of p–u. Scale bars, 50 mm.

Fig. 8d; n 5 10/13). We then electroporated DLL1 under the control of the neural-crest-specific promoter in the neural tube and verified that this resulted in the overexpression of DLL1 protein in neural crest cells (Supplementary Fig. 9a–c). We observed a significant increase of MYF5 expression (Fig. 4d and Supplementary Fig. 8e; n 5 9/13). Loss of DLL1 function was achieved by electroporating a dominantnegative form of DLL1 (DN DLL1)26 and with siRNAs against chick DLL1. DN DLL1 protein was expressed in neural crest cells (Supplementary Fig. 9k–m) and the siRNA construct efficiently reduced the endogenous DLL1 mRNA (Supplementary Fig. 9n–p) and protein (Supplementary Fig. 9q–s) levels. Both the DN DLL1 (n 5 17/17) and the DLL1 siRNA (n 5 10/11) resulted in a significant reduction in MYF5 staining (Fig. 4e, f and Supplementary Fig. 8f, g). Overexpression of DLL1 in neural crest resulted in a robust activation of chick HES1 mRNA expression (Supplementary Fig. 9d–f) and of the NOTCH reporter activity in somites (67%; Supplementary Fig. 9i, j), whereas electroporation of DTA or DN DLL1 led to a near loss of NOTCH reporter activity (1.6% and 1.8%, respectively, Supplementary Fig. 9g, h, j; controls: 11%, Fig. 1c, d; Supplementary Fig. 2l), strongly supporting the hypothesis that NOTCH ligands presented Figure 4 | Neural crest regulates myogenesis in somites through NOTCH signalling. a, Mosaic expression of chick DLL1 (in blue, yellow arrowheads) within the HNK1-positive (in red) neural crest population. b–f, Confocal stacks of neural tube, neural crest and somites in dorsal view, 24 h after electroporation of one half of the neural tube with U2-DTA (b), CAGGSDVLDDEP (c), U3-DLL1 (d), U2-DN DLL1 (e) (see Methods for details) and siRNA against chick DLL1 (f). In green (f) native RFP; in green (b–e) GFP immunostaining; in red, MYF5 and in blue, HNK1. Dotted lines in b–f indicate the level of transverse sections shown in Supplementary Fig. 8c–g. Scale bars, 50 mm.

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LETTER RESEARCH by neural crest cells modulate NOTCH signalling in somites. When DTA, DLL1 or DN DLL1 was electroporated into neural crest, MYOD expression was affected similarly to MYF5 (Supplementary Fig. 10), indicating that the two major molecular players of the early myogenic program are similarly regulated by DLL1 from neural crest. The proliferation of progenitors within the DML was not significantly changed in these experiments (Supplementary Fig. 11a–l), further supporting the hypothesis (Fig. 3t, u) that NOTCH signalling regulates the progressive differentiation of the muscle progenitor pool within the DML. Because neural crest emigrates from the neural tube during a limited time period (about 24 h from when migration initiates), the neuralcrest-mediated regulation of muscle growth is limited to the initial phases of myotome formation. However, this may have long-term consequences on muscle growth, as we observed significant changes in myotome growth 48 h after electroporation of DTA, DLL1 or DN DLL1 into the neural crest, that is, 24 h after crest migration has ceased (Supplementary Fig. 12a–s). As controls, we verified that the neural crest manipulations did not affect the expression of the known modulators of myotome formation in the dorsal neural tube, that is, WNT1, WNT3A and BMP4 (Supplementary Fig. 13a–l). It is unclear whether the same regulatory mechanisms are used in mouse. Hypomorph Dll1 mouse mutants displayed an enlarged primary myotome20. However, as Dll1 is expressed in both paraxial mesoderm and neural crest in early mouse embryo, the source of Notch signalling that engenders this phenotype remains to be defined. To examine this question, the inhibition of Dll1 activity in specific cell types of the mouse embryo will be required. Our model suggests an additional role of the NOTCH pathway during myogenesis whereby, within a population of DML cells all exposed to uniform gradients of myogenic activating factors, only those cells that transiently activate the NOTCH pathway undergo myogenesis. Transient NOTCH signalling is triggered by the NOTCH ligand DLL1 carried and presented by migrating neural crest cells in a ‘kiss and run’ mode of signalling transduction (Supplementary Movie 2). This links the timing of myotome formation to that of neural crest migration, providing a mechanistic link for the concurrence of these two events (Supplementary Fig. 14a–g). The ability of migrating cells to influence cell fate in neighbouring tissues may reveal a general principle for generating pulses of signal activation that result in the differentiation of a defined subset of cells within a stem or progenitor pool.

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METHODS SUMMARY Electroporation, vectors, time-lapse experiments and confocal analyses. Further details can be found in Methods. The somite electroporation technique has been described elsewhere6,27. Time-lapse experiments were performed essentially as described27 on transverse slices of embryos. Quantifications and statistical analyses. On average, more than 2,300 cells were counted per point to compute the corresponding quantifications shown in Figs 1–3 and Supplementary Figs 2–6. Statistical analyses were performed using the GraphPad Prism software. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Received 6 September 2010; accepted 24 February 2011. Published online 15 May 2011. 1. 2. 3.

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Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank N. Rosenthal and P. Currie for critical reading of the manuscript. This study was funded by grants from the Agence Nationale pour le Recherche (ANR), and by the EU 6th Framework Programme Network of Excellence MYORES. The help of P. Weber, S. Firth, C. Johnson and I. Harper from Imaging Facilities (IBDML, Marseille and MMI, Monash University) is acknowledged. Author Contributions A.C.R. and C.M conceived the experiments. A.C.R. predominantly performed the work with the help of O.S. D.S. designed the animation. C.M. supervised the project and wrote the paper. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Correspondence and requests for materials should be addressed to C.M. ([email protected]).

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RESEARCH LETTER METHODS Electroporation and confocal analysis. The somite electroporation technique that was used throughout this study has been described elsewhere6,27. Briefly, we targeted the expression of various constructs to the dorsomedial portion of newly formed interlimb somites of Hamburger–Hamilton (HH) stage 15–16 chick embryos (24–28 somite)28. We have previously shown that this technique allows the specific expression of cDNA constructs in the DML of the dermomyotome6. To target the neural crest population, we electroporated the dorsal neural tube of HH stage 13–14 chick embryos at the level of the presomitic mesoderm. The following constructs have been previously published: HES1–d2EGFP and the HES1–EGFP29 (provided by R. Kageyama) contain the mouse Hes1 promoter region upstream of destabilized or stable GFP. The CAGGS-H2B–RFP (provided by S. Tajbakhsh) contains a fusion of histone 2B with RFP downstream of the CAGGS strong ubiquitous promoter (CMV/chick b-actin promoter/enhancer). The CAGGS–EGFP30 contains the CAGGS promoter followed by the EGFP reporter gene. The pCAB-HA-NICD-IRES-GFP (provided by N. Daudet) contains an HA-tagged NICD under the control of the CAGGS promoter31. The doxycyclin inducible system is composed of two plasmids that are co-electroporated: first, the pCIRX-rtTA-IRES-DsRed32 (provided by O. Pourquie´) contains a Tet-On Advanced transactivator (rtTA, Clontech) downstream of the CAGGS promoter. The IRES-DsRed-Express allows the detection of electroporated cells. Second, the pBI-HANICD-EGFP is the response plasmid (Clontech) in which the HA-tagged constitutively active form of NOTCH, NICD, was cloned. The bidirectional tetracyclin-response element drives the expressions of EGFP (which serves as an internal control of the induced response, see Supplementary Fig. 2a, b) and HANICD. pCLGFP-DVLDDEP contains a mutated form of Xenopus Dishevelled that lacks the DEP domain, driven by the CAGGS promoter25. This construct contains also EGFP driven by its own SV40 promoter. The siRNA directed against chick NOTCH1 has been described elsewhere18 We made new constructs for this study: to construct the HES1 nVENUS-PEST, a destabilized nuclear Venus GFP variant33 was inserted downstream of the mouse Hes1 promoter region29. The CAGGS-DN MAML1–EGFP contains a truncated, dominant-negative form of the human Mastermind (DN MAML1), fused with EGFP17 downstream of the CAGGS promoter. The pCAB-HA-NICD was constructed by removing the EGFP reporter from pCAB-HA-NICD-IRES-GFP. The U2- and U3-EGFP were made by inserting the U2 and U3 evolutionary conserved Sox10 enhancer sequences34 in the TK-EGFP35 plasmid, that contains the thymidine kinase minimal promoter upstream of the EGFP. The diphtheria toxin gene36, the chick DLL1 or a dominant-negative form of this gene37 were inserted in the U2 or the U3-TK-EGFP in place of the EGFP to obtain the U2-DTA, the U2DN DLL1 and the U3-DLL1 electroporation vectors. To detect electroporated cells, those plasmids were electroporated with a pCAGGS-EGFP. We have constructed two RNA interference plasmids as described previously18 that each express two siRNAs directed against chick DLL1. Sequences TCACAGCGATA ACTCCGATAAA and TGCAGGAGTTTGTCAACAAGAA were inserted in siRNA chick DLL1 A, whereas sequences GATTCAGTATATTCCACTTCAA and CCGGCACCTTCTCGCTCATCAT were inserted in siRNA chick DLL1 B. Electroporation of plasmids A, B, or A together with B efficiently decreased the endogenous expression of chick DLL1 mRNA and protein, whereas the electroporation of siRNA directed against luciferase had no effect on chick DLL1 expression. An RFP reporter gene is inserted in the same constructs to detect electroporated cells. Antibody stainings and BrdU labelling. For BrdU labelling, embryos were incubated for 30 min with 50 ml of a 1 mg ml21 BrdU (Sigma) solution. Whole-mount antibody stainings were performed as described25. The following antibodies were used: rabbit polyclonals directed against chick myogenic regulatory factors MYF5 and MYOD38; chick DLL139; and anti-RFP (Abcam); chicken polyclonals against EGFP (Abcam); rat polyclonals against the HA tag and anti-BrdU (Abcam). We also used monoclonals against the dermomyotome and dorsal neural tube marker PAX7 and against terminal myogenic differentiation marker MyHC (MF20) (obtained from the Developmental Studies Hybridoma Bank); and the neuralcrest-specific monoclonal antibody HNK1 (provided by A. Eichmann). In situ hybridization. The following probes were used: chick HES1/cHairy2 (ref. 40) and chick37 DLL1 and chick LFNG (provided by O. Pourquie´), and 400 bp cDNA clones coding for fragments of chick WNT1, WNT3A and a 1 kb chick BMP4 probe41.

Doxycyclin-mediated induction of NOTCH signalling. Eight hours after electroporation of pCIRX-rtTA-IRES-DsRed and pBI-HANICD-EGFP, doxycyclin (300 ml of a 0.1 mg ml21 solution) was added onto the embryos, and it was either washed off after one hour with PBS for transitory upregulation of NICD, or left overnight, for permanent expression of this molecule. We verified that the response plasmid is completely silent before doxycyclin addition (that is, no EGFP expression, Supplementary Fig. 6a) while it is strongly and rapidly activated 6 h after doxycyclin addition (Supplementary Fig. 6b) Time-lapse experiments and confocal analyses. Time-lapse experiments were performed essentially as described27 on transverse slices (250 mm) of embryos. Embryo slices were filmed for 11 h at 37 uC with a confocal inverted Leica SP5 microscope equipped with a resonant scanner, at the rate of one image stack per ten minutes. Confocal images were acquired transversally over a thickness of 100 mm; Supplementary Movie 1 corresponds to a fraction (10 mm thick) of the acquired images. Dorsal views shown in Figs 1–4 are projections of stacks of confocal images. Confocal stacks of images were visualized and analysed with the Imaris software suite. Cell countings were performed using the Improvision Volocity software suite. Quantifications and statistical analyses. Electroporation results in the transfection of a portion of the targeted cell population, which is variable from embryo to embryo. To precisely evaluate the phenotypes obtained after electroporation of cell-autonomously acting cDNA constructs, the number of positive cells was compared to the total number of electroporated cells, recognized by an internal fluorescent reporter construct. On average, more than 2,300 cells were counted per point and the corresponding quantifications are shown in Figs 1–3 and Supplementary Figs 2–6. This mode of quantification could not be applied when constructs were electroporated in one tissue while the effects were evaluated in another, such as in experiments shown in Fig. 4 and Supplementary Figs 8–11. In this case, we report the number of embryos in which we observed a phenotype similar to the one that is illustrated in the figures, over the total number of electroporated embryos. Statistical analyses were performed using the GraphPad Prism software. Mann–Whitney non-parametric two-tail testing was applied to populations to determine the P values indicated in the figures. In each graph, columns correspond to the mean and error bars correspond to the standard deviation. ***P value , 0.0001. 28. Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of the chick embryo. Dev. Dyn. 195, 231–272 (1992). 29. Ohtsuka, T. et al. Visualization of embryonic neural stem cells using Hes promoters in transgenic mice. Mol. Cell. Neurosci. 31, 109–122 (2006). 30. Tobiume, M. et al. Inefficient enhancement of viral infectivity and CD4 downregulation by human immunodeficiency virus type 1 Nef from Japanese long-term nonprogressors. J. Virol. 76, 5959–5965 (2002). 31. Daudet, N. & Lewis, J. Two contrasting roles for Notch activity in chick inner ear development: specification of prosensory patches and lateral inhibition of hair-cell differentiation. Development 132, 541–551 (2005). 32. Iimura, T. & Pourquie, O. Collinear activation of Hoxb genes during gastrulation is linked to mesoderm cell ingression. Nature 442, 568–571 (2006). 33. Nagoshi, E. et al. Circadian gene expression in individual fibroblasts: cellautonomous and self-sustained oscillators pass time to daughter cells. Cell 119, 693–705 (2004). 34. Werner, T., Hammer, A., Wahlbuhl, M., Bosl, M. R. & Wegner, M. Multiple conserved regulatory elements with overlapping functions determine Sox10 expression in mouse embryogenesis. Nucleic Acids Res. 35, 6526–6538 (2007). 35. Uchikawa, M., Ishida, Y., Takemoto, T., Kamachi, Y. & Kondoh, H. Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev. Cell 4, 509–519 (2003). 36. Maxwell, I. H., Maxwell, F. & Glode, L. M. Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. Cancer Res. 46, 4660–4664 (1986). 37. Henrique, D. et al. Maintenance of neuroepithelial progenitor cells by Delta–Notch signalling in the embryonic chick retina. Curr. Biol. 7, 661–670 (1997). 38. Manceau, M. et al. Myostatin promotes the terminal differentiation of embryonic muscle progenitors. Genes Dev. 22, 668–681 (2008). 39. Henrique, D. et al. cash4, a novel achaete-scute homolog induced by Hensen’s node during generation of the posterior nervous system. Genes Dev. 11, 603–615 (1997). 40. Jouve, C. et al. Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm. Development 127, 1421–1429 (2000). 41. Marcelle, C., Stark, M. R. & Bronner-Fraser, M. Coordinate actions of BMPs, Wnts, Shh and noggin mediate patterning of the dorsal somite. Development 124, 3955–3963 (1997).
