Pri peptides are mediators of ecdysone for the temporal control of development


Animal development fundamentally relies on the precise control, in space and time, of genome expression. Whereas we have a wealth of information about spatial patterning, the mechanisms underlying temporal control remain poorly understood. Here we show that Pri peptides, encoded by small open reading frames, are direct mediators of the steroid hormone ecdysone for the timing of developmental programs in Drosophila. We identify a previously uncharacterized enzyme of ecdysone biosynthesis, GstE14, and find that ecdysone triggers pri expression to define the onset of epidermal trichome development, through post-translational control of the Shavenbaby transcription factor. We show that manipulating pri expression is sufficient to either put on hold or induce premature differentiation of trichomes. Furthermore, we find that ecdysone-dependent regulation of pri is not restricted to epidermis and occurs over various tissues and times. Together, these findings provide a molecular framework to explain how systemic hormonal control coordinates specific programs of differentiation with developmental timing.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: GstE14 encodes a component of the ecdysone pathway.
Figure 2: The ecdysone pathway controls epidermal differentiation.
Figure 3: The ecdysone hormone is required for pri expression and Svb maturation in trichome cells.
Figure 4: The ecdysone pathway drives epidermal remodelling through the regulation of pri transcription.
Figure 5: Svb specifies the differentiation of trichomes in the pupal notum.
Figure 6: Pri mediates ecdysone-dependent temporal control of epidermal trichome differentiation.
Figure 7: Ecdysone controls pri expression at the larval–pupal developmental transition.
Figure 8: Role of pri in mediating the action of ecdysone for the temporal control of morphogenesis.


  1. 1

    Pauli, A., Rinn, J. L. & Schier, A. F. Non-coding RNAs as regulators of embryogenesis. Nat. Rev. Genet. 12, 136–149 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Ulitsky, I. & Bartel, D. P. lincRNAs: genomics, evolution, and mechanisms. Cell 154, 26–46 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3

    Aspden, J. L. et al. Extensive translation of small ORFs revealed by Poly-Ribo-Seq. eLife e03528 (2014).

  4. 4

    Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Hanada, K. et al. Small open reading frames associated with morphogenesis are hidden in plant genomes. Proc. Natl Acad. Sci. USA 110, 2395–2400 (2013).

    CAS  Article  PubMed  Google Scholar 

  6. 6

    Magny, E. G. et al. Conserved regulation of cardiac calcium uptake by peptides encoded in small open reading frames. Science 341, 1116–1120 (2013).

    CAS  Article  PubMed  Google Scholar 

  7. 7

    Slavoff, S. A. et al. Peptidomic discovery of short open reading frame-encoded peptides in human cells. Nat. Chem. Biol. 9, 59–64 (2013).

    CAS  Article  PubMed  Google Scholar 

  8. 8

    Savard, J., Marques-Souza, H., Aranda, M. & Tautz, D. A segmentation gene in tribolium produces a polycistronic mRNA that codes for multiple conserved peptides. Cell 126, 559–569 (2006).

    CAS  Article  PubMed  Google Scholar 

  9. 9

    Galindo, M. I., Pueyo, J. I., Fouix, S., Bishop, S. A. & Couso, J. P. Peptides encoded by short ORFs control development and define a new eukaryotic gene family. PLoS Biol. 5, e106 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Pueyo, J. I. & Couso, J. P. The 11-aminoacid long Tarsal-less peptides trigger a cell signal in Drosophila leg development. Dev. Biol. 324, 192–201 (2008).

    CAS  Article  PubMed  Google Scholar 

  11. 11

    Kondo, T. et al. Small peptide regulators of actin-based cell morphogenesis encoded by a polycistronic mRNA. Nat. Cell Biol. 9, 660–665 (2007).

    CAS  Article  PubMed  Google Scholar 

  12. 12

    Chanut-Delalande, H., Ferrer, P., Payre, F. & Plaza, S. Effectors of tridimensional cell morphogenesis and their evolution. Semin. Cell Dev. Biol. 23, 341–349 (2012).

    CAS  Article  PubMed  Google Scholar 

  13. 13

    Payre, F., Vincent, A. & Carreno, S. ovo/svb integrates Wingless and DER pathways to control epidermis differentiation. Nature 400, 271–275 (1999).

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Sucena, E., Delon, I., Jones, I., Payre, F. & Stern, D. L. Regulatory evolution of shavenbaby/ovo underlies multiple cases of morphological parallelism. Nature 424, 935–938 (2003).

    CAS  Article  PubMed  Google Scholar 

  15. 15

    Delon, I. & Payre, F. Evolution of larval morphology in flies: get in shape with shavenbaby. Trends Genet. 20, 305–313 (2004).

    CAS  Article  PubMed  Google Scholar 

  16. 16

    McGregor, A. P. et al. Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448, 587–590 (2007).

    CAS  Article  PubMed  Google Scholar 

  17. 17

    Frankel, N. et al. Morphological evolution caused by many subtle-effect substitutions in regulatory DNA. Nature 474, 598–603 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Menoret, D. et al. Genome-wide analyses of Shavenbaby target genes reveals distinct features of enhancer organization. Gen. Biol. 14, R86 (2013).

    Article  CAS  Google Scholar 

  19. 19

    Chanut-Delalande, H., Fernandes, I., Roch, F., Payre, F. & Plaza, S. Shavenbaby couples patterning to epidermal cell shape control. PLoS Biol. 4, e290 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Andrew, D. J. & Baker, B. S. Expression of the Drosophila secreted cuticle protein 73 (dsc73) requires Shavenbaby. Dev. Dyn. 237, 1198–1206 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Fernandes, I. et al. Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes. Dev. Cell 18, 64–76 (2010).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Kondo, T. et al. Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis. Science 329, 336–339 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Thummel, C. S. Molecular mechanisms of developmental timing in C. elegans and Drosophila. Dev. Cell 1, 453–465 (2001).

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Yamanaka, N., Rewitz, K. F. & O’Connor, M. B. Ecdysone control of developmental transitions: lessons from Drosophila research. Annu. Rev. Entomol. 58, 497–516 (2013).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Delanoue, R., Slaidina, M. & Leopold, P. The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells. Dev. Cell 18, 1012–1021 (2010).

    CAS  Article  PubMed  Google Scholar 

  26. 26

    Colombani, J. et al. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science 310, 667–670 (2005).

    CAS  Article  PubMed  Google Scholar 

  27. 27

    Andersen, D. S., Colombani, J. & Leopold, P. Coordination of organ growth: principles and outstanding questions from the world of insects. Trends Cell Biol. 23, 336–344 (2013).

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Yamanaka, N. et al. Neuroendocrine control of Drosophila larval light preference. Science 341, 1113–1116 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Saisawang, C., Wongsantichon, J. & Ketterman, A. J. A preliminary characterization of the cytosolic glutathione transferase proteome from Drosophila melanogaster. Biochem. J. 442, 181–190 (2012).

    CAS  Article  PubMed  Google Scholar 

  30. 30

    Board, P. G. & Menon, D. Glutathione transferases, regulators of cellular metabolism and physiology. Biochim. Biophys. Acta 1830, 3267–3288 (2013).

    CAS  Article  PubMed  Google Scholar 

  31. 31

    Yoshiyama, T., Namiki, T., Mita, K., Kataoka, H. & Niwa, R. Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth. Development 133, 2565–2574 (2006).

    CAS  Article  PubMed  Google Scholar 

  32. 32

    Yoshiyama-Yanagawa, T. et al. The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme. J. Biol. Chem. 286, 25756–25762 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Ono, H. et al. Spook and Spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera. Dev. Biol. 298, 555–570 (2006).

    CAS  Article  PubMed  Google Scholar 

  34. 34

    Niwa, R. et al. Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway. Development 137, 1991–1999 (2010).

    CAS  Article  PubMed  Google Scholar 

  35. 35

    Warren, J. T. et al. Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: A P450 enzyme critical in ecdysone biosynthesis. Insect Biochem. Mol. Biol. 34, 991–1010 (2004).

    CAS  Article  PubMed  Google Scholar 

  36. 36

    Chavez, V. M. et al. The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development 127, 4115–4126 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Petryk, A. et al. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. Proc. Natl Acad. Sci. USA 100, 13773–13778 (2003).

    CAS  Article  PubMed  Google Scholar 

  38. 38

    Huang, X., Warren, J. T. & Gilbert, L. I. New players in the regulation of ecdysone biosynthesis. J. Genet. Genomics 35, 1–10 (2008).

    Article  PubMed  Google Scholar 

  39. 39

    Nusslein-Volhard, C., Wieschaus, E. & Kluding, H. Mutations affecting the pattern of larval cuticle in Drosophila Melanogaster. I. zygotic loci on the seconde chromosome. Roux Arch. Dev. Biol. 193, 267–282 (1984).

    CAS  Article  Google Scholar 

  40. 40

    Rewitz, K. F., O’Connor, M. B. & Gilbert, L. I. Molecular evolution of the insect Halloween family of cytochrome P450s: phylogeny, gene organization and functional conservation. Insect Biochem. Mol. Biol. 37, 741–753 (2007).

    CAS  Article  PubMed  Google Scholar 

  41. 41

    Warren, J. T. et al. Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster. Proc. Natl Acad. Sci. USA 99, 11043–11048 (2002).

    CAS  Article  PubMed  Google Scholar 

  42. 42

    Talamillo, A. et al. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis. PLoS Genet. 9, e1003473 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43

    Chavoshi, T. M., Moussian, B. & Uv, A. Tissue-autonomous EcR functions are required for concurrent organ morphogenesis in the Drosophila embryo. Mech. Dev. 127, 308–319 (2010).

    CAS  Article  PubMed  Google Scholar 

  44. 44

    Moussian, B. Recent advances in understanding mechanisms of insect cuticle differentiation. Insect Biochem. Mol. Biol. 40, 363–375 (2010).

    CAS  Article  PubMed  Google Scholar 

  45. 45

    Payre, F. Genetic control of epidermis differentiation in Drosophila. Int. J. Dev. Biol. 48, 207–215 (2004).

    CAS  Article  PubMed  Google Scholar 

  46. 46

    Frankel, N. et al. Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466, 490–493 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47

    Koelle, M. R. et al. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell 67, 59–77 (1991).

    CAS  Article  PubMed  Google Scholar 

  48. 48

    Yao, T. P. et al. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366, 476–479 (1993).

    CAS  Article  PubMed  Google Scholar 

  49. 49

    Ruaud, A. F., Lam, G. & Thummel, C. S. The Drosophila nuclear receptors DHR3 and βFTZ-F1 control overlapping developmental responses in late embryos. Development 137, 123–131 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. 50

    Carney, G. E. & Bender, M. The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis. Genetics 154, 1203–1211 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Cherbas, L., Hu, X., Zhimulev, I., Belyaeva, E. & Cherbas, P. EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue. Development 130, 271–284 (2003).

    CAS  Article  PubMed  Google Scholar 

  52. 52

    Kamimura, M. et al. Fungal ecdysteroid-22-oxidase, a new tool for manipulating ecdysteroid signaling and insect development. J. Biol. Chem. 287, 16488–16498 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53

    Shlyueva, D. et al. Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin. Mol. Cell 54, 180–192 (2014).

    CAS  Article  PubMed  Google Scholar 

  54. 54

    Kozlova, T. & Thummel, C. S. Essential roles for ecdysone signaling during Drosophila mid-embryonic development. Science 301, 1911–1914 (2003).

    CAS  Article  PubMed  Google Scholar 

  55. 55

    Riddiford, L. M. in The Development of Drosophila Melanogaster (ed Martinez-Arias, M. B.a.A.) 899–939 (Cold Spring Harbor Laboratory Press, 1993).

    Google Scholar 

  56. 56

    Fluegel, M. L., Parker, T. J. & Pallanck, L. J. Mutations of a Drosophila NPC1 gene confer sterol and ecdysone metabolic defects. Genetics 172, 185–196 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57

    Horner, M. A. et al. The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis. Genes Dev. 23, 2711–2716 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. 58

    Enya, S. et al. A Halloween gene noppera-bo encodes a glutathione S-transferase essential for ecdysteroid biosynthesis via regulating the behaviour of cholesterol in Drosophila. Sci. Rep. 4, 6586 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Poodry, C. A. & Schneiderman, H. A. The ultrastructure of the developing leg of Drosophila melanogaster. Roux Arch. Dev. Biol. 166, 1–44 (1970).

    Article  Google Scholar 

  60. 60

    McLean, P. F. & Cooley, L. Protein equilibration through somatic ring canals in Drosophila. Science 340, 1445–1447 (2013).

    CAS  Article  PubMed  Google Scholar 

  61. 61

    Pueyo, J. I. & Couso, J. P. Tarsal-less peptides control Notch signalling through the Shavenbaby transcription factor. Dev. Biol. 355, 183–193 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. 62

    Pi, H. et al. Identification of 11-amino acid peptides that disrupt Notch-mediated processes in Drosophila. J. Biomed. Sci. 18, 42 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. 63

    Delon, I., Chanut-Delalande, H. & Payre, F. The Ovo/Shavenbaby transcription factor specifies actin remodelling during epidermal differentiation in Drosophila. Mech. Dev. 120, 747–758 (2003).

    CAS  Article  PubMed  Google Scholar 

  64. 64

    Andres, A. J. & Thummel, C. S. Methods for quantitative analysis of transcription in larvae and prepupae. Methods Cell Biol. 44, 565–573 (1994).

    CAS  Article  PubMed  Google Scholar 

  65. 65

    Founounou, N., Loyer, N. & Le Borgne, R. Septins regulate the contractility of the actomyosin ring to enable adherens junction remodeling during cytokinesis of epithelial cells. Dev. Cell 24, 242–255 (2013).

    CAS  Article  PubMed  Google Scholar 

  66. 66

    Dunne, J. C., Kondylis, V. & Rabouille, C. Ecdysone triggers the expression of Golgi genes in Drosophila imaginal discs via broad-complex. Dev. Biol. 245, 172–186 (2002).

    CAS  Article  PubMed  Google Scholar 

Download references


We are grateful to FlyBase and the Bloomington, Vienna and Kyoto stock centres, as well as R. Niwa, M. Kamimura and J. Colombani for providing flies, and H. Bellen for bacterial artificial chromosome constructs. We thank B. Ronsin (Toulouse RIO Imaging) for help with microscopy and O. Bohner for technical assistance. We also thank A. Khila, A. Vincent, P. Leopold and E. France for critical reading of the manuscript, and are indebted to R. Niwa for sharing unpublished results. This work was supported by ANR (smORFpeptides and Chrononet), Association pour la Recherche sur le Cancer (12011669), Azm & Saade Association, JST PRESTO program, MEXT KAKENHI (21115007) and Fondation RITC.

Author information




Y.K. and F.P. conceived and directed the project. Y.H. initiated the project and H.C-D. carried out most experiments presented here. A.D., J.B., K.N., S.I., L.D., P.V. and C.P. conducted experiments and gave further helpful insights. H.C-D., Y.H., A.P-M., T.K., Y.L., R.S., B.M., S.K., K.P.W., S.P., Y.K. and F.P. designed the experiments, analysed data and contributed to data interpretation. H.C-D., Y.H., Y.K. and F.P. prepared the figures and wrote the manuscript. All authors helped write and revise the paper.

Corresponding authors

Correspondence to Yuji Kageyama or François Payre.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 GstE14 is required for Dusky-like expression in trichome cells.

A. Schematic representation of the second chromosome of Drosophila melanogaster, focusing on the cytogenetic position 49F10-F13 and associated genes (blue arrows). From all lines we tested in this screen (see Supplementary Table 1), we observed a complete absence of Dyl staining only in the two overlapping deletions Df(2R)BSC273 and Df(2R)Exel7124(dark red). A neighbouring deletion with unaffected Dyl expression (Df(2R)ED2311) is in dark green. A secondary screening with a smaller deficiency, Df(2R)BSC272, restricted the genetic interval to 9 genes. To identify the responsible gene(s), we generated a series of transgenic lines carrying BAC genomic constructs (see Supplementary Table 3) and assayed their rescuing activity when reintroduced in the Df(2R)BSC272 background. While BAC-126C02 (red box) did not restore Dyl staining, BAC-157I07, -146O12 and -83L02 (light green boxes) fully rescued Dyl expression in Df(2R)BSC272 embryos. Since the three latter regions share a single gene, GstE14, we generated a construct narrowed down to a 4,6kb DNA fragment encompassing only this locus (P[GstE14]). B. As observed for rescuing BACs, P[GstE14] was sufficient to fully rescue Dyl expression within trichomes, as seen in stage-15 embryos (ventral views). Of note, P[GstE14]also suppressed the embryonic lethality observed for homozygous Df(2R)BSC272 mutants. Rescuing assays have been carried out in at least three independent experiments. Scale bars are 100 μm (whole embryo) and 20 μm for closeup pictures.

Supplementary Figure 2 GstE14 encodes an insect-specific Glutathione S transferase.

A. Alignment of GstE14 protein sequences across Drosophila species. Dmel, Drosophila melanogaster; Dsec, Drosophila sechellia; Dyak, Drosophila yakuba; Dsim, Drosophila simulans; Dere, Drosophila erecta; Dana, Drosophila ananassae; Dper, Drosophila persimilis; Dpse, Drosophila pseudoobscura; Dvir, Drosophila virilis; Dmoj, Drosophila mojavensis; Dgrim, Drosophila grimshawi; Dwil, Drosophila willistoni. B. Cladogram showing the distribution of GstE14 sequences within Drosophila species. The GstD1 protein from Drosophila melanogaster was introduced as outgroup. Protein sequences were extracted from flybase (, multiple alignment, curation, phylogenetic tree reconstruction and rendering were processed using ClustalW2 (, and MUSCLE, Gblocks, PhyML, TreeDyn packages available at

Supplementary Figure 3 GstE14 functions in cholesterol metabolism.

A. Cuticle preparation of Df(GstE14) and spo mutant embryos incubated in Schneider’s medium supplemented with either 20E, ecdysone or cholesterol during mid-embryogenesis. Incubation with Schneider’s medium alone (mock) was used as control. All three compounds significantly suppressed embryonic lethality, as well as rescued epidermal differentiation, that is cuticle differentiation and trichome formation, for Df(GstE14) mutants. In contrast, spo mutants were rescued by the exogenous addition only of 20E and ecdysone, but not by cholesterol, consistently with the documented requirement of spo activity for the transformation of 7-dehydro-cholesterol to ketodiol33. Scale bar, 100 μm. B. Schematic representation of the successive steps of the biosynthetic pathway leading to ecdysone production from dietary sterols. As deduced from rescuing experiments, GstE14 activity is required for the very early stages of the pathway, since its lack can be rescued by cholesterol. C. High cholesterol diet of parental flies suppresses the embryonic lethality of GstE14 mutants, allowing a marked increase in life span. Df(GstE14)/CyoDfdYFP and spo/TM3DfdYFP heterozygous flies were fed for two days with high cholesterol diet, or regular food medium for control, and transferred to egg collection devices. Parental high-cholesterol diet led to the survival of approx 10% of Df(GstE14) mutants, which hatched into viable L1 larvae. The experiments have been made four times independently. The total number of mutant embryos analysed is 422 individuals for GstE14 and > 1,000 for spo. Rescued larvae displayed no obvious morphological defects when compared with wild type larvae. Although these animals remained alive for several days (up to 7 days), they failed to proceed for pupariation, or even larval stage transitions, and instead remained long-lived L1 larvae as deduced from the examination of mouth hooks, a phenotypical marker of larval stages. Arrows highlight the number of mouth hook teeth in wild type, which displays a characteristic increase across larval stages. The chart plot means values, for three independent experiments. Errors bars are s.d., scale bar is 25 μm. D. Inactivation of GstE14 impinges on whole body cholesterol levels, both in embryos and in larvae. The sterol content of Df(GstE14) mutant embryos, and larvae driving UAS–dsRNA–GstE14 (line #1: HMJ21555; line #2 v1018884) in the ring gland (phm-Gal4) was assessed using a commercial assay. When compared with wild type controls, GstE14 embryos display higher levels of sterol (P value = 0.0028). The same was true for phm > dsRNA–GstE14larvae (P value = 0.0006), showing that GstE14 activity in the ring gland is required for maintaining proper cholesterol levels. Extracts were made from hand-counted embryos or larvae, with 1 to 5 independent samples of the same genotype per experiment. All experiments have been repeated independently three times. The graph shows all data points. Statistical tests used two-tailed Mann Whitney tests, error bars are s.d. (blue), means are indicated by a red dotted line.

Supplementary Figure 4 Regulatory interactions within the ecdysone signalling pathway.

A. phmE7 mutant embryos that are defective in 20E production (see Fig. 2b) show a strong down-regulation in the epidermal expression of sha and primRNAs. In contrast, svb mRNA remains expressed at normal-looking levels in phmE7 mutants. B. In situ hybridization showing that GstE14 activity is required for the embryonic expression of early ecdysone-responsive genes, such as Blimp-1 and Hr46. These defects mimic the reduction of Blimp-1 and Hr46expression observed in phmE7 mutant embryos. Scale bars are 100 μm.

Supplementary Figure 5 Ecdysone signalling is required for trichome formation.

A. Expression of EcRDN driven by ptc–Gal4 in epidermal cells represses pri expression (right panel) compared with wild type embryos (left panel). White arrows highlight the reduction of pri expression in ptccells. B. Cuticle of first instar larvae expressing EcRDN alone (left), or in combination with pri (right), throughout embryonic epidermal cells (using the e22cGal4 driver). Pri over-expression allows a significant suppression of EcRDN-induced epidermal defects, including the rescue of misshapen trichomes. Upper panels are lateral view of whole larvae, lower panels ventral views of A3-A4 segments. C. The enzymatic inactivation of ecdysone in epidermal cells, using UAS-E22oxidasedriven by ptc–Gal4, prevents trichome formation in corresponding cells (red arrows). Scale bars are 100 μm for pictures of whole embryos (A) and cuticles (B), and 10 μm for higher magnification (B and C).

Supplementary Figure 6 pri is an early ecdysone-responsive gene.

A. Snapshots of genomic regions encompassing the ecdysone-responsive genes Hr46, Blimp-1 and ftz-f1, showing in vivo EcR binding events (4 h APF) visualized by the intensity of ChiP-seq signal (brown). Genomic coordinates and gene position are indicated within an approx 150kb window. B. Dynamics of relative mRNA levels, extracted from modENCODE Temporal expression Data (mRNA-Seq). Throughout the Drosophila life cycle, pri displays temporal variations that strikingly parallels the ecdysone-responsive Hr46 gene, and correlates to a lesser extend to Blimp-1. In contrast, the temporal dynamics of ftz-f1mRNA levels appears clearly delayed, when compared with pri expression. C. In situ hybridization to Hr46, Blimp-1 and primRNAs in wild type embryos, from stage-11 to stage-16. While their expression is restricted to a limited number of cell patches in early stages (stage-11), the three genes display a concomitant onset of their expression in embryonic epidermal cells at stage-14. Later on, the expression fades and only residual signal is detected at stage-16. All embryos are shown at the same magnification. Scale bar is 100 μm.

Supplementary Figure 7 Premature expression of trichome effectors during embryogenesis.

In situhybridization to pri and dyl mRNA show dynamics of their epidermal expression in wild type embryos, with an onset at stage-13 and stage-14/15, respectively. The precocious expression of pri, triggered by the early pnr–Gal4 driver, induces premature dyl expression in pnr dorsal cells, showing that pri controls the temporal onset of trichome effectors in epidermal cells. Similar results were observed when driving a constitutively activated form of Svb (SvbCA), further demonstrating that pri expression normally times the onset of Svb activation, and thereby, the whole program of trichome formation. Of note, this artificial advance in the onset of trichome effector expression was nevertheless not sufficient to induce premature trichomes, indicating that embryonic epidermal cells at stage-13 are yet not competent to engage morphological differentiation. Therefore, while Svb defines the spatial pattern and pri the temporal onset of epidermal trichomes, their formation can occur only once epidermal cells have reached a competent stage, likely relying on independent factors involved in the general differentiation of the embryonic epidermis. Such general factors known for their role in epidermal differentiation can include transcription factors (for example, Grh, Vri, Ribbon, Ttk, and/or Gata factors)44, as well as regulators of apico-basal polarity, cell junctions, vesicle trafficking or secretion (reviewed in44,45). All pictures are at the same magnification. Scale bar is 100 μm.

Supplementary Figure 8 Effectors of embryonic trichome formation are required for the differentiation of adult trichomes in the notum.

Scanning Electron Micrographs of trichomes in the adult notum, showing consequences of the inactivation of three genes: singed (sn), forked (f) and miniature (m), which are direct targets of the Svb transcription factor during embryonic epidermal differentiation19,21. When compared to wild type, the notum trichomes of sn3,f36A andm1 mutants display characteristic alterations of their shape and improper organization. Scale bars are 3 μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2104 kb)

Supplementary Table 1

Supplementary Information (XLSX 55 kb)

Supplementary Table 2

Supplementary Information (XLSX 41 kb)

Supplementary Table 3

Supplementary Information (XLSX 9 kb)

Supplementary Table 4

Supplementary Information (XLSX 55 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chanut-Delalande, H., Hashimoto, Y., Pelissier-Monier, A. et al. Pri peptides are mediators of ecdysone for the temporal control of development. Nat Cell Biol 16, 1035–1044 (2014).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing