Evolution of the gene regulatory network controlling trunk segmentation in insects

The study of pattern formation in insects is the main source of our current understanding of the genetic processes underlying the development of an organism. Ontogeny has been thoroughly studied in the model organism Drosophila melanogaster, where a set of transcription factors and signaling molecules pattern the fly embryo through a segmentation gene cascade. Over the past 20 years, this model has been compared to different organisms throughout the Metazoa. Here I describe the functional analysis of genes and gene regulatory network controlling segmentation in the short germ beetle Tribolium castaneum. The hunchback gene is one of the major early determinants in the Drosophila segmentation cascade, where it serves an instructive role in patterning the entire body plan. In several insects, the role of hb in patterning body compartments (cardinal regions) is conserved. However, in hemimetabolous insects developing as short germs hb role has been reported to differ from the canonical gap function described in holometabolous insects. In the first chapter I describe the role of hb in Tribolium, a holometabolous insect developing as short germ. This analysis revealed that Tc'hb has an indirect effect in segmentation, mediated by other gap genes like giant, and a most likely a direct effect in the segment identity specification, by setting the anterior border of thoracic and abdominal Hox genes. This finding suggests an ancestral role of hb as a cardinal gene within insects and allows the reinterpretation of the canonical gap phenotype described in the fly. The expression analysis of ESTs in Tribolium identified a putative non-coding RNA showing a gap-like expression pattern during segmentation. In the second chapter I describe the functional analysis of this gene, named mille-pattes. This analysis defined Tc'mlpt as a novel segmentation gene in Tribolium, which controlls trunk segmentation in a cross-regulatory network among gap genes and regulates the expression domains of Hox genes. Strikingly, mlpt does not code for a transcription factor, but instead, encodes several small peptides, which are conserved among mlpt homologues in various insects. As a model, the gene regulatory network controlling segmentation in Drosophila has been thoroughly tested in many other organisms, revealing a surprising plasticity of the developmental mechanism controlling segmentation among insects. In order to identify the regulatory interactions among the gap genes that are controlling segmentation in Tribolium, I further characterized the gap gene cross-regulatory network in Tribolium and their interaction with pair rule genes. This analysis provided a powerful data set on the regulatory interactions among gap genes and their interactions with pair rule genes in Tribolium. Finally, the concomitant characterization of segmentation genes presented in this thesis allowed the reinterpretation of the role of hunchback among insects, specially the canonical gap phenotype described for Tribolium and Drosophila. Furthermore, by studying the interactions between gap and Hox genes in Tribolium it was possible to propose a model for the regulation and function of Tc'Antp and for the regulation of the Hox genes along the AP axis in Tribolium

Oncopeltus fasciatus zen is essential for serosal tissue function in katatrepsis

AbstractUnlike most Hox cluster genes, with their canonical role in anterior–posterior patterning of the embryo, the Hox3 orthologue of insects has diverged. Here, we investigate the zen orthologue in Oncopeltus fasciatus (Hemiptera:Heteroptera). As in other insects, the Of-zen gene is expressed extraembryonically, and RNA interference (RNAi) experiments demonstrate that it is functionally required in this domain for the proper occurrence of katatrepsis, the phase of embryonic movements by which the embryo emerges from the yolk and adjusts its orientation within the egg. After RNAi knockdown of Of-zen, katatrepsis does not occur, causing embryos to complete development inside out. However, not all aspects of expression and function are conserved compared to grasshopper, beetle, and fly orthologues. Of-zen is not expressed in the extraembryonic tissue until relatively late, suggesting it is not involved in tissue specification. Within the extraembryonic domain, Of-zen is expressed in the outer serosal membrane, but unlike orthologues, it is not detectable in the inner extraembryonic membrane, the amnion. Thus, the role of zen in the interaction of serosa, amnion, and embryo may differ between species. Of-zen is also expressed in the blastoderm, although this early expression shows no apparent correlation with defects seen by RNAi knockdown

The Drosophila dead ringer gene is required for early embryonic patterning through regulation of argos and buttonhead expression

Copyright © 1999 by Company of BiologistsThe dead ringer (dri) gene of Drosophila melanogaster is a member of the recently discovered ARID-box family of eukaryotic genes that encode proteins with a conserved DNA binding domain. dri itself is highly conserved, with specific orthologs in the human, mouse, zebrafish and C. elegans genomes. We have generated dri mutant alleles to show that dri is essential for anterior-posterior patterning and for muscle development in the embryo. Consistent with the mutant phenotype and the sequence-specific DNA-binding properties of its product, dri was found to be essential for the normal early embryonic expression pattern of several key regulatory genes. In dri mutant embryos, expression of argos in the terminal domains was severely reduced, accounting for the dri mutant head phenotype. Conversely, buttonhead expression was found to be deregulated in the trunk region, accounting for the appearance of ectopic cephalic furrows. Curiously, dri was found also to be required for maintenance of expression of the ventrolateral region of even-skipped stripe four. This study establishes dri as an essential co-factor in the regulated expression of specific patterning genes during early embryogenesis

Evolution der translationalen Repression von caudal in höheren Insekten

Der anterior lokalisierte Entwicklungsfaktor bicoid (bcd) steuert the translationale Repression von caudal (cad) in Drosophila Embryos. Das bcd Gen kommt im Gegensatz zu cad nur in einer abgeleiteten Gruppe von Diptera vor. Die Expression von cad in anderen Insektenarten muss daher auf andere Weise reguliert sein. Interessanterweise kann BCD die Translation des Tribolium castaneum cad Homologs (Tc cad) reprimieren, wenn es in transgenen Drosophila Embryos exprimiert ist. Der Bereich an dem BCD in der Tc cad mRNA bindet, liegt wahrscheinlich in der 3 UTR des Transkripts und es stellt sich die Frage, ob BCD an ein konserviertes Element in den unterschiedlichen mRNAs bindet. Mit Hilfe eines in vivo Sensor-Assays wurden kurze Regionen in den 3 untranslatierten Regionen (3 UTR) von Drosophila und der Bremse Haematopota pluvialis identifiziert, die in Anwesenheit von BCD die translationale Repression von Sensortranskripten herbeiführen. Diese RNA Regionen weisen Ähnlichkeiten in ihren Sekundärstrukturen auf und könnten die Grundlage für ein konserviertes BCD-Bindungselement darstellen. Die direkte Bindung der BCD-Homeodomäne an diese RNA-Regionen wurde mit Hilfe eines elektrophoretischen Mobilitäts-Assays nachgewiesen. Die BCD-Bindungsregion der Drosophila 3 UTR überlappt mit einer putativen Bindungsstelle der microRNA miR-308. Mutationen in dieser Region verhindern einerseits die miR-308-Bindung und andererseits die BCD-abhängige translationale Repression des Sensors. Des Weiteren können auch verschiedene Isoformen des BCD Proteins die Translation von Sensortranskripten verhindern. Zusammenfassend weisen diese Ergebnisse auf das Vorhandensein von alternativen Mechanismen der cadDrosophila hin und könnten auch in anderen Insektenarten vorhanden sein.The anterior patterning factor bicoid (bcd) mediates translational repression of caudal (cad) in Drosophila embryos and is an evolutionary novelty present only in higher dipterans. Therefore other insect species must follow different strategies to restrict cad expression. Interestingly, BCD translationally represses the mRNA of the cad homologue of Tribolium castaneum (Tc cad) when expressed in Drosophila embryos. The region to which BCD binds has been speculated to be in the Tc cad 3 UTR and raised the question whether BCD may recognize regulatory element(s) that are conserved between Drosophila, Tribolium and other insect species. By establishing an in vivo sensor for BCD-mediated translational repression I was able to identify small regions in the cad 3 UTR of Drosophila and the horsefly Haematopota pluvialis that mediate BCD-dependent translational repression. These elements show similarities in their predicted secondary structures, which could be the basis for a conserved BCD-binding element. Using electrophoretic mobility shift assays I could show direct binding of the BCD homeodomain to these 3 UTR regions. The BCD-binding region of the Dm cad 3 UTR co-localizes with a target site of the microRNA miR-308 and mutations in this region abolish miRNA-binding and BCD-mediated translational repression. Furthermore, different BCD isoforms are able to mediate translational repression of sensors carrying BCD-binding regions. Taken together, these findings suggest that alternative mechanism(s) for the translational repression of cad mRNA are likely to exist in Drosophila and may also be present in other insect species