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

Nature Cell Biology volume 16, pages 1035–1044 (2014)Cite this article

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Abstract

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.

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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.

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Acknowledgements

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

Author notes

  1. Takefumi Kondo

    Present address: Present address: RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.,

  2. Hélène Chanut-Delalande and Yoshiko Hashimoto: These authors contributed equally to this work.

  3. Yvan Latapie: The authors would like to dedicate this article to the memory of Y. Latapie (1980–2014), whose life was taken by an avalanche in the Pyrenees.

Authors and Affiliations

  1. Centre de Biologie du Développement, Université de Toulouse, UPS, 31062 Toulouse cedex 9, France

    Hélène Chanut-Delalande, Anne Pelissier-Monier, Azza Dib, Jérôme Bohère, Yvan Latapie, Laurence Dubois, Philippe Valenti, Cédric Polesello, Serge Plaza & François Payre

  2. CNRS, UMR5547, Centre de Biologie du Développement, Toulouse, 31062 cedex 9, France

    Hélène Chanut-Delalande, Anne Pelissier-Monier, Azza Dib, Jérôme Bohère, Yvan Latapie, Laurence Dubois, Philippe Valenti, Cédric Polesello, Serge Plaza & François Payre

  3. Okazaki Institute for Integrative Bioscience, National institutes of Natural Sciences Okazaki, Aichi 444-8787, Japan

    Yoshiko Hashimoto, Takefumi Kondo & Satoru Kobayashi

  4. Institute for Genomics and Systems Biology and Department of Human Genetics, University of Chicago, Illinois 60637, USA

    Rebecca Spokony & Kevin P. White

  5. Research Center for Environmental Genomics, Organization of Advanced Science and Technology, Kobe University, Kobe 657-850, Japan

    Kaori Niimi, Sachi Inagaki & Yuji Kageyama

  6. Animal Genetics, Interfaculty Institute for Cell Biology, University of Tübingen, 72076 Tübingen, Germany

    Bernard Moussian

  7. Department of Biology, Graduate School of Science, Kobe University, Kobe 657-850, Japan

    Yuji Kageyama

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  1. Hélène Chanut-Delalande
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Contributions

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.

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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 (http://flybase.org), multiple alignment, curation, phylogenetic tree reconstruction and rendering were processed using ClustalW2 (www.ebi.ac.uk), and MUSCLE, Gblocks, PhyML, TreeDyn packages available at www.phylogeny.fr.

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.

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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). https://doi.org/10.1038/ncb3052

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