Serge Plaza

Small Peptides Switch the Transcriptional Activity of Shavenbaby During Drosophila Embryogenesis

Transcription On and sORF Eukaryotic transcriptomes include numerous RNAs that are presumed noncoding because they include only short open reading frames (sORFs). However, some sORF RNAs actually produce small peptides with unknown activity. Now Kondo et al. (p. 336; see the Perspective by Rosenberg) report the function of peptides containing 11 to 32 amino acids that are encoded by the gene polished-rice (pri). pri triggers N-terminal truncation of the transcription factor Shavenbaby that controls epidermal differentiation in Drosophila. Following pri expression, Shavenbaby is converted from a transcriptional repressor to an activator. Thus, sORF peptides can control transcriptional programs during embryonic development. A conserved locus in insects encodes four small peptides that shift a transcription factor from repressor into activator. 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.

Molecular basis for the inhibition of Drosophila eye development by Antennapedia

Hox genes encoding homeodomain transcriptional regulators are known to specify the body plan of multicellular organisms and are able to induce body plan transformations when misexpressed. These findings led to the hypothesis that duplication events and misexpression of Hox genes during evolution have been necessary for generating the observed morphological diversity found in metazoans. It is known that overexpressing Antennapedia (Antp) in the head induces antenna‐to‐leg as well as head‐to‐thorax transformation and eye reduction. At present, little is known about the exact molecular mechanism causing these phenotypes. The aim of this study is to understand the basis of inhibition of eye development. We demonstrate that Antp represses the activity of the eye regulatory cascade. By ectopic expression, we show that Antp antagonizes the activity of the eye selector gene eyeless. Using both in vitro and in vivo experiments, we demonstrate that this inhibitory mechanism involves direct protein–protein interactions between the DNA‐binding domains of EY and ANTP, resulting in mutual inhibition.

Zona Pellucida Domain Proteins Remodel the Apical Compartment for Localized Cell Shape Changes

The zona pellucida domain (ZPD) defines a conserved family of membrane-anchored matrix proteins that are, as yet, poorly characterized with respect to their functions during development. Using genetic approaches in flies, we show here that a set of eight ZPD proteins is required for the localized reorganization of embryonic epidermal cells during morphogenesis. Despite varying degrees of sequence conservation, these ZPD proteins exert specific and nonredundant functions in the remodeling of epidermal cell shape. Each one accumulates in a restricted subregion of the apical compartment, where it organizes local interactions between the membrane and the extracellular matrix. In addition, ZPD proteins are required to sculpture the actin-rich cell extensions and maintain appropriate organization of the apical compartment. These results on ZPD proteins therefore reveal a functional subcompartmentalization of the apical membrane and its role in the polarized control of epithelial cell shape during development.

Fascin is required for blood cell migration during Drosophila embryogenesis

Fascin is well characterized in vitro as an actin-bundling protein and its increased expression is correlated with the invasiveness of various cancers. However, the actual roles and regulation of Fascin in vivo remain elusive. Here we show that Fascin is required for the invasive-like migration of blood cells in Drosophila embryos. Fascin expression is highly regulated during embryonic development and, within the blood lineage, is specific to the motile subpopulation of cells, which comprises macrophage-like plasmatocytes. We show that Fascin is required for plasmatocyte migration, both as these cells undergo developmental dispersal and during an inflammatory response to epithelial wounding. Live analyses further demonstrate that Fascin localizes to, and is essential for the assembly of, dynamic actin-rich microspikes within plasmatocyte lamellae that polarize towards the direction of migration. We show that a regulatory serine of Fascin identified from in vitro studies is not required for in vivo cell motility, but is crucial for the formation of actin bundles within epithelial bristles. Together, these results offer a first glimpse into the mechanisms regulating Fascin function during normal development, which might be relevant for understanding the impact of Fascin in cancers.

The YPWM motif links Antennapedia to the basal transcriptional machinery.

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.

Functional divergence between eyeless and twin of eyeless in Drosophila melanogaster

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.

Cross-regulatory protein–protein interactions between Hox and Pax transcription factors

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.

From A to Z: apical structures and zona pellucida-domain proteins

The terminal differentiation of epithelial cells involves changes in the apical compartment, including remodeling of the cytoskeleton and junctions to modify its three-dimensional organization. It also often triggers the building of specialized extracellular matrices, the function of which remains poorly understood. Hundreds of extracellular matrix proteins expressed in a variety of epithelia possess a conserved region called the zona pellucida-domain (ZP domain). There is evidence to suggest that ZP-domains mediate the polymerization of proteins into fibrils or matrices and that mutation of ZP-domains can result in severe pathologies, such as infertility, deafness, and cancer. Recent work in worms and flies demonstrates that ZP-domain proteins play a crucial role in organizing and shaping highly specialized apical structures in epithelial cells.

Fascin is required for blood cell polarity and migration during Drosophila embryogenesis.

Fascin is well characterized in vitro as an actin-bundling protein and its increased expression is correlated with the invasiveness of various cancers. However, the actual roles and regulation of Fascin in vivo remain elusive. Here we show that Fascin is required for the invasive-like migration of blood cells in Drosophila embryos. Fascin expression is highly regulated during embryonic development and, within the blood lineage, is specific to the motile subpopulation of cells, which comprises macrophage-like plasmatocytes. We show that Fascin is required for plasmatocyte migration, both as these cells undergo developmental dispersal and during an inflammatory response to epithelial wounding. Live analyses further demonstrate that Fascin localizes to, and is essential for the assembly of, dynamic actin-rich microspikes within plasmatocyte lamellae that polarize towards the direction of migration. We show that a regulatory serine of Fascin identified from in vitro studies is not required for in vivo cell motility, but is crucial for the formation of actin bundles within epithelial bristles. Together, these results offer a first glimpse into the mechanisms regulating Fascin function during normal development, which might be relevant for understanding the impact of Fascin in cancers.

Fascin promotes filopodia formation independent of its role in actin bundling

Mutation of a critical residue of fascin eliminates the protein’s actin-bundling activity but maintains its positive role in filopodia formation