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

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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  PubMed  Google Scholar  Download references 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A. Vincent, P. Leopold and E. France for critical reading of the manuscript, and are indebted to R. Niwa for sharing unpublished results.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 * Takefumi Kondo Present address: Present address: RIKEN Center for Developmental Biology, Kobe 650-0047, Japan., * Hélène Chanut-Delalande and Yoshiko

Hashimoto: These authors contributed equally to this work. * 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 * 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 * 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 * Okazaki Institute for Integrative Bioscience, National institutes of Natural Sciences Okazaki, Aichi 444-8787, Japan Yoshiko Hashimoto, Takefumi Kondo 

& Satoru Kobayashi * Institute for Genomics and Systems Biology and Department of Human Genetics, University of Chicago, Illinois 60637, USA Rebecca Spokony & Kevin P. White *

Research Center for Environmental Genomics, Organization of Advanced Science and Technology, Kobe University, Kobe 657-850, Japan Kaori Niimi, Sachi Inagaki & Yuji Kageyama * Animal

Genetics, Interfaculty Institute for Cell Biology, University of Tübingen, 72076 Tübingen, Germany Bernard Moussian * Department of Biology, Graduate School of Science, Kobe University, Kobe

657-850, Japan Yuji Kageyama Authors * Hélène Chanut-Delalande View author publications You can also search for this author inPubMed Google Scholar * Yoshiko Hashimoto View author

publications You can also search for this author inPubMed Google Scholar * Anne Pelissier-Monier View author publications You can also search for this author inPubMed Google Scholar *

Rebecca Spokony View author publications You can also search for this author inPubMed Google Scholar * Azza Dib View author publications You can also search for this author inPubMed Google

Scholar * Takefumi Kondo View author publications You can also search for this author inPubMed Google Scholar * Jérôme Bohère View author publications You can also search for this author

inPubMed Google Scholar * Kaori Niimi View author publications You can also search for this author inPubMed Google Scholar * Yvan Latapie View author publications You can also search for

this author inPubMed Google Scholar * Sachi Inagaki View author publications You can also search for this author inPubMed Google Scholar * Laurence Dubois View author publications You can

also search for this author inPubMed Google Scholar * Philippe Valenti View author publications You can also search for this author inPubMed Google Scholar * Cédric Polesello View author

publications You can also search for this author inPubMed Google Scholar * Satoru Kobayashi View author publications You can also search for this author inPubMed Google Scholar * Bernard

Moussian View author publications You can also search for this author inPubMed Google Scholar * Kevin P. White View author publications You can also search for this author inPubMed Google

Scholar * Serge Plaza View author publications You can also search for this author inPubMed Google Scholar * Yuji Kageyama View author publications You can also search for this author

inPubMed Google Scholar * François Payre View author publications You can also search for this author inPubMed Google Scholar 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. 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 (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-Gal_4) 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–GstE14_larvae (_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. _phm__E_7 mutant embryos that are defective in 20E

production (see Fig. 2b) show a strong down-regulation in the epidermal expression of _sha_ and _pri_mRNAs. In contrast, _svb_ mRNA remains expressed at normal-looking levels in _phm__E_7

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 _Hr46_expression observed in _phm__E_7 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 _ptc_cells. 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-E22oxidase_driven 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-f1_mRNA levels appears clearly delayed, when compared with _pri_

expression. C. _In situ_ hybridization to _Hr46_, _Blimp-1_ and _pri_mRNAs 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 situ_hybridization 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 _sn__3_,_f__36A_ and_m__1_ 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 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). https://doi.org/10.1038/ncb3052 Download citation * Received: 07 April 2014 *

Accepted: 15 September 2014 * Published: 26 October 2014 * Issue Date: November 2014 * DOI: https://doi.org/10.1038/ncb3052 SHARE THIS ARTICLE Anyone you share the following link with will

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