Claudio R. Alonso

The regulation of Hox gene expression during animal development

Hox genes encode a family of transcriptional regulators that elicit distinct developmental programmes along the head-to-tail axis of animals. The specific regional functions of individual Hox genes largely reflect their restricted expression patterns, the disruption of which can lead to developmental defects and disease. Here, we examine the spectrum of molecular mechanisms controlling Hox gene expression in model vertebrates and invertebrates and find that a diverse range of mechanisms, including nuclear dynamics, RNA processing, microRNA and translational regulation, all concur to control Hox gene outputs. We propose that this complex multi-tiered regulation might contribute to the robustness of Hox expression during development.

Genome-wide analysis of mRNA decay patterns during early Drosophila development

BackgroundThe modulation of mRNA levels across tissues and time is key for the establishment and operation of the developmental programs that transform the fertilized egg into a fully formed embryo. Although the developmental mechanisms leading to differential mRNA synthesis are heavily investigated, comparatively little attention is given to the processes of mRNA degradation and how these relate to the molecular programs controlling development.ResultsHere we combine timed collection of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally and zygotically encoded factors to mRNA degradation, and the ways in which mRNA decay profiles relate to gene function, mRNA localization patterns, translation rates and protein turnover. We also detect cis-regulatory sequences enriched in transcripts with common degradation patterns and propose several proteins and microRNAs as developmental regulators of mRNA decay during early fruit fly development. Finally, we experimentally validate the effects of a subset of cis-regulatory sequences and trans-regulators in vivo.ConclusionsOur work advances the current understanding of the processes controlling mRNA degradation during early Drosophila development, taking us one step closer to the understanding of mRNA decay processes in all animals. Our data also provide a valuable resource for further experimental and computational studies investigating the process of mRNA decay.

The molecular elements that underlie developmental evolution

Abundant evidence indicates that developmental evolution, the foundation of morphological evolution, is based on changes in gene function. Over the past decade a consensus has developed that transcriptional regulation, acting through enhancer sequences, is the primary level of evolutionarily significant change. Here we propose that other regulatory levels are probably as important as enhancers in developmental evolution. We also explain why these alternative regulatory levels might have been neglected, and briefly discuss ways to test our hypothesis.

Drosophila miniature and dusky encode ZP proteins required for cytoskeletal reorganisation during wing morphogenesis.

We have characterised the function of two Drosophila genes, miniature and dusky, that are required for the morphological reorganisation of the apical membrane during wing epidermis differentiation. These genes encode transmembrane proteins containing a ZP (zona pellucida) domain and are homologous to several vertebrate and invertebrate apical matrix components. miniature and dusky are only expressed in tissues secreting a cuticle, and the Min protein localises to the apical membrane during the early stages of cuticle formation. We propose that Min and Dusky form a novel subfamily within the ZP domain proteins and are specifically involved in the interactions between the apical membrane, the cytoskeleton and the forming cuticle.

Developmental RNA processing of 3′UTRs in Hox mRNAs as a context-dependent mechanism modulating visibility to microRNAs

The Drosophila Hox gene Ultrabithorax (Ubx) controls the development of thoracic and abdominal segments, allocating segment-specific features to different cell lineages. Recent studies have shown that Ubx expression is post-transcriptionally regulated by two microRNAs (miRNAs), miR-iab4 and miR-iab8, acting on target sites located in the 3′ untranslated regions (UTRs) of Ubx mRNAs. Here, we show that during embryonic development Ubx produces mRNAs with variable 3′UTRs in different regions of the embryo. Analysis of the resulting remodelled 3′UTRs shows that each species harbours different sets of miRNA target sites, converting each class of Ubx mRNA into a considerably different substrate for miRNA regulation. Furthermore, we show that the distinct developmental distributions of Ubx 3′UTRs are established by a mechanism that is independent of miRNA regulation and therefore are not the consequence of miR-iab4/8-mediated RNA degradation acting on those sensitive mRNA species; instead, we propose that this is a hard-wired 3′UTR processing system that is able to regulate target mRNA visibility to miRNAs according to developmental context. We show that reporter constructs that include Ubx short and long 3′UTR sequences display differential expression within the embryonic central nervous system, and also demonstrate that mRNAs of three other Hox genes suffer similar and synchronous developmental 3′UTR processing events during embryogenesis. Our work thus reveals that developmental RNA processing of 3′UTR sequences is a general molecular strategy used by a key family of developmental regulators so that their transcripts can display different levels of visibility to miRNA regulation according to developmental cues.

Drosophila Upf1 and Upf2 loss of function inhibits cell growth and causes animal death in a Upf3-independent manner

Nonsense-mediated RNA decay (NMD) is a surveillance mechanism that degrades transcripts containing nonsense mutations, preventing the translation of truncated proteins. NMD also regulates the levels of many endogenous mRNAs. While the mechanism of NMD is gradually understood, its physiological role remains largely unknown. The core NMD genes upf1 and upf2 are essential in several organisms, which may reflect an important developmental role for NMD. Alternatively, the lethality of these mutants might arise from their function in NMD-independent processes. To analyze the developmental importance of NMD, we studied Drosophila mutants of the other core NMD gene, upf3. We compare the resulting upf3 phenotype with those defects observed in upf1 and upf2 loss-of-function mutants, as well as with flies expressing a mutant Upf2 protein unable to bind Upf3. Our results show that Upf3 is an NMD effector in the fly but, unlike Upf1 and Upf2, plays a peripheral role in the degradation of most NMD targets and is not required for development or viability. Furthermore, Upf1 and Upf2 loss-of-function inhibits cell growth and induces apoptosis through a Upf3-independent pathway. Accordingly, disruption of Upf2-Upf1 interaction causes death, while the Upf2-Upf3 complex is dispensable for viability. Our findings suggest that NMD is essential for cell growth and animal development, and that the lethality of upf1 and upf2 mutants is not due to disrupting their roles during NMD-independent processes, but to their function in the degradation of specific mRNAs by the NMD pathway. Furthermore, our results show that Upf3 is not always essential in NMD.

Alternative Splicing Modulates Ubx Protein Function in Drosophila melanogaster

The Drosophila Hox gene Ultrabithorax (Ubx) produces a family of protein isoforms through alternative splicing. Isoforms differ from one another by the presence of optional segments—encoded by individual exons—that modify the distance between the homeodomain and a cofactor-interaction module termed the “YPWM” motif. To investigate the functional implications of Ubx alternative splicing, here we analyze the in vivo effects of the individual Ubx isoforms on the activation of a natural Ubx molecular target, the decapentaplegic (dpp) gene, within the embryonic mesoderm. These experiments show that the Ubx isoforms differ in their abilities to activate dpp in mesodermal tissues during embryogenesis. Furthermore, using a Ubx mutant that reduces the full Ubx protein repertoire to just one single isoform, we obtain specific anomalies affecting the patterning of anterior abdominal muscles, demonstrating that Ubx isoforms are not functionally interchangeable during embryonic mesoderm development. Finally, a series of experiments in vitro reveals that Ubx isoforms also vary in their capacity to bind DNA in presence of the cofactor Extradenticle (Exd). Altogether, our results indicate that the structural changes produced by alternative splicing have functional implications for Ubx protein function in vivo and in vitro. Since other Hox genes also produce splicing isoforms affecting similar protein domains, we suggest that alternative splicing may represent an underestimated regulatory system modulating Hox gene specificity during fly development.

Hox Proteins: Sculpting Body Parts by Activating Localized Cell Death

Hox proteins shape animal structures by eliciting different developmental programs along the anteroposterior body axis. A recent study reveals that the Drosophila Hox protein Deformed directly activates the cell-death-promoting gene reaper to maintain the boundaries between distinct head segments.

MicroRNA-encoded behavior in Drosophila

The relationship between microRNA (miRNA) regulation and the specification of behavior is only beginning to be explored. We found that mutation of a single miRNA locus (miR-iab4/iab8) in Drosophila larvae affects the animal’s capacity to correct its orientation if turned upside down (self-righting). One of the miRNA targets involved in this behavior is the Hox gene Ultrabithorax, whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants, whereas thermogenetic manipulation of SRN activity results in changes in self-righting behavior. Our work thus reveals a miRNA-encoded behavior and suggests that other miRNAs might also be involved in behavioral control in Drosophila and other species. In Drosophila larvae, mutation of a single microRNA locus affects the animal’s ability to correct its orientation if turned upside down. MicroRNAs that control behavior MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity. They repress expression through complementary base pairing interactions with target messenger RNAs. MiRNAs are involved in regulating many cell and developmental processes. Picao-Osorio et al. find that miRNAs can also control behavior in the fruit fly Drosophila. A specific miRNA locus regulates the self-righting response in larva that ate tipped over onto their backs. The miRNA locus targets a gene required for the normal activity of two neurons involved in the self-righting response. Science, this issue p. 815

Integration of RNA processing and expression level control modulates the function of the Drosophila Hox gene Ultrabithorax during adult development

Although most metazoan genes undergo alternative splicing, the functional relevance of the majority of alternative splicing products is still unknown. Here we explore this problem in the Drosophila Hox gene Ultrabithorax (Ubx). Ubx produces a family of six protein isoforms through alternative splicing. To investigate the functional specificity of the Ubx isoforms, we studied their role during the formation of the Drosophila halteres, small dorsal appendages that are essential for normal flight. Our work shows that isoform Ia, which is encoded by all Ubx exons, is more efficient than isoform IVa, which lacks the amino acids coded by two small exons, in controlling haltere development and regulating Ubx downstream targets. However, our experiments also demonstrate that the functional differences among the Ubx isoforms can be compensated for by increasing the expression levels of the less efficient form. The analysis of the DNA-binding profiles of Ubx isoforms to a natural Ubx target, spalt, shows no major differences in isoform DNA-binding activities, suggesting that alternative splicing might primarily affect the regulatory capacity of the isoforms rather than their DNA-binding patterns. Our results suggest that to obtain distinct functional outputs during normal development genes must integrate the generation of qualitative differences by alternative splicing to quantitative processes affecting isoform protein expression levels.