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To achieve a highly differentiated state, cells undergo multiple transcriptional processes whose coordination and timing are not well understood. In Drosophila embryonic epidermal cells, polished-rice (Pri) smORF peptides act as temporal mediators of ecdysone to activate a transcriptional program leading to cell shape remodeling. Here, we show that the ecdysone/Pri axis concomitantly represses the transcription of a large subset of cuticle genes to ensure proper differentiation of the insect exoskeleton. The repression relies on the transcription factor Ken and persists for several days throughout early larval stages, during which a soft cuticle allows larval crawling. The onset of these cuticle genes normally awaits the end of larval stages when the rigid pupal case assembles, and their premature expression triggers abnormal sclerotization of the larval cuticle. These results uncovered a temporal switch to set up distinct structures of cuticles adapted to the animal lifestyle and which might be involved in the evolutionary history of insects.

Adult stem cells must continuously fine-tune their behavior to regenerate damaged organs and avoid tumors. While several signaling pathways are well known to regulate somatic stem cells, the underlying mechanisms remain largely unexplored. Here, we demonstrate a cell-intrinsic role for the OvoL family transcription factor, Shavenbaby (Svb), in balancing self-renewal and differentiation of Drosophila intestinal stem cells. We find that svb is a downstream target of Wnt and EGFR pathways, mediating their activity for stem cell survival and proliferation. This requires post-translational processing of Svb into a transcriptional activator, whose upregulation induces tumor-like stem cell hyperproliferation. In contrast, the unprocessed form of Svb acts as a repressor that imposes differentiation into enterocytes, and suppresses tumors induced by altered signaling. We show that the switch between Svb repressor and activator is triggered in response to systemic steroid hormone, which is produced by ovaries. Therefore, the Svb axis allows intrinsic integration of local signaling cues and inter-organ communication to adjust stem cell proliferation versus differentiation, suggesting a broad role of OvoL/Svb in adult and cancer stem cells., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators., (Copyright © 2015, American Association for the Advancement of Science.)

In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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

Background: Developmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes., Results: We defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity., Conclusions: Our results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program.

A substantial proportion of eukaryotic transcripts are considered to be noncoding RNAs because they contain only short open reading frames (sORFs). Recent findings suggest, however, that some sORFs encode small bioactive peptides. Here, we show that peptides of 11 to 32 amino acids encoded by the polished rice (pri) sORF gene control epidermal differentiation in Drosophila by modifying the transcription factor Shavenbaby (Svb). Pri peptides trigger the amino-terminal truncation of the Svb protein, which converts Svb from a repressor to an activator. Our results demonstrate that during Drosophila embryogenesis, Pri sORF peptides provide a strict temporal control to the transcriptional program of epidermal morphogenesis.

Homeotic Hox selector genes encode highly conserved transcriptional regulators involved in the differentiation of multicellular organisms. Ectopic expression of the Antennapedia (ANTP) homeodomain protein in Drosophila imaginal discs induces distinct phenotypes, including an antenna-to-leg transformation and eye reduction. We have proposed that the eye loss phenotype is a consequence of a negative posttranslational control mechanism because of direct protein-protein interactions between ANTP and Eyeless (EY). In the present work, we analyzed the effect of various ANTP homeodomain mutations for their interaction with EY and for head development. Contrasting with the eye loss phenotype, we provide evidence that the antenna-to-leg transformation involves ANTP DNA-binding activity. In a complementary genetic screen performed in yeast, we isolated mutations located in the N terminus of the ANTP homeodomain that inhibit direct interactions with EY without abolishing DNA binding in vitro and in vivo. In a bimolecular fluorescence complementation assay, we detected the ANTP-EY interaction in vivo, these interactions occurring through the paired domain and/or the homeodomain of EY. These results demonstrate that the homeodomain supports multiple molecular regulatory functions in addition to protein-DNA and protein-RNA interactions; it is also involved in protein-protein interactions.

HOX genes specify segment identity along the anteroposterior axis of the embryo. They code for transcription factors harbouring the highly conserved homeodomain and a YPWM motif, situated amino terminally to it. Despite their highly diverse functions in vivo, HOX proteins display similar biochemical properties in vitro, raising the question of how this specificity is achieved. In our study, we investigated the importance of the Antennapedia (Antp) YPWM motif for homeotic transformations in adult Drosophila. By ectopic overexpression, the head structures of the fly can be transformed into structures of the second thoracic segment, such as antenna into second leg, head capsule into thorax (notum) and eye into wing. We found that the YPWM motif is absolutely required for the eye-to-wing transformation. Using the yeast two-hybrid system, we were able to identify a novel ANTP-interacting protein, Bric-à-brac interacting protein 2 (BIP2), that specifically interacts with the YPWM motif of ANTP in vitro, as well as in vivo, transforming eye to wing tissue. BIP2 is a TATA-binding protein associated factor (also known as dTAFII3) that links ANTP to the basal transcriptional machinery.

It is well established that developmental programs act during embryogenesis to determine animal morphogenesis. How these developmental cues produce specific cell shape during morphogenesis, however, has remained elusive. We addressed this question by studying the morphological differentiation of the Drosophila epidermis, governed by a well-known circuit of regulators leading to a stereotyped pattern of smooth cells and cells forming actin-rich extensions (trichomes). It was shown that the transcription factor Shavenbaby plays a pivotal role in the formation of trichomes and underlies all examined cases of the evolutionary diversification of their pattern. To gain insight into the mechanisms of morphological differentiation, we sought to identify shavenbaby's downstream targets. We show here that Shavenbaby controls epidermal cell shape, through the transcriptional activation of different classes of cellular effectors, directly contributing to the organization of actin filaments, regulation of the extracellular matrix, and modification of the cuticle. Individual inactivation of shavenbaby's targets produces distinct trichome defects and only their simultaneous inactivation prevent trichome formation. Our data show that shavenbaby governs an evolutionarily conserved developmental module consisting of a set of genes collectively responsible for trichome formation, shedding new light on molecular mechanisms acting during morphogenesis and the way they can influence evolution of animal forms., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila, two Pax6 genes function in a pathway in which twin of eyeless (toy) directly regulates eyeless (ey), which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, we used different truncated forms of toy and small eyes (sey), and tested their capacity to induce ectopic eye development in an ey-independent manner. Even though the Paired domains of TOY and SEY have DNA-binding properties that differ from those of the Paired domain of EY, they all are capable of inducing ectopic eye development in an ey mutant background. We also show that one of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, we show that only the homeodomain (HD) of EY is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of TOY and EY, and significant evolutionary divergence between the two Drosophila Pax6 genes.

Cnidarians are the simplest animals in which distinct eyes are present. We have previously suggested that cnidarian Pax-Cam might represent a precursor of the Pax-6 class. Here we show that when expressed in Drosophila imaginal discs, Pax-Cam chimeric proteins containing the C-terminal region of EY were capable of eye induction and driving expression of a reporter gene under the control of a known EY target (the sine oculis gene). Whilst these results are consistent with a Pax-6-like function for Pax-Cam, in band shift experiments we were unable to distinguish the DNA-binding behaviour of the Pax-Cam Paired domain from that of a second Acropora Pax protein, Pax-Bam. The ability of a Pax-Bam/EY chimera to also induce eye formation in leg imaginal discs, together with the in vitro data, cast doubt on previously assumed direct relationships between cnidarian Pax genes and the Pax-6 and Pax-2/5/8 classes of bilateral animals., (Copyright 2003 Wiley-Liss, Inc.)

Diversification of Drosophila segmental and cellular identities both require the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that we used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated EY proteins. Apart from eye loss, these new eyeless alleles lead to defects in the adult olfactory appendages: the maxillary palps and antennae. In support of these observations, both ey and pb are expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either PB or EY proteins. A combination of in vivo and in vitro approaches indicates that PB suppresses EY transactivation activity via protein-protein contacts of the PB homeodomain and EY Paired domain. The direct functional antagonism between PB and EY proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis.

Drosophila eye development is under the control of early eye specifying genes including eyeless (ey), twin of eyeless (toy), eyes absent (eya), dachshund (dac) and sine oculis (so). They are all conserved between vertebrates and insects and they interact in a combinatorial and hierarchical network to regulate each other expression. so has been shown to be directly regulated by ey through an eye-specific enhancer (so10). We further studied the regulation of this element and found that both Drosophila Pax6 proteins namely EY and TOY bind and positively regulate so10 expression through different binding sites. By targeted mutagenesis experiments, we disrupted these EY and TOY binding sites and studied their functional involvement in the so10 enhancer expression in the eye progenitor cells. We show a differential requirement for the EY and TOY binding sites in activating so10 during the different stages of eye development. Additionally, in a rescue experiment performed in the so(1) mutant, we show that the EY and TOY binding sites are required for compound eye and ocellus development respectively. Altogether, these results suggest a differential requirement for EY and TOY to specify the development of the two types of adult visual systems, namely the compound eye and the ocellus.