Françoise Simon

Anne Ramat

Sophie Louvet-Vallée

Jérôme Lacoste

Angélique Burg

Agnès Audibert

Michel Gho
email: michel.gho@sorbonne-universite.fr

Shaping of Drosophila neural cell lineages through coordination of cell proliferation and cell fate by the BTB-ZF transcription factor Tramtrack-69

Keywords: Ttk69, Cyclin-E, Hamlet, Precursor cells, Sensory organs

Cell diversity in multicellular organisms relies on coordination between cell proliferation and the acquisition of cell identity. The equilibrium between these two processes is essential to assure the correct number of determined cells at a given time at a given place. Using genetic approaches and correlative microscopy, we show that Tramtrack-69 (Ttk69, a BTB-ZF transcription factor ortholog of the human PLZF factor) plays an essential role in controlling this balance. In the Drosophila bristle cell lineage, producing the external sensory organs composed by a neuron and accessory cells, we show that ttk69 loss of function leads to supplementary neural-type cells at the expense of accessory cells. Our data indicate that Ttk69

) regulates cell fate acquisition and terminal differentiation by downregulating the expression of hamlet and upregulating that of Suppressor of Hairless, two transcription factors involved in neural-fate acquisition and accessory-cell differentiation, respectively. Thus, Ttk69 plays a central role in shaping neural cell lineages by integrating molecular mechanisms that regulate progenitor cell-cycle exit and cell-fate commitment.

INTRODUCTION

Organisms are composed of morphologically and functionally distinct cell types. Such cell diversity is generated from a restricted set of precursor cells producing a limited number of differentiated cells. Division of precursor cells gives rise to daughter cells that differentiate and acquire specific fates. The transit from a proliferative to cell-cycle arrested state during this process is tightly regulated and requires changes in transcriptional programs.

Disentangling the molecular mechanisms that control the balance between proliferation and differentiation is essential for understanding the formation and maintenance of organisms, as well as human diseases, such as cancer, in which this process is disturbed. BTB-ZF transcription factors are involved in a wide variety of biological processes [START_REF] Kelly | POZ for effect -POZ-ZF transcription factors in cancer and development[END_REF]. They include Drosophila Broad-complex factors (BR-C), Bric-à-brac (Bab), and several pox virus zinc-finger proteins [START_REF] Chaharbakhshi | Broad-complex, tramtrack, and bric-à-brac (BTB) proteins: Critical regulators of development[END_REF]. All possess a protein/protein interaction motif (BTB/POZ) at the N-terminus that allows protein homo and multimerization and one or several zinc-finger DNA-binding motifs [START_REF] Bonchuk | Drosophila BTB/POZ Domains of "ttk Group" Can Form Multimers and Selectively Interact with Each Other[END_REF].

These proteins are conserved from Saccharomyces cerevisiae to Homo sapiens and act as transcriptional repressors or activators, depending on the BTB domain [START_REF] Siggs | The BTB-ZF transcription factors[END_REF]. The founding BTB-ZF members are all Drosophila transcriptional repressors that regulate processes such as metamorphosis, ovary development, and neurogenesis [START_REF] Karim | The Drosophila Broad-Complex plays a key role in controlling ecdysone-regulated gene expression at the onset of metamorphosis[END_REF][START_REF] Guo | tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS[END_REF][START_REF] Sahut-Barnola | Drosophila ovary morphogenesis: analysis of terminal filament formation and identification of a gene required for this process[END_REF]. In vertebrates, the human BTB-ZF, promyelocytic leukemia zinc finger (PLZF), acts as a tumor-suppressor maintaining cell growth inhibition and quiescence by transcriptional repression of the c-myc proto-oncogene [START_REF] Mcconnell | Growth Suppression by Acute Promyelocytic Leukemia-Associated Protein PLZF Is Mediated by Repression of c-myc Expression[END_REF]. Accordingly, plzf loss of function has been correlated with prostate and lung cancer [START_REF] Nenseth | Role of PLZF as a tumor suppressor in prostate cancer[END_REF]. Moreover, this factor regulates organogenesis by controlling the balance between self-renewal and differentiation of neural stem cells [START_REF] Sobieszczuk | A feedback loop mediated by degradation of an inhibitor is required to initiate neuronal differentiation[END_REF][START_REF] Gaber | PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors[END_REF]. Overall, BTB-ZF proteins have fundamental and conserved roles during development, controlling cell proliferation and differentiation.

The Drosophila ortholog of PLZF, Tramtrack (Ttk), also plays multiple roles during development, including cell proliferation and cell-fate decisions in the nervous system, intestinal stem cells, photoreceptors, and tracheal cells [START_REF] Giesen | Glial development in the Drosophila CNS requires concomitant activation of glial and repression of neuronal differentiation genes[END_REF][START_REF] Lai | Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development[END_REF][START_REF] Badenhorst | Tramtrack controls glial number and identity in the Drosophila embryonic CNS[END_REF][START_REF] Araujo | Tramtrack regulates different morphogenetic events during Drosophila tracheal development[END_REF][START_REF] Wang | Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages[END_REF][START_REF] Liu | Concise Review: Balancing Stem Cell Self-Renewal and Differentiation with PLZF[END_REF]. In particular, Ttk is a key regulator of cell fate in the peripheral nervous system, in which it promotes non-neural instead of neural fates [START_REF] Guo | tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS[END_REF]. Ttk is considered to be a transcriptional repressor from the initial studies on even skipped and fushi tarazu genes (Harrison andTravers 1988, 1990;[START_REF] Brown | Repression of the Drosophila fushi tarazu (ftz) segmentation gene[END_REF]. The ttk locus encodes two proteins, Ttk69 and Ttk88, via alternative splicing [START_REF] Read | Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities[END_REF]. Both isoforms share a common conserved Nterminal BTB/POZ domain but contain divergent C-terminal zinc-finger C2H2-type domains for DNA binding, conferring specific DNA binding and probably independent functions for each isoform [START_REF] Read | Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities[END_REF]. In spite of these differences, Ttk69 appears to have a broader spectrum of functions than Ttk88. For example, Ttk69, but not Ttk88, promotes specific non-neuronal fates, such as cone cells, during eye development [START_REF] Lai | Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development[END_REF] and enteroblast cells during intestine development [START_REF] Wang | Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages[END_REF]. Ttk has been also shown to be involved in cell-cycle regulation. It has been shown that overexpression of Ttk69, but not Ttk88, causes the complete loss of mitosis in the eye disc morphogenetic furrow through the repression of String, the positive regulator of the G2/M transition [START_REF] Baonza | Pointed and Tramtrack69 establish an EGFR-dependent transcriptional switch to regulate mitosis[END_REF].

Similarly, Ttk69 negatively regulates intestinal stem-cell proliferation [START_REF] Wang | Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages[END_REF].

Finally, in mechanosensory organs, ttk loss of function leads to complete transformation of sensory cells into neurons [START_REF] Guo | tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS[END_REF] and the loss of Ttk69 alone also induces cell proliferation [START_REF] Audibert | Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage[END_REF]. In order to dig into the mechanisms by which Ttk69 control cell determination and division, we focus on how Ttk69 controls the balance between cell proliferation and the acquisition of cell fate in the bristle system. To this end, we extensively used the ttk 1e11 allele, a P-element insertion flanked by a deletion in the ttk locus [START_REF] Xiong | Tramtrack is a transcriptional repressor required for cell fate determination in the Drosophila eye[END_REF], which is considered to be a specific null allele for ttk69 [START_REF] Lai | Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development[END_REF].

The Drosophila external mechanosensory organs, or bristles, are an excellent model system to study the balance between proliferative and determined states of progenitor cells [START_REF] Fichelson | Cell cycle and cell-fate determination in Drosophila neural cell lineages[END_REF]. Each bristle is composed of a shaft and an annular cuticular structure, called the socket, at its base. Bristles are formed during pupal stage from four specialized cells, with a common origin: two outer cells, the socket and shaft cells, and two inner cells, the neuron and sheath cell [START_REF] Hartenstein | Development of adult sensilla on the wing and notum of Drosophila melanogaster[END_REF]. Each cell differs from the other by its size, relative position, and expression of specific markers (Figure 1A, Movie S1). They arise from the division of a primary precursor cell (or pI) after a stereotypical sequence of four asymmetric cell divisions (the bristle cell lineage). In the dorsal thorax (notum), pI cells divide to generate a posterior secondary precursor cell (pIIa) and an anterior secondary precursor cell (pIIb). The division of pIIa leads to the formation of the outer cells (the pIIa sub-lineage), whereas the pIIb cell gives rise to the inner cells (the pIIb sub-lineage), following two rounds of division. First, pIIb divides to give rise to a glial cell that enters apoptosis shortly after birth and a tertiary precursor cell, pIIIb. Then, pIIIb divides to produce the sheath and the neuron [START_REF] Gho | Revisiting the Drosophila microchaete lineage: a novel intrinsically asymmetric cell division generates a glial cell[END_REF][START_REF] Fichelson | The glial cell undergoes apoptosis in the microchaete lineage of Drosophila[END_REF]. At each of these divisions, the Notch (N) pathway is differentially activated in only one daughter cell. This differential activation ensures the acquisition of different fates by both daughter cells [START_REF] Guo | Control of daughter cell fates during asymmetric division: interaction of Numb and Notch[END_REF]. As such, the N-pathway does not specify particular identities, but its activation triggers different outcomes depending on the cellular context, likely in cooperation with other factors that specify cell fate [START_REF] Ramat | Escargot and Scratch regulate neural commitment by antagonizing Notch activity in Drosophila sensory organs[END_REF]. Only some of these specific factors are known in the bristle lineage. Two are Sequoia (Seq) and Hamlet (Ham), two zinc-finger transcription factors, expressed in pIIb sub-lineage cells, that have a critical role in the acquisition of inner cell identity [START_REF] Moore | hamlet, a binary genetic switch between singleand multiple-dendrite neuron morphology[END_REF][START_REF] Moore | Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification[END_REF][START_REF] Andrews | Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila[END_REF]. Indeed, sensory organs (SO) in ham and seq mutants are composed of external cells only, due to respecification of the inner cells. Moreover, it has also been shown that Seq controls ham expression, indicating that these factors are related in a complex regulatory network of transcription factors [START_REF] Andrews | Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila[END_REF].

Here, we use the bristle lineage to explore how Ttk69 coordinates terminal cell determination and cell-cycle arrest. We show that loss of ttk69 leads to the production of supernumerary progenitor cells and the re-specification of cell fate identity. Notably, we observed a cell transformation in which an outer cell acquired an inner cell precursor identity, precisely the pIIa shaft cell adopts a pIIIb cell fate. Since outer and inner cells are cousin cells, this kind of transformation has been previously named cousin-cousin cell transformation [START_REF] Moore | Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification[END_REF]. We identified the CycE gene, encoding the essential cyclin required for entry into S-phase, as a Ttk69 downstream gene. In addition, we show that Ttk69 regulates cell-fate acquisition and terminal differentiation by controlling the expression of ham and Suppressor of Hairless (Su(H)), which encodes the transducing transcription factor of N-receptor signaling. We propose that Ttk69 is a central node of a transcriptional regulatory network that assures cell lineage completion by controlling the acquisition of terminal cell fates and the arrest of cell proliferation.

MATERIALS AND METHODS

CycE reporter constructions

To establish transgenic fly strains bearing CycE transcriptional reporters, the eve promoter of the eve.p-LacZ.attB construct [START_REF] Liberman | Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence[END_REF]

Fly strains

Somatic clones were obtained using the FLP/FRT recombination system [START_REF] Xu | Analysis of genetic mosaics in developing and adult Drosophila tissues[END_REF]. The y, w; FRT82B ttk 1e11 /CyO^SM5 line (41754 Bloomington) was crossed with the y, w, Ubx-FLP; FRT82B ubi-nls::GFP line (gift of J. Knoblich) to generate ttk69-null somatic clones. Somatic ham and seq clones were generated using the y, w; FRT40A ham 1 /CyO^SM5 The GAL4/UAS expression system [START_REF] Brand | Targeted gene expression as a means of altering cell fates and generating dominant phenotypes[END_REF] was used to express the following UAS-constructions in the mechanosensory bristle cell lineage using, as a GAL4 driver, the line neuralized p72 -Gal4 (neur p72 ) [START_REF] Bellaïche | Frizzled regulates localization of cell-fate determinants and mitotic spindle rotation during asymmetric cell division[END_REF] Genotypes used in each figures are recapitulated in Supplementary Table S1.

Immunohistology

Pupal nota were dissected at 17-32 h APF and processed as previously described [START_REF] Gho | Subcellular localization of Suppressor of Hairless in Drosophila sense organ cells during Notch signalling[END_REF]

Quantification and statistical analyses

Quantifications of immunostaining were done by calculating the correlated total cell fluorescence (CTCF) with Fiji [START_REF] Schindelin | Fiji: an opensource platform for biological-image analysis[END_REF]: CTCF = integrated density -(area of selected cells x mean fluorescence of background). For figures 1F, 1G, 4D, 4E, 5B, 5C, to compare different experiments, individual quantifications in control conditions were normalized to the mean of similar quantifications in Ttk69-mutant SOs. As such, a ratio of 1 indicates that immunostaining in control and Ttk69-mutant SOs is similar, less than one that measurements in control conditions are lower than those observed in Ttk69-mutant cells and conversely when the ratio was higher than one. To compare quantification of Ttk69 immunostaining presented in Figure S3 in different experiments under a given condition, individual quantifications of Ttk69 immunostaining were normalized to the mean of quantifications of Su(H) accumulation. In Figure 6, to compare different experiments under a given condition, individual quantifications of immunostaining were normalized to the mean of similar quantifications of ELAV staining observed in neurons.

Statistical significance is calculated by Mann-Whitney test, ***: p < 0,001, ns: not significant.

Analyses and graph were done using KaleidaGraph software. Error bars represent SEM.

Time lapse microscopy

We performed live imaging of SOs in neur-H2B::GFP; FRT82B ttk 1e11 pupae following protocols described previously [START_REF] Simon | Notch and Prospero repress proliferation following cyclin E overexpression in the Drosophila bristle lineage[END_REF][START_REF] Sallé | CycA is involved in the control of endoreplication dynamics in the Drosophila bristle lineage[END_REF]. The construction neur-H2B::GFP (line 22A4, gift of F. Schweisguth) allows the following of SO cells throughout the progression of the bristle lineage. White pupae were collected and aged until 20 h APF at 25°C in a humid chamber before dissection and mounting for imaging. Live imaging data were collected using a spinning disk coupled to an Olympus BX-41 microscope (Roper Scientific, 40X, NA 0.75 objective, CoolSnapHQ2 camera). The temperature of the recording chamber was carefully controlled (±0.1°C) using a homemade Peltier device temperature controller fixed to the microscope stage. Systems were driven by Metamorph software (Universal Imaging). Z-stacks of images were acquired every 3 min and assembled using ImageJ software (NIH). At the end of the movies, pupae were dissected and immunostaining were carried out as described previously. Imaged cells were unambiguously identified by their relative position, nuclear size, and order of birth.

Tramtrack69 pull down

Experiments were performed either using a batch of E. coli-expressed Ttk69::ZF or 20 h old white Drosophila embryo protein extracts. Ttk69::ZF was expressed from BL21 (DE3) bacteria transformed with a pET15 vector in which the C-terminal fragment (318-641) of Ttk69 protein, containing zinc fingers, was cloned in-frame with a histidine tag (gift from A.

Travers). Non-denatured E. coli extracts were prepared after a 2 h induction in 0.1mM IPTG and embryo extracts were obtained as described by Wordarz [START_REF] Wodarz | Extraction and immunoblotting of proteins from embryos[END_REF] (1 mg devitellinized embryos was always extracted in 5 µl lysis buffer to calibrate extraction). DNA templates were generated by PCR using 5' biotinylated primers. As controls, a ftz template was obtained using 5'GGGAGTTGCGCACTTGCTTG and 5'GTGCACGCAACGCTGGTGAG primers, which correspond to the portion of the fushitarazu (ftz) promoter bearing the canonical AGGAC Ttk69 binding sites and a RP49 template devoid of this sequence was obtained by PCR using 5' TGTACTTGGCATCCGCGAG and 5' CACCAGCACTTCTCCAACAC. Two CycE templates were obtained using two set of primers 5' GCAAGATTATGAATATCTAT -5' GTGTGCGCGCATGCGCAACG and 5' Protein extracts and beads were prepared separately according to the manufacturer's instructions. Beads were washed four times with 100 mM NaCl/25 mM NaH 2 PO 4 . Extracts were divided equally and added to the beads. At the end of each incubation, the beads were pelleted and precipitated proteins were analyzed by Western blot. Ttk69::ZF was revealed using mouse anti-Penta-Histidine (1:1000; Qiagen; 34660), whereas Ttk69 protein from embryo extracts was revealed using rabbit anti-Ttk69 (1:4000 gift from A. Travers).

Specificity of the Ttk69 antibody was tested by analyzing the immuno-detection of protein extracts from pools of ten 20 h old ttk 1e11 /TM6 and w 1118 embryos. Anti-β tubulin staining

(1:10000, Amersham) was used as a loading control. Visualization was performed using horseradish peroxidase coupled to anti-mouse or anti-rabbit (1:10000, Promega) antibodies coupled to the Super Signal Western blotting detection system (Pierce) according to the manufacturer's instructions.

RESULTS

Ttk69 loss of function leads to the formation of SOs with extra inner cells and only one type of outer cells

To precisely determine the involvement of Ttk69 in cell-cycle progression and cell-fate determination, we studied somatic clones of ttk 1e11 , which specifically disrupts Ttk69, hereafter called Ttk69 clones [START_REF] Lai | Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development[END_REF]. SOs inside Ttk69 clones (called Ttk69mutant SOs) were devoid of the shaft and presented only sockets externally (Figure 1B). At the cellular level, 82% (n = 30) of the mutant organs were composed of more than four cells (up to eight cells) at 28 h after pupal formation (APF) (Figures 1C arrowheads andD).

Among the SO cells, one (67%) or two (33%) cells were Pdm1-positive outer cells (n = 30)

and, in all cases, they expressed Su(H), a landmark of socket cells (Figures 1D andE). These data show that the absence of the shaft structure is associated with the lack of a cell expressing a shaft signature (Pdm1 positive, Su(H) negative). We observed that in Ttk69mutant SO socket cells, although present, did not have a normal shape (Figure 1B, insets b, c).

It was previously shown that the normal socket cell differentiation is dependent of the Notch transcription factor Su(H) auto-amplification. Thus, Su(H) is first induced in presumptive socket cells in response to the N-pathway and subsequently boosted via its binding to a 3'enhancer (ASE5, [START_REF] Barolo | A Notch-Independent Activity of Suppressor of Hairless Is Required for Normal Mechanoreceptor Physiology[END_REF] The remaining cells in Ttk69-mutant SOs expressed inner-cell markers. Immunostaining against ELAV and Prospero (Pros) revealed the presence of one to four neurons and one or no sheath cell (Figure 1H). We also observed up to three cells per cluster (n = 35) that were positive for both ELAV and Pros immunostaining (Figures 1H andI). This suggests the presence of either additional pIIIb precursor cells or post-mitotic cells in which the fate was not yet well resolved or not at all [START_REF] Ramat | Escargot and Scratch regulate neural commitment by antagonizing Notch activity in Drosophila sensory organs[END_REF]. Thus, the Ttk69-mutant lineage is

probably not yet completed at 28h APF. Overall, these data show that loss of function of Ttk69 leads to the formation of SOs composed of extra inner cells and only socket cells as the outer cell type.

Ttk69 promotes cell-cycle arrest and triggers terminal cell-fate identities

Several non-exclusive explanations can account for the presence of SOs with supplementary cells in Ttk69-mutant SOs. One is that the glial cells do not die but divide and produce extra terminal cells. We examined this possibility by studying cell death in Ttk69mutant SOs. TUNEL assays showed that glial cells undergo apoptosis in Ttk69-mutant SOs at the same time as in control organs located outside the mutant clone (Figure 2A, n = 3). Thus, the supplementary cells do not originate from glial cells that resume proliferation. It is also possible that supplementary cells arise from bristle cells that do not properly exit from the cell cycle and continue to proliferate. We explored this possibility by searching for metaphasic cells using phospho-ser10 histone-3 (PH3) immunoreactivity at 28 h APF, when control SO cells are already post-mitotic. Ttk69-mutant SOs containing four or more cells harbored sensory cells positive for PH3 (Figure 2B, arrowheads, 20% of SOs, n = 25). As these clusters contained the terminal number of cells, these data show that the metaphasic cells were not due to delayed divisions. Thus, these data indicate that Ttk69-mutant SOs harbor supplementary cells due to additional mitoses.

To unambiguously define the origin of the supernumerary cells, we used correlative microscopy that combines live imaging to record the entire pattern of cell divisions in Ttk69mutant SOs followed by immunolabeling to identify cell identities. During time-lapse recording, sensory cells were identified by the expression of GFP under the control of the neuralized promoter (neur-GFP).!At the end of each recording, the imaged notum was fixed and immunolabeled with anti-Su(H), as well as anti-ELAV and anti-Pros to highlight outer and inner cells, respectively. Imaged SOs could be unambiguously recognized within the fixed nota by their relative position with respect to the midline, the position of the macrochaetae, or the rows of microchaetae [START_REF] Fichelson | Mother-daughter precursor cell fate transformation after Cdc2 down-regulation in the Drosophila bristle lineage[END_REF]. We confirmed the presence of extra cell divisions and revealed an unexpected cell transformation event. First, there was a supplementary division in a pIIa daughter cell, identified as the future socket cell by its position in the cluster (Movie S2 and Figure 2C, panels 4 and 5). This extra division was symmetric, leading to two Su(H)-positive socket cells (Figure 2D, D'). We also observed socket cells in mitosis, identified by Su(H) and PH3 immunoreactivity, in fixed material (Figure 2E, arrowhead, n = 3). Thus, future socket cells undergo an extra division in the absence of Ttk69 (Figure 2F). In addition, the anteriorly located pIIa daughter cell, the presumptive shaft cell that normally does not divide, underwent repetitive cell divisions (Movie S3, Figure 3A, panels 3 and 5). Surprisingly, immunostaining of the resulting clusters showed that cells arising from these extra divisions acquired a neural fate, as they expressed Pros and ELAV (Figure 3B, B'), two markers expressed in sheath and neuron cells, respectively. This suggests that presumptive shaft cells underwent cousin-cousin cell transformation in which outer cells acquired an inner cell fate (Figure 3C). Consistent with this possibility, we also observed cells with weak expression of Pdm1 associated with weak expression of Pros in fixed material, suggesting that they were midway through transformation (arrowhead in Figures 3D-D"). Furthermore, we detected clusters harboring two Pros-positive cells, of which one was dividing (PH3 positive, arrowhead in Figures 3E-E"). These different lines of evidence led us to conclude that the presumptive shaft cells underwent cell fate re-specification and acquired a pIIIb precursor cell identity. We never observed by in vivo recordings cell lineages in which both pIIa daughter cells entered division. This is likely due to the low probability of such cases. We do not favor the possibility that the division of pIIa daughter cells is mutually exclusive, as using fixed material, we observed SOs composed of more than five cells and harboring two socket cells, a situation that required ectopic division of both pIIa daughter cells.

The re-specification of presumptive shaft cells into inner precursor cells can explain, in part, the observation that Ttk69-mutant sensory clusters harbored up to six neural (ELAV/Pros positive) cells, showing that extra inner-cells originate at the expense of outer cells. Overall, these results suggest that Ttk69 promotes cell precursor exit, acting both by arresting cell proliferation and triggering terminal cell fate identity.

Ttk69 induces cell cycle exit via transcriptional repression of CycE expression.

Our results suggest that Ttk69 regulates cell proliferation. We have already shown that Ttk69-mutant sensory cells strongly accumulate CycE protein [START_REF] Audibert | Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage[END_REF]. We studied whether supplementary mitosis related to the accumulation of ectopic CycE by assessing whether a reduction in the dose of CycE could revert the phenotype of supplementary cell divisions observed in Ttk69-mutant SOs. As already described, we observed that most SOs (85%, n = 42) inside Ttk69-mutant clones contained more than the normal four cells (Figure 4A). This dropped to 18% when the clones were induced in a CycE AR95/+ heterozygous background (n = 35). In another set of experiments to study the number of socket cells, 45% of Ttk69-mutant SOs harbored duplicated Su(H)-positive socket cells (n = 55), whereas only 11% did so in the CycE AR95/+ heterozygous background (n = 51).

These results show that reducing the dose of CycE is sufficient to markedly reduce the number of supplementary divisions observed in Ttk69-mutant SOs. This strongly suggests that the supplementary mitoses observed in Ttk69-mutant SOs are mainly driven by the increase in CycE levels induced after Ttk69 loss of function.

We analyzed the role of Ttk69 in the transcriptional expression of CycE by first testing the capacity of Ttk69 to bind the CycE promoter. It has been previously shown that a 4.6 Kb proximal fragment of the whole CycE promoter is able to recapitulate zygotic CycE expression in the embryonic peripheral nervous system (Figure S2A, [START_REF] Jones | Tissue-specific regulation of cyclin E transcription during Drosophila melanogaster embryogenesis[END_REF]. We have identified eight AGGAC canonical Ttk binding sites in this 4.6 Kb fragment organized in two clusters: three in region I and five in region II (Figure 4B) (Harrison andTravers 1988, 1990). The binding of Ttk69 to these regions was assessed by a DNA-mediated Ttk pull-down assay using the Ttk69 C-terminal domain, containing the C2H2-type zinc-finger. The Ttk69 zinc-finger domain behaved as expected, since it was efficiently retained on beads coated with DNA fragments bearing canonical Ttk-binding sites ftz, as well as on regions I or II (Figure 4B, lanes 1, 3, and 5). In contrast, we observed low-level retention when beads were coated with DNA free of Ttk binding sites (rp49, Figure 4B, compare lanes 1 and 2); quantification showed that binding to the rp49 probe was reduced to 11% (n = 5) of that observed using the ftz probe. We also observed low-level retention when the beads were coated with regions I or II in which the Ttk binding sites were mutated (Im and IIm respectively, Figure 4B, compare lane 3 with 4 and lane 5 with 6). Binding to the Im probe was reduced to 17% (n = 5) of that containing region I and binding to the IIm probe was reduced to 23% (n = 5) of that containing region II. As such, this in vitro approach suggests that the control of CycE expression by Ttk69 relies on the direct binding of this factor to canonical Ttk binding sites to the CycE promoter. We then tested this possibility in vivo by lac-Z transcriptional reporter strategy. Firstly, we followed in bristle sensory cells lacZ expression under the control of the 4.6WT fragment. We observed endogenous CycE and β-Gal accumulation in inner cells, whereas both were only barely detected in pIIa daughter cells in 24h APF pupae (arrows in S1C). These data confirm that this region is sufficient to drive Ttk69-mediated CycE regulation. Although, the D-fragment harbors a

Ttk69 binding site at the more distal location, we do not believe that this binding site plays a role on the Ttk69-mediated control of CycE expression since Ttk69 continues to regulate the expression of reporter when this Ttk69 binding site was mutated (see above, Figures 4E,E').

As such, these data suggest that Ttk69 may control CycE expression by binding directly to unknown binding sites located in the D-fragment or indirectly either via another factor that recognizes the D-fragment or via the regulation of the expression of CycE transcriptional regulators.

Ttk69 binds to the CycE-promoter independently of canonical sites

To address these possibilities, we divided the D-fragment devoid of the Ttk69 binding site in three (D1-D3) and assessed their role in Ttk69 binding by performing DNA-mediated Ttk pull-down assays (Figure 5D). As expected, a high and a low amount of Ttk69 precipitated with beads coated with ftz and rp49 probes respectively (Figure 5E, lanes 1 and 2). When we assessed the binding of the Ttk69 zinc-finger domain to the D-fragments, we observed that only a low amount of Ttk69 precipitated (Figure 5E, top panel, lanes 3 to 5). This signal was comparable to the one obtained with beads coated with a fragment devoid of Ttk69 binding sites (Figure 5E, rp49-probe (lane 2), n = 2). These data indicate that the D-fragments do not retain theTtk69 zinc-finger domain. Next, we tested whether endogenous Ttk69 binds these CycE promoter fragments in association with other partners, by performing similar DNAmediated Ttk pull-down assays but using embryonic extracts and a specific Ttk69 antibody (Figures 5E andF). Our data show that endogenous Ttk69 was not retained by the D2 CycEpromoter fragment (Figure 5E bottom panel, lane 3, quantification showed that binding to the D2 probe was similar to that observed using the rp49 probe (n = 3)). Remarkably, it was retained on beads coated with the D1 and D3 fragments (Figure 5E, bottom panel, lanes 2 to 5, signals using the D1 probe was 15 times and using the D3 probe was 13 times of that observed for the rp49 probe (n = 3)). These data show that the binding of Ttk69 to the D1 and D3 CycE promoter fragments required either the native form of Ttk69 or other proteins present in embryonic extract.

Ttk69 downregulates hamlet expression

We wished to identify Ttk target genes involved in cell fate regulation in SOs. We focused on two candidates, hamlet (ham) and sequoia (seq) [START_REF] Moore | Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification[END_REF][START_REF] Andrews | Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila[END_REF], because the phenotype associated with ham and seq loss of function, inner to outer cell transformation, is similar to that of ttk gain of function. In addition, ham and seq genes are expressed in patterns that are complementary to the expression pattern of ttk during the bristle lineage (Figures 1A and6A, see supplementary Figure 4 in [START_REF] Andrews | Sequoia regulates cell fate decisions in the external sensory organs of adult Drosophila[END_REF]. We thus studied the potential epistatic interactions between these three factors.

We first studied ttk expression in SOs when ham and seq were either overexpressed or downregulated. We used a temperature conditional driver to overexpress ham or seq late in development to avoid potential interference with outer-to-inner cell transformations induced by seq or ham-overexpression. Under these conditions, we detected no cell fate transformations, assessed by Su(H) immunoreactivity as a marker of outer socket fate (Figures S3A-C, Su(H) panels). This analysis revealed that Ttk69 expression was unaffected when either ham or seq were overexpressed (Figures S3A-C, Ttk panels. Quantification in Figure S1D). Reciprocally, we failed to observe modifications in the number of Ttk-positive cells early during development in ham or seq mutant SOs (20 and 22 h APF, Figures S3D-G).

We observed sensory clusters harboring four Ttk69-positive cells only late in the bristle lineage in seq or ham mutant clones (24 h APF in 12% and 34% of clusters in ham and seq mutant clones, respectively, Figures S3D-G). These data suggest that supernumerary Ttk cells in ham and seq loss of function are due to cell transformation induced by the loss of function of seq or ham, rather than direct deregulation of ttk69 expression. Thus, we conclude that ttk expression is independent of Ham and Seq.

Next, we analyzed whether ham or seq expression are controlled by Ttk69. We thus overexpressed Ttk69 in neurons, where it is never detected, and analyzed Seq and Ham protein accumulation. We used a similar strategy as before to overexpress Ttk69 late in development and observed no cell-fate transformation as shown by ELAV immunoreactivity (Figures 6B andC, ELAV panels). Under these conditions, Seq accumulated at the same level as in the control situation, whereas Ham immunoreactivity was strongly reduced (compare the right panels in Figures 6B andC for Seq and Ham detection; respectively. Quantification in Figure S1E,F). The observed effects were not due to changes in cell fate, as ELAV expression was unaltered. As such, we conclude that Ttk69 does not affect seq expression, whereas it downregulates ham expression.

Ttk69 maintained non-neural cell fate via repression of hamlet expression.

Our results show that Ttk69 downregulates ham expression. This suggests that ham is repressed in Ttk69 expressing cells, in particular in pIIa precursor cells and their progeny. It is thus expected that ham would be ectopically expressed in pIIa cells in Ttk69-mutant SOs.

Indeed, we observed the presence of three to four Ham-positive cells in 50% of Ttk69-mutant SOs analyzed (SOs inside Ttk69 clones) at 24 h APF, whereas there were no more than two in the control SOs (SOs outside Ttk69 clones) (Figures 7A andB). However, ectopic expression of ham could be due to the deregulation of ham expression per se or to the cousin-cousin cell transformation already described. We assessed ham expression in Ttk69-mutant SOs at early stages to determine the mechanism behind its ectopic expression. We observed three Hampositive cells in Ttk69-mutant SOs composed of four cells as early as 20 h APF, even before the completion of the bristle lineage (Figures 7A andB). These data show that the ectopic expression of ham in the absence of Ttk69 is an early event during the cousin-cousin cell transformation. Moreover, we occasionally observed cells positive for both Ham and Pdm1, a specific marker of pIIa descendent cells, in Ttk69-mutant sensory clusters (arrowhead in Figures 7C,C',n = 4). This signature is consistent with the cells undergoing transformation from outer to inner cells.

These data suggest that the de-repression of ham in pIIa cells in Ttk69-mutants drives cousin-cousin cell transformation, in which pIIa shaft cells adopt a pIIIb cell fate. We tested this possibility by studying whether the reduction of ham expression in Ttk69-mutant SOs could restore shaft identity. We analyzed cell identities in Ttk69-mutant SOs, namely SOs inside Ttk69 clones, in flies that were otherwise ham heterozygous. This analysis was preformed in pupae at 28 h APF, when this cousin-cousin transformation has already taken place in Ttk69-mutant SOs. Under these conditions, in 4 over 60 Ttk69-mutant SOs analyzed, we observed a Pdm1 positive/Su(H) negative cells, a specific sign of shaft cells (Figures 7D-D''', arrowhead). These rescues were significant since the shaft cell type was never observed in Ttk69-mutant SOs under ham normal conditions (see Fig. 1D). Attempts to increase the percentage of rescue were aborted since further reduction of ham starts to affect cell fate identities per se. Our results show that a reduction in ham expression can rescue the formation of shaft cells in the absence of Ttk69. Thus, Ttk69 maintains a non-neural cell fate in pIIa daughter cells by inhibiting the adoption of the inner precursor fate via the repression of ham expression.

DISCUSSION

An important goal in developmental biology is to understand the mechanisms by which cell proliferation and cell-fate acquisition are coupled during organogenesis. Here, we show that Ttk69, a member of the evolutionarily conserved BTB-ZF transcription factors acts as a link between these two processes. We found that Ttk69 is essential for exiting the proliferative progenitor state and conferring a non-neural fate to the progeny during the formation of mechanosensory bristles. Indeed, using mainly clonal analysis, we show that ectopic cell divisions occur independently of changes in cell fate. In addition, we observed that Ttk69-mutant SOs harbor supplementary terminal cells due to cell transformation that generates extra neural progenitor cells. This was associated with upregulation of CycE, required for S-phase entry, and the ectopic expression of hamlet, a neural determinant. As such, the BTB-ZF transcriptional factor, Ttk69, links cell proliferation and cell fate acquisition in the bristle cell lineage (Figure 8).

Ttk69 acts as a dual factor linking cell proliferation and cell fate acquisition.

Our results showed that the loss of Ttk69 leads to SOs harboring up to eight terminal cells.

Supplementary terminal cells arose from two different mechanisms: an extra division of socket cells and, more importantly, several rounds of extra division due to the re-specification of the presumptive shaft cell into a pIIIb precursor cell. It was previously shown that SOs in complete ttk null pupae are composed of only four neurons [START_REF] Guo | tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS[END_REF]. The fact that no ectopic cells were generated when all Ttk isoforms were absent may reflect different kinetics between cell cycle arrest and cell differentiation. ttk null cells rapidly acquired an arrested cell cycle and neuronal terminal fate, rather than entering a proliferative precursor state, as after Ttk69 loss of function alone. Thus, the specific effects of Ttk69 on the cell cycle were masked in the ttk null mutant. Use of the Ttk69 loss-of-function mutant made it possible to reveal intermediate cell fates, prior to the acquisition of terminal-cell identities. Moreover, the study of mutants that exclusively affect Ttk69 allowed decoupling of the acquisition of cell cycle arrest and cell fate.

The effects of Ttk69 on the core cell cycle machinery were revealed by the ectopic divisions of socket cells observed under Ttk69 loss of function conditions. These cells are already committed to acquire a terminal identity, indicating that such ectopic divisions are not associated with changes in cell fate. This shows that Ttk69 impedes cell cycle progression per se. Accordingly, we show that the control of cell-cycle progression by Ttk69 involves transcriptional downregulation of CycE expression. Such negative control of CycE expression by Ttk69 appears to be a general effect, as it has also been observed in proliferating glia cells [START_REF] Badenhorst | Tramtrack controls glial number and identity in the Drosophila embryonic CNS[END_REF]). Moreover, it has also been shown that Ttk69 represses the expression of string, which encodes the phosphatase (Cdc25) essential for G2/M transition in the imaginal eye disc [START_REF] Baonza | Pointed and Tramtrack69 establish an EGFR-dependent transcriptional switch to regulate mitosis[END_REF]. This suggests that Ttk69 represses cell-cycle progression at different phases of the cell cycle. Thus, the induction of ectopic cell divisions in the absence of Ttk69 is probably due to the multiple effects of Ttk69 on cell-cycle progression. The diverse targets of Ttk69 in the cell cycle machinery could explain the involvement of this factor in the transition between different modes of the cell cycle [START_REF] Jordan | Notch signaling through tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells[END_REF][START_REF] Sun | Regulation of the endocycle/gene amplification switch by Notch and ecdysone signaling[END_REF]. In SOs, pIIa terminal cells underwent endocycles that require the fine-tuned control of CycE expression. Indeed, we have already shown that endocycles are abolished in CycE null mutants, occur at very low CycE levels, and become mitotic cycles at high CycE levels [START_REF] Simon | Notch and Prospero repress proliferation following cyclin E overexpression in the Drosophila bristle lineage[END_REF][START_REF] Sallé | CycA is involved in the control of endoreplication dynamics in the Drosophila bristle lineage[END_REF]. We show here that Ttk69 is involved in a mechanism that limits CycE levels. Thus, Ttk69 is probably involved in the transition from mitotic cell cycles to endocycles in pIIa terminal cells. Similar transitions between two cycling states associated with two different levels of Ttk69 have been observed in ovary epithelial follicular cells during the transition between endocycles to gene amplification [START_REF] Jordan | Notch signaling through tramtrack bypasses the mitosis promoting activity of the JNK pathway in the mitotic-to-endocycle transition of Drosophila follicle cells[END_REF][START_REF] Sun | Regulation of the endocycle/gene amplification switch by Notch and ecdysone signaling[END_REF]. We propose that Ttk69 contributes to the dampening of CycE levels, allowing cells to transit throughout different modes of the cell cycle.

In addition to cell proliferation, both pIIa daughter cells were differentially affected under Ttk69-mutant conditions, at the level of terminal differentiation for socket cells and determination for shaft cells. For socket cells, although SOs of adult Ttk69-mutants contain socket cuticular structures, auto-amplification of Su(H) expression is impaired, leading to misshapen sockets [START_REF] Barolo | A Notch-Independent Activity of Suppressor of Hairless Is Required for Normal Mechanoreceptor Physiology[END_REF]. For shaft cells, presumptive shaft cells are respecified and acquire a neural progenitor identity due to the mis-expression of ham. Ham is normally expressed in pIIIb precursor cells and its overexpression induces the formation of SOs bearing supernumerary cells expressing both ELAV and Pros, such as in pIIIb cells (see Figure 4 in [START_REF] Moore | Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification[END_REF])). In addition, ham loss of function induces the conversion of terminal inner cells into outer cells, suggesting that Ham is essential to acquire the neural precursor fate [START_REF] Moore | Conversion of neurons and glia to external-cell fates in the external sensory organs of Drosophila hamlet mutants by a cousin-cousin cell-type respecification[END_REF]). Moreover, we observed that a reduction of ham levels in Ttk69-mutant clones decreased shaft re-specification, in agreement with the fact that Ham is an essential regulator of neural precursor fate. Finally, we show that Ttk69 repressed ham expression. Overall, these data suggest that re-specification of shaft cells is due to the ectopic expression of ham as a consequence of the loss of function of Ttk69. ham was also mis-expressed in socket cells, but did not lead to cell transformation. This apparent contradiction may be related with the differential activation of the N-pathway between these two sister cells. Indeed, Ttk69 loss of function induces a cell fate change in shaft cells, a Noff cell. In contrast, Ttk69 loss of function in socket cells, in which the N-pathway is activated as soon as the cells are formed [START_REF] Remaud | S-phase favours notch cell responsiveness in the Drosophila bristle lineage[END_REF], impaired only their late differentiation. These results suggest that early activation of the N-pathway prevents cell fate transformation.

We conclude that Ttk69 is required in terminal cells to repress the neural precursor state by inhibiting both proliferative capacity, by repressing CycE expression, and neural fate, by repressing ham. This shows that Ttk69 is a central actor in the coordination between cell cycle arrest and cell fate acquisition.

Tt69 regulates its downstream genes in several ways.

In this study, we identified three genes CycE, ham, and Su(H) whose expression was deregulated in Ttk69-mutant clones. We show here that Ttk69 also regulates CycE. Indeed, we have identified a region in the 4.6 kb CycE promoter fragment where Ttk acts to turn off CycE expression. The analysis of this region revealed an unexpected feature. Indeed, our structure-function analysis failed to identify a region responsible for turning on CycE expression in sensory organ cells. Thus, the reporter was always expressed in these cells in all deletions tested and none of these fragments seem to be specifically required to turn on CycE expression. One possibility is that the 0.4 kb most proximal region of CycE promoter (dark pink in Figures 4,5) drives CycE expression. Alternatively, there are multiple regions scattered along the 4.6 fragments that control CycE expression. Further experiments are required to understand how the 4,6 kb promoter turns on CycE in this system. In contrast, Ttk69-mediated CycE down-regulation was driven through a promoter domain (fragment D) independently of the canonical AGGAC Ttk69 binding sites [START_REF] Brown | Repression of the Drosophila fushi tarazu (ftz) segmentation gene[END_REF]. Moreover, we showed that the Ttk69 zinc-finger domain does not bind subdomains of fragment D (D1, D3), whereas the native Ttk69 protein, present in a late embryonic extract, does. There are two non-exclusive explanations for this observation. Either an uncharacterized Ttk69 domain outside the zinc-finger domain binds directly to the D fragment through a non-canonical binding site or Ttk69 binds indirectly to the CycE promoter via an interaction with trans-acting factors. The first explanation is formally possible, but no DNA binding domain has been described in the N-terminal portion of the Ttk69 protein. Nevertheless, it is well known that Ttk69 may bind to other noncanonical binding sites. This is the case for the GTCCTG and TTATCCG sequences in eve and ftz promoters respectively [START_REF] Harrison | The tramtrack gene encodes a Drosophila finger protein that interacts with the ftz transcriptional regulatory region and shows a novel embryonic expression pattern[END_REF][START_REF] Read | Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities[END_REF].

However, we observed that Ttk69 continued to downregulate CycE expression in the absence of these non-canonical sites, making this explanation unlikely. In contrast, several lines of evidence support the second explanation. It is known that the activity of Ttk69 can be influenced by the presence of other DNA-binding factors. Thus, the repressive action of Ttk69 depends on interactions with MEP1 and Mi-2 proteins, which recruit the ATPdependent chromatin-remodeling complex (NuRD) [START_REF] Reddy | Drosophila transcription factor Tramtrack69 binds MEP1 to recruit the chromatin remodeler NuRD[END_REF]). Moreover, it has been shown that, although Ttk could bind directly to eve promoter repressing eve expression (see above), Ttk69 represses eve expression independently of their direct binding to DNA, by interacting with GAGA factors through its BTB/POZ domain. When bound to DNA, GAGA zinc-finger factors (Trithorax-like, Trl) activate the transcriptional machinery, but this transcription is inhibited when it is complexed with Ttk69 [START_REF] Pagans | Repression by TTK69 of 29 GAGA-mediated Activation Occurs in the Absence of TTK69 Binding to DNA and Solely Requires the Contribution of the POZ/BTB Domain of TTK69[END_REF]. In bristle sensory cells, RNAi-mediated loss of function of the MEP1, Mi-2, and Trl genes did not affect cell number or cell fates, even when these loss-of-function mutations were analyzed in a sensitized ttk69 heterozygous background (Figure S4). Despite these results, we favor a model in which another factor is required for Ttk69 to bind the D fragment of the CycE promoter. Furthermore, we did not find Ttk binding close to the CycE promoter using genome-wide Ttk binding profiles from 0 to 12-h old embryos, published by the modENCODE project. Moreover, CycE up-regulation was not observed in genome-wide expression experiments performed in S2 cells treated with dsRNA directed against Ttk69 [START_REF] Reddy | Drosophila transcription factor Tramtrack69 binds MEP1 to recruit the chromatin remodeler NuRD[END_REF]. These data suggest that Ttk69 does not regulate CycE expression during early embryonic stages. Thus, Ttk-mediated downregulation of CycE expression late in development probably requires cell specific trans-acting factors.

The involvement of trans-acting factors would explain the diversity of the Ttk response of particular cells at specific developmental stages. Such diversity mediated by trans-acting factors allows Ttk to regulate the expression of a broad spectrum of genes in bristle sensory cells in response to N-pathway activation (Figure 8). This is true not only for genes related to cell proliferation, such as CycE, but also those controlling cell fate, such as ham and Su(H).

Our data suggest that Ttk69 represses ham in pIIa sublineage cells and activates the Su

  was replaced with part of the CycE promoter from the 16,4 lacZ construct engineering by H Richardson[START_REF] Jones | Tissue-specific regulation of cyclin E transcription during Drosophila melanogaster embryogenesis[END_REF] that contained the full length CycE promoter region. 4,6WT fragment covers the Kpn1-Xho1 proximal region of the full-length CycE promoter. All other constructs derived from the 4,6WT construct, with deletion of the Kpn1-BssHII and the Ale1-Nco1 fragments in the ΔAC-lacZ construct; deletion of the BssHII-Ale1 fragment in the ΔB-lacZ construct and deletion of the Nco1-Xho1 fragment in the ΔD construct. To generate 4,6m-lacZ promoter bearing mutated Ttk69 binding sites, we used the QuickChange Multi Site-Directed Mutagenesis kit (Stratagene). Experiments were performed following the manufacturer instructions. For each Ttk binding site, specific primer carrying ACTGC sequence (underlined in the sequence) to replace the canonical AGGAC sequence were generated and are '. Clones obtained were sequenced and WT fragments of the 4,6WT-lacZ construct were replaced after enzymatic digestion by mutated fragments to generate the 4,6m-lacZ construct. Transgenic flies were generated by BestGene Inc. (Chino Hills, CA). All constructs were inserted at the same locus, attP40 on chromosome II, using ΦC31 integrase-based tools, to avoid expression variations due to genomic environment.

GTTGGATTAACCCTTTCTGG - 5 '

 5 AGGATTTAAGTCTCAACTC to cover fragments I and II, respectively, which correspond to the proximal part of the promoter bearing the canonical Ttk69 AGGAC binding site. Im and IIm CycE mutated promoters, in which all canonical AGGAC sequences were replaced by a ACTGC sequences, were obtained using the same primers as for fragments I and II of the CycE promoter. The three CycE templates D3 fragment. Biotinylated DNA was coupled to streptavidin-coated magnetic beads (M280, Dynal Biotechnology) with 0.1 mg beads per 200 ng DNA, overnight at 4°C. The beads were washed three times in B&W buffer (as recommended by the supplier) and streptavidin-immobilized DNA saturated for 1 h in PBS-20% horse serum before incubation for 1 h with the protein extract in PBS-Triton (0.15%).

Figures

  Figures 4C-C", S2B). These data confirm that the expression of the 4.6 Kb CycE transgenic reporter mirrors the expression of the endogenous CycE protein[START_REF] Audibert | Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage[END_REF]. Then, we analyzed lacZ expression under the control of a wild-type 4.6 CycE promoter fragment (4.6WT) and a 4.6 CycE fragment in which all 8 Ttk69 binding sites were mutated (4.6m in which the AGGAC sequence was replaced by the ACTGC sequence) (Figure4D, E). As expected, the expression of the 4.6WT construct was upregulated in Ttk69-mutant SOs. Thus, we observed a particularly high level of β-Gal accumulation in both pIIa daughter cells (Figures4D, D' arrowheads. Quantifications in FigureS1B). Surprisingly, the expression pattern of the 4.6m transcriptional reporter, where the eight Ttk69 binding sites has been mutated, was not modified in wild-type SOs (Figures 4E, E' arrow and S2C, C'). Moreover, expression of the 4.6m-lacZ construct was still upregulated in Ttk69-mutant SOs (Figures 4E, E', arrowheads. Quantifications in FigureS1B). As such, although in vitro experiments show that Ttk69 binds CycE promoter, the in vivo results indicate that repression of CycE expression in sensory cells is not mediated through binding to the canonical AGGAC Ttkbinding sites present in the 4.6 fragment.To accurately define which part of the CycE promoter is required for Ttk69 regulation, we divided the 4.6Kb CycE cis-regulatory fragment into four regions (A to D) and monitored the regulatory activity of constructs with these regions deleted in bristle sensory cells (Figures5A and S2A). All constructs were inserted at the same locus (using ΦC31 integrase-based tools, except line D-lacZ) to avoid expression variations due to genomic environment. A CycE promoter construct bearing a deletion of region A and C (ΔAC-lacZ) as well as a deletion of region B (ΔB-lacZ) showed an expression pattern similar to that of the 4.6Kb-lacZ construct, indicating that the deletion of these regions did not remove the Ttk69 regulatory domain (FiguresS2D, D', E, E'). In contrast, deletion of region D (ΔD-lacZ) led to high levels of βGal accumulation in all sensory cells under control conditions (Figures5B, B', arrows and S2F, F'). Moreover, the expression pattern was similar in Ttk69-mutant SOs and control SOs outside of the clones (Figures5B, B', arrowheads. Quantifications in FigureS1C). These data show that the cis-regulatory sequence required for Ttk69-mediated down-regulation of CycE expression is located in the D-sequence. Next, we analyzed lacZ expression under the control of the fragment D (D-lacZ) in wild-type and in Ttk69-mutant SOs. Although a low level of β-Gal accumulation was observed with the D-lacZ construct, this construct behaves similarly as the 4.6WT-lacZ construct. β-Gal was barely detected in pIIa control cells (Figures5C, C' arrow and S2G, G') while a high level of β-Gal accumulation was observed in mutant SOs

  Su(H) is positively regulated by Ttk69, as revealed by its down-regulation in socket cells in the Ttk69-mutant context. The action of Ttk69 on Su(H) enhancer may occur in two different ways. Either Ttk69 acts directly as a transcriptional activator or it represses the expression of an unknown factor, as Ttk69 has always been described as a transcriptional repressor. The time required to express this putative relay factor is consistent with the observation that Ttk69 loss of function affected only late socket cell differentiation. Furthermore, we showed that such Ttk69-mediated regulation occurs through the Su(H) 3'end auto-regulatory enhancer, ASE5. It is interesting to note that, the long-lasting high-level of Su(H) expression mediated by the auto-regulatory ASE5 loop does not require N-pathway signaling[START_REF] Liu | An Enhancer Composed of Interlocking Submodules Controls Transcriptional Autoregulation of Suppressor of Hairless[END_REF]. As such, once again Ttk69 affected Notchindependent processes. Further experiments are required to elucidate how Ttk69 activates the auto-amplification of Su(H) expression.

  (H) autoregulatory loop in socket cells. CycE and ham regulation occur earlier in this lineage and likely involves the binding of Ttk69 to gene promoters as we have shown for CycE. In contrast, Su(H) regulation takes place late in the lineage, implying the probable repression of intermediary relay factors. Trans-acting factors would allow cell-specific responses to Ttk69, whereas intermediary relay factors would allow diversification over time. Thus, since Ttk69 is a N-effector, the mechanism of action of this factor increases the spatial and temporal diversity of the N-pathway cell response.

FIGURE LEGENDS Figure 1 .

 LEGENDS1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Extra mitoses in Ttk69-mutant socket cells. (A, B, E) Ttk69 clones, outlined by a white line are detected by the absence of GFP (green) in fixed tissues. (A) Apoptosis occurred at the same time in mutant (arrowhead) and control (arrow) SO. Sensory cells (blue); apoptotic cells (TUNEL staining, red). (B) Extra mitoses (PH3 immunoreactivity (blue), arrowheads) in Ttk69-mutant SO composed of four cells (red); control cluster in the inset. (C,

Figure 5 .

 5 Figure 5. Ttk69 binds indirectly to the CycE promoter. (A) Diagram of CycE promoter depicting the four specific regions analyzed (A to D). Black dots, canonical AGGAC Ttkbinding sites. ΔD CycE and D CycE transcriptional reporters bearing a deletion of the D region and only the D region respectively. (B, C) Expression pattern of the ΔD-lacZ (B, B') and D-lacZ (C, C') in control SO (arrows) and Ttk69-mutant SO (arrowheads). Ttk69 clones outlined by a white line were detected by the absence of GFP (green). Sensory cells (Cut immunoreactivity, blue) and expression of CycE transcriptional reporters (β-Gal immunoreactivity, red and bottom panels). (D) As in (A) showing the regions of the fragment D analyzed (D1 to D3). (E) DNA-mediated pull-down assay using Ttk69-his-zinc-finger (top) and whole Ttk69 protein from an embryo protein extract (bottom). Magnetic beads were coated with: lanes 1 and 2, as in Figure 4B; and lanes 3, 4, and 5, the D1, D2, and D3 regions, respectively, of the CycE promoter. (F) Detection of Ttk69 protein from a protein extract of one control (white) and one Ttk69-mutant embryo. Tubulin, loading control. Scale bars: 10 µm for B and C.

Figure 6 .

 6 Figure 6. Ttk69 downregulates hamlet but not sequoia expression. (A) Complementary expression pattern of Ttk69 and Ham proteins in bristle sensory cells. Ttk69 and Ham expression in bristle sensory cells at progressive stages of development from 18 to 28 h APF. Sensory cells are shown in blue (YFP staining), Ttk69-positive cells in red (shown as a separate channel in the middle panels), and Ham-positive cells in green (shown as a separate channel in the bottom panels). The sensory cells shown are the precursor cells: pIIb, pIIa, and pIIIb and the terminal cells: glial cells (g), sheath cells (s), neurons (n), shaft cells (sh), and socket cells (so). Ttk69 is first detected in pIIa cells and their daughter cells and later in the sheath cells. Ham is first detected in pIIIb cells and their daughter cells, before disappearing from the sheath cell when Ttk69 appears in this cell. (B, C) Ttk69 over-expression represses hamlet but not sequoia expression. Analysis of Seq (B) and Ham (C) protein accumulation after specific expression of Ttk69 in sensory cells. Sensory cells (green); neurons (blue), Ham and Seq (red). ELAV (middle panels), Ham, and Seq channels (right panels) are shown in inverted color. Scale bars: 5 µm in A and B, 10 µm in C.

Figure 7 .

 7 Figure 7. Ttk69 represses hamlet expression to maintain the non-neural cell fate. Ttk69 clones outlined by a white line were detected by the absence of GFP (green). (A) ham expression (red) in Ttk69-mutant SO at 20 and 24 h APF. Sensory cells in blue. (B) Histogram showing the percentage of SO harboring one (white bars), two (black bars), three (dark grey bars), or four (pale grey bars) Ham-positive cells in control and Ttk69-mutant SO at 20 and 24 h APF. (C) Cell transformation of a Pdm1-positive cell to a Ham-expressing cell. Arrowhead, Ttk69-mutant cell having both Pdm1 (blue in C') and Ham (red in C') immunoreactivities. Pupae at 22 h APF. (C') Higher magnification of the Ttk69-mutant cluster outlined in C. Merged and separate Pdm1 and Ham channels. (D) Recovery of shaft cells. Arrowhead, a Pdm1 positive, Su(H) negative Ttk69-mutant cell in a ham heterozygous mutant background. ham +/-pupae at 28 h APF. D' -D"', separate Pdm1, Su(H) an GFP channels. Scale bars: 10 µm.

Figure 8 .

 8 Figure 8. Ttk69 as a central node of a transcriptional regulatory network coordinating terminal cell fate acquisition and the arrest of cell proliferation. In response to N-pathway activation (Guo et al. 1995), Ttk69 (1) downregulates CycE expression inducing transition from a mitotic to endocycle mode of the cell cycle and (2) downregulates ham and upregulates Su(H) expression. Downregulation of ham expression prevents the acquisition of

Table S1 .

 S1 Fly genotypes. Fly genotypes used in each figure.

ACKNOWLEDGEMENTS

We specially thank YN. Jan, J. Knoblich, H. Bellen, H. Richardson and A. Travers for antibodies. The fly community for fly strains. Heather McLean for critical reading.

Funding was provided by the Centre National de la Recherche Scientifique and the Sorbonne Université. Anne Ramat was financed by grants from the Ministère de l'Education Nationale et de la Recherche and the Association pour la Recherche sur le Cancer, France. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

Supplemental material available at Figshare : https://figshare.com/s/1b83fb1c25b97f9210ea

D) Correlative 4D live imaging and lineage analysis showing an extra division of socket cells.

A Ttk69 clone was identified by the lack of GFP expression in epithelial cells and SO inside the clones were imaged. (C) Representative frames (1-6), depicted in inverted fluorescence, from a time-lapse recording of one Ttk69-mutant SO at 19 h APF. Glial cell outlined in blue, pIIb and its progeny in orange, the shaft cell in green, and the socket cell and its daughter in purple. Frames 4 and 5, division of the socket cell. Apoptosis of the glial cell between frames

Supplementary Movies:

Movie S1. In vivo imaging of a control SO during 19 h period beginning at 16 h after pupal formation (APF). Sensory cells identified by the expression of H2B-YFP. Anterior is to the left and the view is dorsal. Each frame was obtained by combining a z-stack (composed of optical sections separated by 1µm) acquired every 3 minutes.

Movie S2.

In vivo imaging of a SO inside a Ttk69-mutant clone during 10.5h period beginning at 19h after pupal formation (APF). The clone was identified by the lack of GFP expression in epithelial cells. Sensory cells identified by the expression of H2B-YFP.

Anterior is to the left and the view is dorsal. Each frame was obtained by combining a z-stack (composed of optical sections separated by 1µm) acquired every 3 minutes. Note supplementary divisions of the posterior pIIa daughter cell corresponding to the presumptive shaft cell (Arrow).

Movie S3.

In vivo imaging of a SO inside a Ttk69-mutant clone during 9h period beginning at 21h after pupal formation (APF). The clone was identified by the lack of GFP expression in epithelial cells. Sensory cells identified by the expression of H2B-YFP.

Anterior is to the left and the perspective is dorsal. Each frame was obtained by combining a z-stack (composed of optical sections separated by 1µm) acquired every 3 minutes. Note supplementary divisions of the anterior pIIa daughter cell corresponding to the presumptive shaft cell (Arrows).

Genotype used

Figure 1 y, w, UbxFLP ; FRT82Bttk 1e11 / FRT82B ubi-nls::GFP y, w, UbxFLP ; Ase(5)::GFP/+; FRT82 ttk 1e11 / FRT82B ubi-nls::GFP