Lucie Vaufrey
email: lucie.vaufrey@laposte.net

Christine Balducci
email: c.balducci@free.fr

René Lafont
email: rene.lafont@upmc.fr

Claude Prigent
email: claude.prigent@univ-rennes1.fr

Stéphanie Le Bras
email: stephanie.lebras@univ-rennes1.fr

Size matters! Aurora A controls Drosophila larval development

Keywords: Drosophila, Aurora A kinase, larval development coordination Graphical

Introduction

Aurora kinases are essential guardians of cell cycle progression with multiple functions in mitosis/meiosis and interphase. They belong to a three member kinase family in mammals that evolved from a single yeast gene (IPL1 in budding yeast and ark1 in fission yeast) [START_REF] Chan | Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast[END_REF][START_REF] Petersen | The S. pombe aurorarelated kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation[END_REF]) that duplicated to give Aurora-A (AurA) and Aurora-B in worms, flies and amphibians. Later on, duplication of Aurora-B gave rise to Aurora-B and Aurora-C in mammals [START_REF] Brown | Evolutionary relationships of Aurora kinases: implications for model organism studies and the development of anti-cancer drugs[END_REF]. Extensive studies in cellular models have revealed the pleiotropic and essential function of AurA during cell cycle. First, AurA plays a key role in the G2 to M transition by locally activating the phosphatase CDC25B and the kinase Plk1 [START_REF] Dutertre | Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2-M transition[END_REF][START_REF] Macurek | Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery[END_REF][START_REF] Seki | Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry[END_REF].

Furthermore, during entry in mitosis, AurA is essential to centrosome maturation a prerequisite to bipolar spindle assembly. Then during mitosis, the main role of the kinase is to control microtubule nucleation to successively assemble the bipolar spindle [START_REF] Pinyol | The role of NEDD1 phosphorylation by Aurora A in chromosomal microtubule nucleation and spindle function[END_REF][START_REF] Sardon | Dissecting the role of Aurora A during spindle assembly[END_REF] and the central spindle [START_REF] Lioutas | Aurora A kinase and its substrate TACC3 are required for central spindle assembly[END_REF][START_REF] Reboutier | Aurora A is involved in central spindle assembly through phosphorylation of Ser 19 in P150Glued[END_REF]. Finally, in G1 during interphase, the kinase participates in cilia resorption by activating the tubulin deacetylase HDAC6 [START_REF] Pugacheva | HEF1dependent Aurora A activation induces disassembly of the primary cilium[END_REF]. Importantly, AurA is an oncogene frequently overexpressed in a large variety of cancers that has rapidly become a priority target for the development of inhibitors for the use in cancer treatment [START_REF] Damodaran | Aurora A Kinase Is a Priority Pharmaceutical Target for the Treatment of Cancers[END_REF].

Therefore, studies of its functions in any organism will impact cancer research due to the conserved nature of this kinase throughout evolution [START_REF] Carmena | The Dawn of Aurora Kinase Research: From Fly Genetics to the Clinic[END_REF].

Studies in organism models have demonstrated that AurA has essential functions during the cell cycle that are required for proper development as described in both vertebrates and invertebrates. In mice, knock-out of AURKA leads to preimplantation lethality with cell cycle defects in mitosis entry timing, bi-polar spindle assembly or chromosome segregation [START_REF] Cowley | Aurora-A kinase is essential for bipolar spindle formation and early development[END_REF][START_REF] Lu | Aurora A is essential for early embryonic development and tumor suppression[END_REF][START_REF] Sasai | Targeted disruption of Aurora A causes abnormal mitotic spindle assembly, chromosome misalignment and embryonic lethality[END_REF]. The fact that knock-out embryos are able to reach the blastocyst stage can be attributed to residual maternal mRNA and/or protein AurA [START_REF] Yao | Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos[END_REF]. Conditional knock-out of AURKA in different embryonic layers and/or at different post-implantation stages induces mitotic delay, increased apoptosis and early embryonic lethality which further supports the essential nature of AurA function during the cell cycle in development [START_REF] Cowley | Aurora-A kinase is essential for bipolar spindle formation and early development[END_REF][START_REF] Yoon | Conditional Aurora A deficiency differentially affects early mouse embryo patterning[END_REF]. Similarly, zfAurora-A depletion in zebrafish embryos also leads to embryonic lethality with similar mitotic failures [START_REF] Jeon | Depletion of Aurora-A in zebrafish causes growth retardation due to mitotic delay and p53-dependent cell death[END_REF]. Outstandingly, mutants are able to achieve embryonic but also larval development in Caenorhabditis elegans (air1) and Drosophila melanogaster (aurA) [START_REF] Furuta | Caenorhabditis elegans Aurora A kinase AIR-1 is required for postembryonic cell divisions and germline development[END_REF][START_REF] Glover | Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles[END_REF] presumably thanks to the maternal contribution, as embryonic RNAi in C. elegans or maternal depletion in drosophila leads to defective mitosis and eventually embryonic lethality [START_REF] Glover | Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles[END_REF][START_REF] Schumacher | A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos[END_REF]. In C.

elegans, mutants reach the adult stage with mitotic defects as reported in the ventral cord and vulva lineages [START_REF] Furuta | Caenorhabditis elegans Aurora A kinase AIR-1 is required for postembryonic cell divisions and germline development[END_REF]. On the contrary, Drosophila mutants do not reach the adult stage and die at the pupal stage during metamorphosis [START_REF] Glover | Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles[END_REF][START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]. The fact that Drosophila aurA loss-offunction (aurA lof ) mutants survive throughout larval development can be explained by larval growth reliance on increasing polyploidy in larval tissues [START_REF] Royzman | Mutations in Drosophila DP and E2F distinguish G1-S progression from an associated transcriptional program[END_REF] and not on mitotic tissues (central nervous system and imaginal discs) of which correct development is eventually essential during pupal metamorphosis [START_REF] Shearn | Genetic control of imaginal disc development in Drosophila[END_REF]. In accordance with this hypothesis, most essential cell cycle regulators have been identified in genetic screens based on late larval/pupal lethality linked to defects in cell division of larval imaginal tissues [START_REF] Gatti | Genes controlling essential cell-cycle functions in Drosophila melanogaster[END_REF]. Surprisingly, the only defects reported in Drosophila aurA lof mutant larvae are in central nervous system neuroblasts (NB) while no defect were observed in other larval tissues included imaginal discs which divide intensively during larval development [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]. Still, aurA lof mutant NB share features of mitotic defects such as delayed mitosis entry, defective centrosomes number and chromosome mis-segregation, a trademark of AurA loss-of-function [START_REF] Caous | Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila[END_REF][START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF].

NB are progenitors of the central nervous system lineages which divide asymmetrically to renew a NB and to produce a ganglion mother cell (GMC) that will divide and produce the multitude of neurons and glial cells (for review see [START_REF] Kang | Control of neural stem cell self-renewal and differentiation in Drosophila[END_REF]). During embryonic development, NB delaminate from the neuroectoderm and divide asymmetrically until they enter quiescence by the end of embryogenesis [START_REF] Lai | Transient nuclear Prospero induces neural progenitor quiescence[END_REF][START_REF] Tsuji | Neuroblast entry into quiescence is regulated intrinsically by the combined action of spatial Hox proteins and temporal identity factors[END_REF]. During larval development, embryonic NB exit quiescence and divide asymmetrically in the central brain and ventral nerve cord while new NB delaminate and first start dividing symmetrically then asymmetrically in the optic lobe. The central brain NB exit from quiescence takes place from the late 1 st instar larval (L1) stage [START_REF] Britton | Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms[END_REF][START_REF] Green | The embryonic development of the Drosophila visual system[END_REF][START_REF] Hartenstein | Early neurogenesis in wild-typeDrosophila melanogaster[END_REF] and is timely controlled in part by glial cells [START_REF] Datta | Control of proliferation activation in quiescent neuroblasts of the Drosophila central nervous system[END_REF][START_REF] Ebens | The Drosophila anachronism locus: a glycoprotein secreted by glia inhibits neuroblast proliferation[END_REF][START_REF] Voigt | Perlecan participates in proliferation of quiescent Drosophila neuroblasts[END_REF] which relay a nutritiondependent fat body derived signal [START_REF] Sousa-Nunes | Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila[END_REF]. Fat-body derived signals controlling systemic growth have recently been identified but it remains to be demonstrated that they actually control NB quiescence exit [START_REF] Koyoma | Growth-blocking peptides as nutrition-sensitive signals for insulin secretion and body size regulation[END_REF][START_REF] Delanoue | Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor[END_REF]. Central brain NB further divide asymmetrically and reach a plateau of extensive division during the 3 rd instar larval (L3) stage until pupariation [START_REF] Ito | Proliferation pattern of postembryonic neuroblasts in the brain of Drosophila melanogaster[END_REF]. In aurA lof mutants (aurA 8839 , aurA 14641 and aurA ST ), brain lobes of wandering L3 larvae reach up to 10 times the volume of control larvae, have too many larval pseudo-NB (cells positive for NB markers and which can divide symmetrically) resulting from defective NB asymmetric cell division and are highly tumorigenic [START_REF] Caous | Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila[END_REF][START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]. Based on these observations, aurA is defined as a brain tumor-suppressor gene in Drosophila. However, other Drosophila brain tumor-suppressor genes are also imaginal disc tumor-suppressor genes [START_REF] Gateff | Tumor suppressor and overgrowth suppressor genes of Drosophila melanogaster: developmental aspects[END_REF], which has not been reported in aurA lof mutants. Therefore, we decided to examine the AurA tumor-suppressor property in other tissues and search for additive phenotypes in aurA lof mutants that could contribute to NB-like over-proliferation. We investigated aurA lof phenotypes from the beginning of larval development as all previous studies were performed at the end of the L3 stage (wandering L3 stage). Here, we revealed that AurA is actually a regulator of different larval development processes including the regulation of larval growth rate and pupariation timing. Furthermore, we could identify that aurA does not act as a tumor-suppressor cellautonomously in larval NB as previously described. Instead, in the absence of AurA function, NB give rise to cells with an intermediate cell-identity between NB and GMC which escape a spatio-temporal control of the number of cell divisions rounds.

Material and Methods

Drosophila strains and genetics.

Origin of all stocks and details of all crosses are listed in Supplementary table 1 and 2.

w 1118 was used as wild-type control for all experiments.

Five aurA alleles, which are all late larval/early pupal lethal (data not shown), were used in this study: aurA 14641 a hypomorph EMS mutant [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]; aurA 8839 a strong hypomorph EMS mutant [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF] The avalanche RNAi experiment was performed at 25°C as previously described [START_REF] Colombani | Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing[END_REF] and with the following genotype: w; elav-GAL80; Rn-GAL4, UAS-Avl dsRNA / TM6b, tub-GAL80. RNAi experiments to study pupariation delay were performed at 25°C

with the following drivers: ubiquitous (69B-GAL4 and arm-GAL4), ring gland-specific (phm-GAL4), peripheral tissue-specific (ppl-GAL4), fat-body specific (lpp-GAL4), neuroblastsspecific (wor-GAL4) and wing-disc specific (pdm2-GAL4 and ser-GAL4); and UAS-dsRNA lines: UAS-aurA dsRNA and UAS-white dsRNA (see supplementary table 1 for genotype details).

RT-PCR

RNA was extracted from 30 central brains of wandering L3 larvae after dissection in PBS and with a RNA isolation kit from Zymo Research. 3 g of total RNA was used for reverse transcriptase reaction performed in a final volume of 20 l during 1h at 50°C using oligodT primer and Superscript III Reverse Transcription (Thermo Fisher Scientific). 2 l of each RT reaction were used for PCR using the following primers:

tubulin, forward 5'-TCCTTGTCGCGTGTGAAACA-3' tubulin, reverse 5'-AGCTTGGACTTCTTGCCGTA-3'

aurA exon 1 to 2, forward 5'-CTGTTAGTTGTCGAGTGCCT-3'

aurA exon 1 to 2, reverse 5'-CAGCAGGCGACCAATATCAA-3' aurA exon 3, forward 5'-TTGATATTGGTCGCCTGCTG-3' aurA exon 3, reverse 5'-AATGTGTTCACTGCGTGTGC-3'

aurA exon 1 to 3, forward 5'-CTGTTAGTTGTCGAGTGCCT-3'

aurA exon 1 to 3, reverse 5'-AATGTGTTCACTGCGTGTGC-3'

AurA antibody, protein production, kinase assay and western blot

Polyclonal anti-AurA antibodies were produced in rabbit against the amino acids 1-15:

MSHPSDHVLRPKENA and ordered from Eurogentec.

Proteins were extracted from wandering L3 larvae as following: 20 brains were dissected in PBS and incubated with vortex for 30min in cold buffer lysis (50 mM Tris-HCl pH 7,4; 150 mM NaCl; 1% TritonX-100; 1.5 mM MgCl 2 ; 0.2 mM Na 3 VO 4 ; 0.5 mM DTT, 4 mg/mL NaF;

5.4 mg/mL β-GlycerolPhosphate, cOmplete™ Protease Inhibitor Cocktail (Roche). After 30min of centrifugation at 13000 rpm, 4°C, supernatant was quantified with the Pierce™ BCA Protein Assay Kit (ThermoFisher). 50 g of proteins diluted in Laemmli buffer was loaded onto Mini-PROTEAN ® TGX™ 4-15% gels (BioRad), transferred onto a PVDF membrane (GE Healthcare) and detected using the following antibodies: rabbit anti-AurA

(1:10000, this paper), rabbit anti-actin (1:1000, Sigma Aldrich) and HRP-conjugated secondary antibodies, 1:5000 (Molecular Probes).

To express recombinant 6xHis-AurA proteins, coding sequences of wild type or mutant aurA were cloned into pET15g plasmid (a gift from Gwenaël Rabut) using the cDNA #LD16949

(cDNA ReSource Center) as a matrix and the following primers: Protein purification and kinase assay with histone H3 were done as previously described [START_REF] Bertolin | A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells[END_REF]. Briefly, BL21(DE3) E.coli strains (New England Biolabs, NEB) are transformed with pET15g-Aurora A plasmid and induced with 0.5 mM isopropyl -D-1thiogalactopyranoside for 16h at 20°C. After lysis and sonication, supernatants were incubated with Nickel-chelating resin (ProBond, Thermo Fisher Scientific) for 1h at 4°C

under gentle rotation and proteins were purified under native conditions. For the kinase assay reaction, 10 pmol of purified Histone H3 and/or 10 pmol of purified wild-type or mutant Aurora-A protein were added to kinase buffer (50 mM Tris-HCl (pH 7.5), 25 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol and 0.01% Triton X-100) with or without 100 μM ATP. The reaction was incubated for 20min at 37°C and 10 L was analyzed by western-blot using rabbit anti-Histone H3 pSer10 (1:1000, Merck Millipore, 06-570). Red Ponceau was used to control the amount of total proteins.

Crosses for larvae measurement, larvae staging and pupariation temporal analyses.

All crosses were performed in parallel with controls on the same standard food Nutri-Fly TM Bloomington Formulation (Flystuff, Genesee Scientific) under constant light at 25°C. For each genotype, 15 virgins of aurA ST / TM6 Tb -Sb -or GAL4 lines were crossed with 5 males of aurA lof / TM6 Tb -Sb -or UAS-dsRNA lines. For all following experiments, embryos were laid for 4h in standard food tube. Tb -was used to distinguish sibling larvae from mutant ones while Sb -marker was used for sibling adult flies.

For larval size measurement and staging, larvae were collected from food at given time points and left in cold PBS until no movement was observed. Larvae pictures were taken with a Leica DFC420 macroscope and staged in respect with anterior and posterior spiracles larval development independently from their size: L1 have white posterior spiracles, L2 have orange posterior spiracles and apparition of club shaped anterior spiracles, early L3 have branched anterior spiracles, late L3 have everted anterior spiracles and wandering L3 are found out of the food [START_REF] Park | Deletion of the ecdysis-triggering hormone gene leads to lethal ecdysis deficiency[END_REF].

For pupariation delay quantification, white pupae were counted every 24 h until no wandering L3 larvae were visible for at least 48h in the laying tube. Pupae were individually collected in a plastic plate with humid paper to allow complete metamorphosis. 100% of pupariation was defined as the total of white pupae counted for one given cross per experiment.

Ecdysteroids quantification by LC-MS/MS

For time course experiments between 72 and 192 h after egg laying (AEL), groups of larvae were collected every 12 hours from 6 to 8 independent crosses and pooled in groups of at least 30 larvae. At given time points, "not wandering" and "wandering" L3 larvae were collected in different tubes and considered as different stage points. For each time and/or stage points, ecdysteroids were quantified from two to four independent pools of larvae with the following protocol adapted from [START_REF] Lavrynenko | The ecdysteroidome of Drosophila: influence of diet and development[END_REF].

Larvae were transferred into CK14 tubes (Ozyme) containing ceramic beads and 500 µL methanol (MeOH) was added. Tubes were inserted in a FastPrep FP120 (Savant Bio101) and agitated for 30 seconds. After centrifugation, pellets were re-extracted with 400 µL MeOH and vortexed. After centrifugation, pellets were once again re-suspended in 400 µL MeOH, vortexed and sonicated for 5min. The three supernatants were mixed, 25 ng of polypodine B (internal standard) was added and the extracts were evaporated until dry using a EZ2

(Genevac) vacuum evaporator without heating for 2 h. Residues were then re-dissolved with with MeOH, and gave a linear curve in the range 5-5000 ng/mL. The limit of quantification (LOQ) was 5 ng/mL. Reference ecdysteroids: 20E, makisterone A and polypodine B (purity > 98%) were obtained from various plant sources and purified by BIOSIPE lab.

Larval tissues immunostaining

The following antibodies (dilution, source) were used: rat anti-Deadpan (1:50, 11D1BC7, Abcam), rat anti-Elav (1:50, 7E8A10, Developmental Studies Hybridoma Bank/DSHB), rabbit anti-pH3 (1:1000, Upstate) and mouse anti-Prospero (1:1000, MR1A, DSHB).

Fluorescently conjugated 488-, 546-and 647-secondary antibodies (Invitrogen) were used at 1:250. Larval tissues from timed and/or staged larvae were dissected in phosphate-buffered saline (PBS) and fixed for 15min in 4% formaldehyde. Tissues were washed in PBS with 0.1% Triton X-100 (PBT) for 10min and incubated overnight with primary antibodies at 4°C under agitation.

Results

Drosophila larval development is temporally affected in the absence of AurA kinase activity.

In order to investigate aurA lof phenotypes during larval development we took advantage of an allelic series: aurA 3A , aurA 8839 , aurA 3C and aurA 14641 and one null mutant, aurA ST (Fig. 1A).

AurA mRNA is detected for all mutants excepted aurA ST (Sup. Fig. 1A), while AurA protein is detectable at the expected size for aurA 8839 , aurA 3C and aurA 14641 and absent for aurA ST and aurA 3A (Fig. 1B). The fact that the antibody we used is able to detect the AurA 3A protein isoform expressed in bacteria, confirms that no AurA protein were detectable in aurA 3A mutants (Sup. Fig. 1B). We further characterized these AurA mutant proteins by measuring their kinase activity in vitro towards histone H3 Serine10, a classical in vitro substrate of AurA [START_REF] Hsu | Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes[END_REF]. We could not detect any kinase activity for any of these AurA mutant forms while we did for the wild-type isoform (Sup. Fig. 1C). Therefore, the allelic series includes mutants with or without AurA proteins but all depleted of detectable AurA kinase activity.

We then analyzed Drosophila early development and strikingly found that all mutants show a delay in pupariation despite the variability observed at different time points for a given genotype (Fig. 1C). Although the expressivity of this phenotype is variable between mutants, they all present a clear delay in pupariation as visible at 144h after egg laying (144h AEL)

when the mean of control genotypes has surpassed 50% of pupariation. We decided to pursue the study with the null allele aurA 3A , which present the strongest delay and has no detectable protein, and the hypomorph aurA 14641 , which has the weakest delay and expresses a kinase dead protein. In physiological conditions, larval development starts at 24h AEL and is temporally stereotyped. It lasts for four days and is divided into four stages of 24h each; 1 st instar (L1), 2 nd instar (L2), early 3 rd instar (early L3) and late 3 rd instar (late L3) including the wandering L3 stage where larvae start crawling prior to pupariation (Fig. 1D). To identify how larval development of aurA lof mutants is temporally affected, we evaluated the larval stages reached every 24h (Fig. 1E). Surprisingly, we first noticed that mutant larvae are predominantly already in L3 stages at 72h AEL (62% for aurA 3A/ST , n total =48 and 72% for aurA 14641/ST , n total =40) when controls are for the most part in L2 stage (75%, n total =138).

Nonetheless, mutant larvae are still in L3 stages at 120h AEL (100% for aurA 3A/ST , n total =59 and 100% for aurA 14641/ST , n total =56) when 36% of controls (n total =183) have already reached the pupal stage. Furthermore, the control shows 62% pupae (n total =214) at 144h AEL while the hypomorph aurA 14641/ST expressing the inactive kinase shows 31% pupae (n total =29) and the null aurA 3A/ST remain in L3 (100%, n total =27). At 168h AEL controls show 100% pupae (n total =121), the hypomorph aurA 14641/ST 88% pupae (n total =34) while the null aurA 3A/ST only 29% (n total =24). These data show that loss of aurA function increases the duration of L3 stage and that the effect is more pronounced with the null mutant (aurA 3A/ST ) compared to the mutant expressing the inactive kinase (aurA 14641/ST ).

aurA loss-of-function affects ring gland development which in turns alters

ecdysteroids production and larval growth rate and speed.

Extension of the L3 stage usually correlates with a phenotype of enlarged larvae due to a longer duration of larval feeding [START_REF] Mcbrayer | Prothoracicotropic hormone regulates developmental timing and body size in Drosophila[END_REF]. As predicted, aurA lof mutant L3 larvae are bigger than control L3 larvae at 120h AEL (Fig. 2A and sup. Fig. 2A). However, larger larvae are already observed in aurA lof mutants at 72h AEL (Fig. 2A and sup. Fig. 2A).

Noteworthy, the majority of control larvae are in L2 stage at 72h AEL while the majority of aurA lof mutants have already reached the L3 stage (Fig. 1E), which could explain the larval size difference. To circumvent this bias of accelerated early larval development in aurA lof , we compared the size of larvae at given larval stages rather than at given developmental time points (Fig. 2B and sup. Fig. 2B). At both L2 and late L3 stages, aurA lof mutant larvae sizes are bigger than the controls (Fig. 2C) which confirm that aurA lof mutant systemic growth is accelerated from the L2 stage.

Early larval growth rate and speed are dependent upon ring gland development and ecdysteroids production. Indeed, defective growth and/or reduction of insulin/Pi3K growthpromoting signaling pathway in ring gland inhibit early release of ecdysteroids required in the fat body to restrict early larval systemic growth [START_REF] Colombani | Antagonistic actions of ecdysone and insulins determine final size in Drosophila[END_REF][START_REF] Mirth | The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster[END_REF].

Furthermore, the precise timing of each molt (L1/L2 and L2/L3), and the onset of metamorphosis, are finely regulated in a stereotyped manner by pulses of ecdysteroid hormones [START_REF] Riddiford | Hormones and Drosophila Development. The Development of Drosophila melanogaster[END_REF]. If the ecdysteroids synthesis pathway is affected, it induces a gradient of phenotypes ranging from delayed-L1 to pupal-lethal through arrested-L3 [START_REF] Danielsen | A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing[END_REF]. We reasoned that both ecdysteroid-related phenotypes aka increased larval body size (Fig. 2B) and pupariation delay (Fig. 1C) could reflect a defect in ecdysteroids production. Using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS), we quantified the temporal profiles of 20E and Maki-A in control and aurA lof mutant larvae from 72h AEL (stage L2) until larvae reach the wandering L3 stage (Sup. Fig. 3A andB). In aurA 3A/ST mutants, we observed a clear deficit in 20E and Maki-A amounts despite the strong variability at each time points. In control larvae, "not wandering"

and "wandering" L3 larvae were present at each time points from 96h AEL which might explain the observed variability of ecdysteroids quantity. Therefore, we discriminated the results of "not wandering" L3 vs "wandering" L3 for control and aurA 3A/ST mutant larvae by pooling our quantification results from 96h AEL (Fig. 2C). Basal levels of ecdysteroids were observed in "not wandering" L3 aurA 3A/ST mutants but the major peak was not observed in "wandering" L3, which confirms that AurA is required for ecdysteroids production.

While ecdysteroid precursors, Ecdysone and 24-methylecdysone, are produced in the prothoracic gland (PG) of the ring gland, they are rapidly converted into the main active molting hormones, 20-Hydroxyecdysone (20E) and Makisterone A (Maki-A) respectively, in peripheral tissues including the fat body (reviewed in [START_REF] Lafont | Ecdysteroid Chemistry and Biochemistry[END_REF]. To identify the organ(s) in which AurA activity is required for this ecdysteroids major peak production, we inactivated aurA in the ring gland or the peripheral tissues. We used the UAS/GAL4 system to induce RNA inactivation (RNAi) specifically in these tissues and search for one of the readouts of defective ecdysteroids production: a pupariation delay. Although we observed a limited effect when inactivating aurA in peripheral tissues [START_REF] Ballard | Gbb/BMP signaling is required to maintain energy homeostasis in Drosophila[END_REF]) using a pumpless (ppl)-GAL4 driver (Sup. Fig. 4B), pupariation was delayed when aurA was inactivated in the PG using a phantom (phm)-GAL4 driver (Fig. 2D). As AurA function appears essential in the ring gland, we looked ring gland larval development in aurA lof mutants. We could not easily dissect the ring gland at early stage due to its smaller size in mutants (not shown) but at 120h AEL, ring gland was measurably smaller in aurA lof mutants compared to controls (Fig. 2E-F and Sup. Fig. 3C). However, it was already reported that it is not the size of the ring gland that matters for ecdysteroids production [START_REF] Colombani | Antagonistic actions of ecdysone and insulins determine final size in Drosophila[END_REF] but rather the endocycle progression of cells in the PG [START_REF] Ohhara | Nutrient-dependent endocycling in steroidogenic tissue dictates timing of metamorphosis in Drosophila melanogaster[END_REF] Consequently, AurA kinase activity is required for the major peak of ecdysteroids production controlling the timing of pupariation.

aurA loss-of-function affects wing disc development rate and speed

Although it has been previously described that imaginal disc development is not altered in wandering L3 larvae of aurA mutants (aurA 8839 , aurA 14641 and aurA ST , [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF])), we wondered if it could be defective at earlier larval stages and if these defects could have been rescued during the prolonged L3 stage and then missed in wandering L3 aurA lof mutants. As wing disc development reaches its maximum speed during L3 stage, we looked at them from the beginning of L3 stage in aurA lof mutants. We were unable to dissect aurA lof mutant wing discs at 96h AEL as we could not identify them with enough confidence due to their small size and their unidentifiable morphology (data not shown). At 120h AEL (Fig. 3A), we observed that wing discs of "not wandering" L3 aurA lof mutant larvae are smaller than "wandering" L3 controls (p=4.72x10 -6 for aurA 3A/ST , n=37 and p=5.26x10 -3 for aurA 14641/ST , n=35), while the difference is mostly abolished at 168h AEL between "wandering" L3 aurA lof mutant and controls (p=2.43x10 -3 for aurA 3A/ST , n=20 and p=0.94 for aurA 14641/ST , n=16). Furthermore, wing disc morphology is also initially altered in aurA 3A/ST mutants as the wing pouch is rarely detected in wing discs before 168h AEL (n=4/37 at 120h AEL and n=17/20 at 168h AEL in mutants, Fig. 3D,G) while it is visible in aurA 14641/ST mutant wing discs (Fig. 3C,F) and control wing discs from 120h AEL (Fig. 3B,E).

These observations demonstrate that loss of aurA reduces wing disc development and that wing discs take advantage of the prolonged L3 stage to recover and resume normal development (size and morphology). Again, as observed for the pupariation delay, the phenotype observed in the mutant aurA 14641/ST (inactive kinase) is less pronounced than that of the mutant aurA 3A/ST (no protein), which further supports the link between aurA loss-offunction and reduction of wing disc growth speed.

The deceleration of wing disc development can result from the reduction of ecdysteroid levels [START_REF] Herboso | Ecdysone promotes growth of imaginal discs through the regulation of Thor in D. melanogaster[END_REF] and/or cell-autonomous defects. Indeed, loss of AurA kinase activity increases cell cycle length by decelerating the entry into and progression through mitosis [START_REF] Caous | Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila[END_REF][START_REF] Lim | Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth[END_REF], which could explain the fact that wing discs need more time to achieve their development in aurA lof mutant larvae. To bypass the ecdysteroids defect effect, we performed a clonal analysis to compare the multiplication rate of aurA ST homozygous mutant cells and wild-type sibling cells, which derive from heterozygous mother cells after induced recombination [START_REF] Xu | Analysis of genetic mosaics in developing and adult Drosophila tissues[END_REF]. We observed that aurA ST homozygous mutant clones were significantly smaller than their wild-type siblings (Fig. 2I' versus 2J' and 2K), which demonstrates that aurA ST wing disc cells divide at a smaller rate than their wild-type siblings. Furthermore, the mitotic index was higher in aurA ST clones than in wild-type clones (Fig. 2G-H), which confirms that aurA loss-of-function affects wing disc development in a cell-autonomous manner.

In case of an alteration of wing disc development, wing disc cells produce an excess of Dilp8 which activates leucine-rich repeat-containing G protein-coupled receptor 3 (LgR3)-neurons to delay pupariation until wing disc development is corrected [START_REF] Colombani | Drosophila Lgr3 Couples Organ Growth with Maturation and Ensures Developmental Stability[END_REF][START_REF] Garelli | Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing[END_REF]. These LgR3 neurons inhibit two pairs of central brain neurons, which innervate the PG and produce the prothoracicotropic hormone required for pupariation-inducing ecdysteroids release in wandering L3 larvae [START_REF] Mcbrayer | Prothoracicotropic hormone regulates developmental timing and body size in Drosophila[END_REF][START_REF] Warren | Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster: correlations with changes in gene activity[END_REF]. To test if the Dilp8-LgR3 pathway could be activated in aurA lof mutants, we specifically inactivated aurA in the wing discs by RNAi using two different GAL4 drivers: serrate (ser) and pdm2. Although we observed wing margin phenotypes (data not shown), inactivation of aurA was not sufficient to induce a pupariation delay and validate this hypothesis (Sup. Fig. 4E,F). Together, these data show that loss of aurA function reduces wing disc development speed by slowing down cell cycle speed and the pupariation delay allows for correction of this developmental defect.

aurA is required for NB-cell lineage differentiation in larval central brain.

Examining AurA function during early Drosophila development helped us to define its pleiotropic roles with multiple phenotypic readouts. Indeed, aurA lof larval developmental rate and speed defects might result indirectly from primary growth defects in the ring gland (Fig.

2) while wing disc development deceleration results from cell-cycle cell-autonomous defects (Fig. 3). It has been previously reported that the volume of the brain lobes of wandering L3 larvae in aurA lof mutants is much greater than that of the controls and is correlated with a huge number of NB [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]. To test if the increased brain lobe size is directly due to the larger size of the aurA lof mutant larvae (Fig. 2B), we measured brain lobe circumferences in another genetic background with enlarged larval size. We took advantage of a validated genetic model in which RNAi of avalanche (avl) in imaginal discs induces a delayed L3 stage and enlarged larvae [START_REF] Colombani | Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing[END_REF]. Although aurA lof mutants and avl dsRNA larvae have the same enlarged larval size at 120h AEL (Sup. Fig. 5A), brain lobe sizes are smaller than controls in both genotypes (Sup.

Fig. 5B). In later stages (144h and 168h AEL) when aur lof and avl dsRNA have reached the wandering stage, brain lobes are still smaller than controls in avl dsRNA while they become much larger in aur lof (Sup. Fig. 5B). Therefore, the enlarged brain lobes phenotype does not appear to be a direct consequence of increased larval growth and/or prolonged L3 stage.

Surprised by the initial smaller size of aur lof brain lobes, we questioned whether the growth rate of brain lobes could be decreased during early larval development as observed in the ring gland. In fact, brain lobe perimeters are significantly smaller in aurA 3A/ST and to a less extent in aurA 14641/ST mutants in early and late L3 stages at 96h AEL (Fig. 4A, p=2.85x10 -7 for aurA 3A/ST , n=33 and p=0.03 for aurA 14641/ST , n=33). Then, brain lobe perimeters progressively increase during aurA lof L3 stage and eventually become bigger than controls, as previously described, in wandering L3 mutant larvae at 192h AEL (Fig. 4A, p=2.85x10 -7 for aurA 3A/ST , n=47 and p=0.03 for aurA 14641/ST , n=8 and Sup. Fig. 6A-G). We confirmed these results with our allelic series: brain lobes sizes are indeed initially smaller in early larval development and become bigger in latest stages independently of the allelic combination which further highlights an early larval function for AurA in brain development (Sup. Fig. 6A). Numerous previous studies have shown that this enlarged brain phenotype of aurA lof wandering larvae is correlated with a huge number of NB in which the length of the cell cycle is increased due to a delay of entry into mitosis [START_REF] Caous | Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila[END_REF][START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Lim | Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]. As central brain NB exit quiescence around 48h AEL at the end of L1 [START_REF] Ding | The Hippo signalling pathway maintains quiescence in Drosophila neural stem cells[END_REF][START_REF] Sousa-Nunes | Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila[END_REF], we looked at the number of cells positive for Deadpan (Dpn), a marker of NB-cell identity, in the central brain from 48h AEL (end of L1). To our surprise, the number of Dpn-positive (Dpn + ) cells appeared higher in aurA 3A/ST brain lobes of L2/L3 stages at 72h AEL (Fig. 4C,F) and increased in such a dramatic way that Dpn + cells cannot be counted unambiguously in L3 stages at 120h AEL (Fig. 4D,G)

when brain lobes are still smaller than the control (Sup. Fig. 6A). Furthermore, the central brain organization is highly affected in aurA 3A/ST mutants and the optic lobe is not always distinguishable at 120h AEL (n= 12/30 central brains, three experiments, data not shown).

Together, these data correlate with a defect in NB-cell lineage differentiation. Indeed, as described for other aurA lof mutant alleles, the number of neurons (marked by Embryonic Lethal Abnormal Visual system, ELAV, a neuron-specific protein) is strongly reduced in aurA 3A/ST brain lobes (Sup. Fig. 6H,I and [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]). Furthermore, we observed a large quantity of cells with a small nucleus positive for both Dpn and Prospero (Dpn + , Pros + ) in aurA 3A/ST brain lobes (Sup. Fig. 6F,G). These cells originate from defective NB asymmetric cell divisions which give rise to two (small nucleus, Dpn + , Pros + ) identical cells [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF][START_REF] Wirtz-Peitz | Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization[END_REF] (referred to as NB-GMClike cells hereafter). To confirm that a complete loss of aurA function impairs NB-cell lineage, we used clonal analysis. In wild-type clone, we can observe one NB which recently divided (large nucleus, Dpn + , Pros + ) is seen with one new GMC (small nucleus, Dpn + , pros + ) and numerous older GMC (small nucleus, Dpn -, Pros + ) (Fig. 4H-H'''). In aurA ST mutant clone, one NB is seen with numerous NB-GMC-like cells (small nucleus, Dpn + , Pros + ) (Fig.

4I-I'''

). Indeed, those NB-GMC-like cells is significantly increased in mutant clones (Fig. 4J, p=1.2x10 -4 , n=14). We also observed a small increase number of GMC (Dpn -, Pros + ) cells in growth rate and speed [START_REF] Caldwell | Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release[END_REF][START_REF] Colombani | Antagonistic actions of ecdysone and insulins determine final size in Drosophila[END_REF][START_REF] Mirth | The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster[END_REF].

Indeed, the hormones fulfill multiple functions such as the repression of larval growth by inhibiting dMyc expression, through its receptor EcR, in the fat body [START_REF] Delanoue | The steroid hormone ecdysone controls systemic growth by repressing dMyc function in Drosophila fat cells[END_REF].

Therefore, the increased larval size that we observed from early larval development might indirectly result from aurA loss-of-function in the ring gland. We could not validate this hypothesis as inactivation of aurA by RNAi using ubiquitous, ring gland-or fat body-specific GAL4 did not produce enlarged larvae (data not shown). Nevertheless, quantification of the two majors ecdysteroids, 20E and Maki-A, showed that although aurA lof mutants can produce basal levels of ecdysteroids, they are unable to produce the major peak required in wandering L3 larvae to pupariate. This result reaffirms our idea that AurA function in the ring gland is necessary for ecdysteroids production peaks and indirectly for early systemic larval growth control. As a consequence, enlarged aurA mutant larvae reaches the drosophila "critical weight" faster, where feeding is no longer necessary (for review see [START_REF] Edgar | How flies get their size: genetics meets physiology[END_REF]), and should pupariate earlier than controls. On the contrary, we observed that aurA lof mutant larvae are characterized by a prolonged L3 stage and consequently a delay in pupariation. This phenotype is directly correlated with the absence of an ecdysteroids peak in wandering L3 mutant larvae. Furthermore, we validated that the pupariation delay can be induced by inactivation of aurA in the ring gland and not in peripheral tissues as central brain, fat body or wing disc (Sup. Fig. 4). Although we could not detect this major ecdysteroids peak, aurA mutant larvae are still able to pupariate. This observation may highlight the limit of ecdysteroids quantification which require a large quantity of larvae to be efficient, however, as aurA lof mutant larvae show a prolonged L3 stage, they are heterogeneous in term of larval timing and the major peak of ecdysteroids might not be present in enough larvae to be detected. Or it may also highlight the fact that this major ecdysteroids peak is not the unique pupariation commitment peak. Indeed, smaller ecdysteroids peaks precede the major one [START_REF] Warren | Studies on the Black Box: incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta[END_REF][START_REF] Lavrynenko | The ecdysteroidome of Drosophila: influence of diet and development[END_REF] and could also act as commitment peak [START_REF] Warren | Studies on the Black Box: incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta[END_REF].

The pupariation delay results in a prolonged L3 stage during which most larval tissue development is undertaken. AurA kinase function is essential in cell cycle regulation and not surprisingly in larval tissue development also. In absence of AurA function, cell cycle speed is cell-autonomously affected in wing disc and inhibits its developmental speed. Indeed, although wing discs are smaller at 120h AEL, they reach a wild type size and morphology in wandering L3 mutant larvae. These results comfort our hypothesis that, in absence of AurA kinase activity, wing discs benefit from the prolonged L3 stage to compensate for being developmentally delayed.

The prolonged L3 stage observed in all aurA lof mutants is also associated with appearance of enlarged brain lobes in delayed L3 larvae. Nonetheless, these brain lobes are actually smaller in early larval stages, which can be explained by defects in NB-cell lineage. Indeed, loss of aurA affects NB asymmetric cell division and give rise to over-proliferative NB-GMC-like cells [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF] which do not provide the normal GMC and/or neurons number to fill the central brain. These cells are probably unable to respond to the microenvironment of the central brain that normally controls the number of NB divisions during larval development [START_REF] Homem | Drosophila neuroblasts: a model for stem cell biology[END_REF][START_REF] Lim | Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth[END_REF] and begin to proliferate rapidly and indefinitely. Indeed, the number of dividing NB-GMC-like cells is remarkably high taking in account that cell cycle length is decreased in Dpn+ cells of aurA lof mutants [START_REF] Caous | Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila[END_REF][START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF]. This over-proliferation phenotype eventually gives rise to enlarged brain lobes correlated with the prolonged L3 stage. Furthermore, when transplanted in the abdomen of wild-type Drosophila hosts, NB-GMC-like cells embedded in a piece of brain become highly tumorigenic [START_REF] Caous | Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila[END_REF][START_REF] Caussinus | Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster[END_REF][START_REF] Rossi | Studying tumor growth in Drosophila using the tissue allograft method[END_REF]. Surprisingly, we could only reproduce partially this NB phenotype by RNAi using a strong NB GAL4 driver (69B) but not with weaker NB-specific GAL4 drivers worniu-or inscutable-GAL4 (data not shown). This suggests that AurA cellautonomous function in NB is not sufficient to explain all its functions in NB asymmetric cell-division and that its functions in other central brain cells such as glial cells [START_REF] Lim | Glial-Specific Functions of Microcephaly Protein WDR62 and Interaction with the Mitotic Kinase AURKA Are Essential for Drosophila Brain Growth[END_REF] or in other tissues might also be involved.

Interestingly, although AurA was initially defined as a tumor-suppressor in Drosophila larvae, it did not appear to be the case in all developing tissues as no over-proliferation were observed in imaginal discs as described for other tumor-suppressor like Brat, disc large or lethal giant larvae [START_REF] Gateff | Tumor suppressor and overgrowth suppressor genes of Drosophila melanogaster: developmental aspects[END_REF]. On the contrary, our results show that in absence of AurA, symmetric cell division is decelerated which decreases larval tissues developmental rate. In mice, it was reported that loss of aurkA increases tumor-incidence after 50 to 60 weeks suggesting a possible tumor-suppressor effect [START_REF] Lu | Aurora A is essential for early embryonic development and tumor suppression[END_REF]. Because of its shorter developmental timing, this cannot be observed in Drosophila aurA lof mutants, which are also pupal-lethal (data not shown and [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Moon | Aurora A kinase negatively regulates Rho-kinase by phosphorylation in vivo[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]). These results can be taken as an indication that AurA could be a tumor-suppressor only in the case of asymmetric cell division. Indeed, AurA has a dual role in NB-asymmetric cell division: being a major regulator of cell division but also a regulator of the asymmetry.

Loss of AurA induces defective asymmetric segregation of Numb (a determinant of GMC cell fate) and aPKC (a central regulator of cell polarity) as well as a defect of central spindle orientation in NB division [START_REF] Lee | Drosophila Aurora-A kinase inhibits neuroblast selfrenewal by regulating aPKC/Numb cortical polarity and spindle orientation[END_REF][START_REF] Wang | Aurora-A acts as a tumor suppressor and regulates self-renewal of Drosophila neuroblasts[END_REF]. Conceptually speaking, it would be interesting to study AurA function in other asymmetric cell divisions, such as adult stem cells, to further validate or invalidate the tumor-suppressor function of aurA in Drosophila.

Figure legends

  aurA 8839 reverse). Gibson Assembly Master Mix (NEB) was used to clone mutant aurA using the following primers for intermediate amplicons: for aurA 3A 5'-CGCGGGAAAAGGAATGTGGTGGCCCTAAAA-3' (forward) and 5'-TTTTAGGGCCACCACATTCCTTTTCCCGCG-3' (reverse); for aurA14641

  . At 120h AEL, cells of the aurA 3A/ST mutant PG are still mitotic while very few mitotic figures are observed in control PG (Sup. Fig 3D). To evaluate the endocycle progression of PG cells, we measured the size of their nucleus (Fig 2H). At 120h AEL, nuclei size density does not follow a Gaussian distribution in late L3 aurA 3A/ST mutants compared to wandering L3 controls. However, at 144h AEL (late L3) and 168h AEL (wandering L3), the nuclei size density distribution of mutants tends to resemble the controls while almost no mitotic figures were observed (Fig 2G). In conclusion, loss of aurA function reduces ring gland developmental speed presumably by slowing down cell cycle speed and thus delaying endocyle progression.

  aurAST mutant clones which suggest that mutant NB divided into NB-GMC-like cells which multiply and eventually give rise and/or differentiate into GMC (Fig. 4K, p=0.069, n=14). In conclusion, AurA loss-of-kinase activity in central brain NB gives rise to cells with an intermediate identity between NB and GMC, which greatly proliferate during central brain larval development. In addition to the prolonged L3 stage, the proliferation of these NB-GMC-like eventually give rise to enlarged brain lobes in aurA lof mutants. Discussion In this study of AurA function during the development of Drosophila larvae, we found that loss of AurA kinase activity results in pleiotropic effects in different tissues (ring gland, imaginal wing disc and central brain) and reduces their growth rate throughout larval development. In early larval development, AurA is first needed in the ring gland to control its growth speed. It is well established that a defect in early ring gland development affects the release of the ecdysteroid precursors by its PG cells and indirectly accelerates early larval

Figure 1 :

 1 Figure 1: Aurora-A loss of kinase activity affects drosophila larval development speed. A) Representation of AurA proteins encoded by the wild-type and allelic series of aurA mutants. B) Western blots of protein extracts from brains using an anti-AurA antibody (top panel) and an actin antibody (lower panel). AurA is not detected in aurA ST and aurA 3A while it is at the expected size in w 1118 , aurA ST/+ , aurA 8839 , aurA 3C and aurA 14641 . C) Graph of percentage of larvae that have pupariated at the given hours after egg laying (AEL). Pupariation is delayed in all aurA mutant genetic backgrounds: aurA 3A/ST (200 larvae, 2 experiments), aurA 8839/ST (191 larvae, 2 experiments), aurA 3C/ST (176 larvae, 2 experiments) and aurA 14641/ST (108 larvae, 2 experiments) compared to w 1118 (977 larvae, 3 experiments). D) Scheme of the larval developmental time scale: L1, L2, early L3, late L3 and wandering L3 are indicated. Morphology of anterior and posterior spiracles to stage the larvae, independently from AEL time points, is represented for L1, L2, early L3 and late L3 stages. E) Percentage of different larval stages and pupae observed in w 1118 , aurA 3A/ST and aurA 8839/ST mutants at given AEL time points during larval development. At each time points, two experiments were at least performed and between 24 and 244 larvae were staged for L1 (purple), L2 (green), early L3 (yellow), late L3 (orange), wandering L3 (red) and pupae (blue).

  Figure 3

  Tissues were washed in PBT and incubated with secondary antibodies at room temperature. When Hoescht was used, tissues were rinsed once in PBT and incubated

	10min with Hoescht (1:100000). Tissues were washed in PBS for 10min and mounted in
	glycerol 90% with 2.5% DABCO. All brains were mounted with dorsal surface up and ventral
	surface down.
	2.7. Confocal Microscopy
	Fluorescent images were acquired with a Leica SP8 confocal equipped with a 20X, 40X or

63X objectives and a laser module (excitation at 405, 488, 561 and 633 nm) at the Microscopy Rennes imaging Center. Z stacks were recorded at 0.5 μm intervals. Confocal and DIC images were acquired and processed using the LAS AF software and further analyzed with ImageJ. Wing disc, ring gland and central brain circumferences were measured using Polygon selection and Measure tools from ImageJ.

2.8. Statistical analyses

Statistics were performed on RStudio using Mann-Whitney test to compare two specific datasets. Multiple comparison within a given dataset (with different aurA lof genotypes or time points) was performed with a Mann-Whitney non-parametric test as the variances of data sets were not equal (tested with a Barlett test), p<0.05 when data of two sets are significantly different, p<0.025 for data of three sets and p<0.0125 data of five sets. Comparison of nuclei density in ring glands of given genotypes was performed with a Two-sample Kolmogorov-Smirnov test, p<0.05 when data of two sets are significantly different.

Acknowledgements

We are indebted to Aurore Dussert for the help with production of aurA CRISPR alleles. We thank Fumio Matsuzaki, Pierre Léopold, Jean-René Hyunh, the Bloomington Stock Center and the DSHB for fly stocks and antibodies. We thank the MRiC (Microscopy Rennes imaging Center). We are grateful to Julien Colombani and Pascal Thérond for helpful discussion on phenotypic analyses, Yann Le Cunff for statistical work, Rebecca Smith for English proof-reading and Céline Burcklé for comments on the manuscript. This work was supported by the CNRS, the University of Rennes 1, the Ligue Nationale Contre la Cancer (équipe labélisée 2014-2017). L.V. was supported by the Ligue Nationale Contre la Cancer and the Région Bretagne.

A) AurA mRNA is detected in all mutants, but in the deletion aurA ST , at the expected size (conventional RT-PCR). B) AurA polyclonal antibody recognizes wild-type AurA protein and bacteria expressed and purified AurA 3A isoform. No protein is detected in aurA 3A mutant brains. C) In vitro AurA kinase assay and corresponding western blot illustrating the abundance of a Ser10-positive band on histone H3 with wild-type AurA and the lack of detectable kinase activity with all mutant isoforms produced in vitro. A) Box plots of larval size at 120h AEL for w 1118 (x, n=45), aurA 3A/ST (◊, n=24) and avalanche (avl) dsRNA (*, n=13) genotypes. Size are similar between aurA 3A/ST and avl dsRNA (p=0.9126) and bigger to controls (aurA 3A/ST , p=7,23.10 -16 ; avl dsRNA , p=7.6x10 -12 ). B) Box plots showing lobe brain perimeters at different developmental time points. At 120h AEL, brains are smaller in aurA 3A/ST (n=80) and avl dsRNA (n=10) compared to w 1118 (n=113, p<2.2x10 -16 and p=1.84x10 -7 , respectively). At 144h AEL and 168h AEL, brains of aurA 3A/ST (n=116 and 80) are now bigger compared to w 1118 (n=65 and 58, p=1.8x10 -4 and 3.25x10 -6 , respectively). On the contrary, brains of avl dsRNA stay smaller (n=10, p=1.9x10 -7 at 144h AEL and n=11, p=2.4x10 -4 at 168h AEL) certainly due to the fact that avl function is essential to development.