A timer gene network is spatially regulated by the terminal system in the Drosophila embryo.

In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the 'timer' genes caudal, Dichaete, and odd-paired, whose expression dynamics correlate with the mode of segmentation. In Drosophila, the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small 'tail' region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless, but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.

The neuroblast timer gene nubbin exhibits functional redundancy with gap genes to regulate segment identity in Tribolium.

The neuroblast timer genes hunchback, Krüppel, nubbin and castor are expressed in temporal sequence in neural stem cells, and in corresponding spatial sequence along the Drosophila blastoderm. As canonical gap genes, hunchback and Krüppel play a crucial role in insect segmentation, but the roles of nubbin and castor in this process remain ambiguous. We have investigated the expression and functions of nubbin and castor during segmentation in the beetle Tribolium. We show that Tc-hunchback, Tc-Krüppel, Tc-nubbin and Tc-castor are expressed sequentially in the segment addition zone, and that Tc-nubbin regulates segment identity redundantly with two previously described gap/gap-like genes, Tc-giant and Tc-knirps. Simultaneous knockdown of Tc-nubbin, Tc-giant and Tc-knirps results in the formation of ectopic legs on abdominal segments. This homeotic transformation is caused by loss of abdominal Hox gene expression, likely due to expanded Tc-Krüppel expression. Our findings support the theory that the neuroblast timer series was co-opted for use in insect segment patterning, and contribute to our growing understanding of the evolution and function of the gap gene network outside of Drosophila.

A revised understanding of Tribolium morphogenesis further reconciles short and long germ development.

In Drosophila melanogaster, the germband forms directly on the egg surface and solely consists of embryonic tissue. In contrast, most insect embryos undergo a complicated set of tissue rearrangements to generate a condensed, multilayered germband. The ventral side of the germband is embryonic, while the dorsal side is thought to be an extraembryonic tissue called the amnion. While this tissue organisation has been accepted for decades and has been widely reported in insects, its accuracy has not been directly tested in any species. Using live cell tracking and differential cell labelling in the short germ beetle Tribolium castaneum, I show that most of the cells previously thought to be amnion actually give rise to large parts of the embryo. This process occurs via the dorsal-to-ventral flow of cells and contributes to germband extension (GBE). In addition, I show that true 'amnion' cells in Tribolium originate from a small region of the blastoderm. Together, my findings show that development in the short germ embryos of Tribolium and the long germ embryos of Drosophila is more similar than previously proposed. Dorsal-to-ventral cell flow also occurs in Drosophila during GBE, and I argue that the flow is driven by a conserved set of underlying morphogenetic events in both species. Furthermore, the revised Tribolium fate map that I present is far more similar to that of Drosophila than the classic Tribolium fate map. Lastly, my findings show that there is no qualitative difference between the tissue structure of the cellularised blastoderm and the short/intermediate germ germband. As such, the same tissue patterning mechanisms could function continuously throughout the cellularised blastoderm and germband stages, and easily shift between them over evolutionary time.

A novel role for Ets4 in axis specification and cell migration in the spider Parasteatoda tepidariorum.

Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos. In spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders. Despite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression. To address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate.

Deep, Staged Transcriptomic Resources for the Novel Coleopteran Models Atrachya menetriesi and Callosobruchus maculatus.

Despite recent efforts to sample broadly across metazoan and insect diversity, current sequence resources in the Coleoptera do not adequately describe the diversity of the clade. Here we present deep, staged transcriptomic data for two coleopteran species, Atrachya menetriesi (Faldermann 1835) and Callosobruchus maculatus (Fabricius 1775). Our sampling covered key stages in ovary and early embryonic development in each species. We utilized this data to build combined assemblies for each species which were then analysed in detail. The combined A. menetriesi assembly consists of 228,096 contigs with an N50 of 1,598 bp, while the combined C. maculatus assembly consists of 128,837 contigs with an N50 of 2,263 bp. For these assemblies, 34.6% and 32.4% of contigs were identified using Blast2GO, and 97% and 98.3% of the BUSCO set of metazoan orthologs were present, respectively. We also carried out manual annotation of developmental signalling pathways and found that nearly all expected genes were present in each transcriptome. Our analyses show that both transcriptomes are of high quality. Lastly, we performed read mapping utilising our timed, stage specific RNA samples to identify differentially expressed contigs. The resources presented here will provide a firm basis for a variety of experimentation, both in developmental biology and in comparative genomic studies.

Toll Genes Have an Ancestral Role in Axis Elongation.

One of the key morphogenetic processes used during development is the controlled intercalation of cells between their neighbors. This process has been co-opted into a range of developmental events, and it also underlies an event that occurs in each major group of bilaterians: elongation of the embryo along the anterior-posterior axis [1]. In Drosophila, a novel component of this process was recently discovered by Paré et al., who showed that three Toll genes function together to drive cell intercalation during germband extension [2]. This finding raises the question of whether this role of Toll genes is an evolutionary novelty of flies or a general mechanism of embryonic morphogenesis. Here we show that the Toll gene function in axis elongation is, in fact, widely conserved among arthropods. First, we functionally demonstrate that two Toll genes are required for cell intercalation in the beetle Tribolium castaneum. We then show that these genes belong to a previously undescribed Toll subfamily and that members of this subfamily exhibit striped expression (as seen in Tribolium and previously reported in Drosophila [3-5]) in embryos of six other arthropod species spanning the entire phylum. Last, we show that two of these Toll genes are required for normal morphogenesis during anterior-posterior embryo elongation in the spider Parasteatoda tepidariorum, a member of the most basally branching arthropod lineage. From our findings, we hypothesize that Toll genes had a morphogenetic function in embryo elongation in the last common ancestor of all arthropods, which existed over 550 million years ago.

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis.

N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum.

In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.

Tribolium embryo morphogenesis: may the force be with you.

Development of multicellular organisms depends on patterning and growth mechanisms encoded in the genome, but also on the physical properties and mechanical interactions of the constituent cells that interpret these genetic cues. This fundamental biological problem requires integrated studies at multiple levels of biological organization: from genes, to cell behaviors, to tissue morphogenesis. We have recently combined functional genetics with live imaging approaches in embryos of the insect Tribolium castaneum, in order to understand their remarkable transformation from a uniform single-layered blastoderm into a condensed multi-layered embryo covered by extensive extra-embryonic tissues. We first developed a quick and reliable methodology to fluorescently label various cell components in entire Tribolium embryos. Live imaging of labeled embryos at single cell resolution provided detailed descriptions of cell behaviors and tissue movements during normal embryogenesis. We then compared cell and tissue dynamics between wild-type and genetically perturbed embryos that exhibited altered relative proportions of constituent tissues. This systematic comparison led to a qualitative model of the molecular, cellular and tissue interactions that orchestrate the observed epithelial rearrangements. We expect this work to establish the Tribolium embryo as a powerful and attractive model system for biologists and biophysicists interested in the molecular, cellular and mechanical control of tissue morphogenesis.

Cell and tissue dynamics during Tribolium embryogenesis revealed by versatile fluorescence labeling approaches.

Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.

Canonical terminal patterning is an evolutionary novelty.

Patterning of the terminal regions of the Drosophila embryo is achieved by an exquisitely regulated signal that passes between the follicle cells of the ovary, and the developing embryo. This pathway, however, is missing or modified in other insects. Here we trace the evolution of this pathway by examining the origins and expression of its components. The three core components of this pathway: trunk, torso and torso-like have different evolutionary histories and have been assembled step-wise to form the canonical terminal patterning pathway of Drosophila and Tribolium. Trunk, torso and a gene unrelated to terminal patterning, prothoraciotrophic hormone (PTTH), show an intimately linked evolutionary history, with every holometabolous insect, except the honeybee, possessing both PTTH and torso genes. Trunk is more restricted in its phylogenetic distribution, present only in the Diptera and Tribolium and, surprisingly, in the chelicerate Ixodes scapularis, raising the possibility that trunk and torso evolved earlier than previously thought. In Drosophila torso-like restricts the activation of the terminal patterning pathway to the poles of the embryo. Torso-like evolved in the pan-crustacean lineage, but based on expression of components of the canonical terminal patterning system in the hemimetabolous insect Acyrthosiphon pisum and the holometabolous insect Apis mellifera, we find that the canonical terminal-patterning system is not active in these insects. We therefore propose that the ancestral function of torso-like is unrelated to terminal patterning and that torso-like has become co-opted into terminal patterning in the lineage leading to Coleoptera and Diptera. We also show that this co-option has not resulted in changes to the molecular function of this protein. Torso-like from the pea aphid, honeybee and Drosophila, despite being expressed in different patterns, are functionally equivalent. We propose that co-option of torso-like into restricting the activity of trunk and torso facilitated the final step in the evolution of this pathway; the capture of transcriptional control of target genes such as tailless and huckebein by this complex and novel patterning pathway.