A1: Publications
Global analysis of dorsoventral patterning in the wasp Nasonia reveals extensive incorporation of novelty in a regulatory network
Pers D., Buchta T., Özüak O., Wolff S., Pietsch J.M., Memon M.B., Roth S. and Lynch J.A., BMC Biology, 2016 14:63, 1. Aug. 2016, https://doi.org/10.1186/s12915-016-0285-y
Background
Gene regulatory networks (GRNs) underlie developmental patterning and morphogenetic processes, and changes in the interactions within the underlying GRNs are a major driver of evolutionary processes. In order to make meaningful comparisons that can provide significant insights into the evolution of regulatory networks, homologous networks from multiple taxa must be deeply characterized. One of the most thoroughly characterized GRNs is the dorsoventral (DV) patterning system of the Drosophila melanogaster embryo. We have developed the wasp Nasonia as a comparative DV patterning model because it has shown the convergent evolution of a mode of early embryonic patterning very similar to that of the fly, and it is of interest to know whether the similarity at the gross level also extends to the molecular level.
Results
We used RNAi to dorsalize and ventralize Nasonia embryos, RNAseq to quantify transcriptome-wide expression levels, and differential expression analysis to identify genes whose expression levels change in either RNAi case. This led to the identification of >100 genes differentially expressed and regulated along the DV axis. Only a handful of these genes are shared DV components in both fly and wasp. Many of those unique to Nasonia are cytoskeletal and adhesion molecules, which may be related to the divergent cell and tissue behavior observed at gastrulation. In addition, many transcription factors and signaling components are only DV regulated in Nasonia, likely reflecting the divergent upstream patterning mechanisms involved in producing the conserved pattern of cell fates observed at gastrulation. Finally, several genes that lack Drosophila orthologs show robust and distinct expression patterns. These include genes with vertebrate homologs that have been lost in the fly lineage, genes that are found only among Hymenoptera, and several genes that entered the Nasonia genome through lateral transfer from endosymbiotic bacteria.
Conclusions
Altogether, our results provide insights into how GRNs respond to new functional demands and how they can incorporate novel components.
Toll Genes Have an Ancestral Role in Axis Elongation
Matthew A. Benton, Matthias Pechmann, Nadine Frey, Dominik Stappert, Kai H. Conrads, Yen-Ta Chen, Evangelia Stamataki, Anastasios Pavlopoulos, Siegfried Roth, Cell Press, June 20, 2016, doi:10.1016/j.cub.2016.04.055
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. 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. 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) 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.
Genome-wide identification of Tribolium dorsoventral patterning genes
Stappert D, Frey N, von Levetzow C, Roth S,
The Company of Biologists Ltd | Development (2016) 143, 2443-2454 doi:10.1242/dev.130641 May-Jun;17(3):198-219. doi: 10.1111/ede.12125.
The gene regulatory network controlling dorsoventral axis formation in insects has undergone drastic evolutionary changes. In Drosophila, a stable long-range gradient of Toll signalling specifies ventral cell fates and restricts BMP signalling to the dorsal half of the embryo. In Tribolium, however, Toll signalling is transient and only indirectly controls BMP signalling. In order to gain unbiased insights into the Tribolium network, we performed comparative transcriptome analyses of embryos with various dorsoventral pattering defects produced by parental RNAi for Toll and BMP signalling components. We also included embryos lacking the mesoderm (produced by Tc-twist RNAi) and characterized similarities and differences between Drosophila and Tribolium twist loss-of-function phenotypes. Using stringent conditions, we identified over 750 differentially expressed genes and analysed a subset with altered expression in more than one knockdown condition. We found new genes with localized expression and showed that conserved genes frequently possess earlier and stronger phenotypes than their Drosophila orthologues. For example, the leucine-rich repeat (LRR) protein Tartan, which has only a minor influence on nervous system development in Drosophila, is essential for early neurogenesis in Tribolium and the Tc-zinc-finger homeodomain protein 1 (Tc-zfh1), the orthologue of which plays a minor role in Drosophila muscle development, is essential for maintaining early Tc-twist expression, indicating an important function for mesoderm specification.
The significance and scope of evolutionary developmental biology: a vision for the 21st century.
Moczek AP, Sears KE, Stollewerk A, Wittkopp PJ, Diggle P, Dworkin I, Ledon-Rettig C, Matus DQ, Roth S, Abouheif E, Brown FD, Chiu CH, Cohen CS, Tomaso AW, Gilbert SF, Hall B, Love AC, Lyons DC, Sanger TJ, Smith J, Specht C, Vallejo-Marin M, Extavour CG.
Evol Dev. 2015 May-Jun;17(3):198-219. doi: 10.1111/ede.12125.
Evolutionary developmental biology (evo-devo) has undergone dramatic transformations since its emergence as a distinct discipline. This paper aims to highlight the scope, power, and future promise of evo-devo to transform and unify diverse aspects of biology. We articulate key questions at the core of eleven biological disciplines—from Evolution, Development, Paleontology, and Neurobiology to Cellular and Molecular Biology, Quantitative Genetics, Human Diseases, Ecology, Agriculture and Science Education, and lastly, Evolutionary Developmental Biology itself—and discuss why evo-devo is uniquely situated to substantially improve our ability to find meaningful answers to these fundamental questions. We posit that the tools, concepts, and ways of thinking developed by evo-devo have profound potential to advance, integrate, and unify biological sciences as well as inform policy decisions and illuminate science education. We look to the next generation of evolutionary developmental biologists to help shape this process as we confront the scientific challenges of the 21st century.
Dynamic BMP signaling polarized by Toll patterns the dorsoventral axis in a hemimetabolous insect
Lena Sachs, Yen-Ta Chen, Axel Drechsler, Jeremy A Lynch, Kristen A Panfilio, Michael Lässig, Johannes Berg, Siegfried Roth, DOI: http://dx.doi.org/10.7554/eLife.05502, Published May 12, 2015, Cite as eLife 2015;4:e05502
Toll-dependent patterning of the dorsoventral axis in Drosophila represents one of the best understood gene regulatory networks. However, its evolutionary origin has remained elusive. Outside the insects Toll is not known for a patterning function, but rather for a role in pathogen defense. Here, we show that in the milkweed bug Oncopeltus fasciatus, whose lineage split from Drosophila's more than 350 million years ago, Toll is only required to polarize a dynamic BMP signaling network. A theoretical model reveals that this network has self-regulatory properties and that shallow Toll signaling gradients are sufficient to initiate axis formation. Such gradients can account for the experimentally observed twinning of insect embryos upon egg fragmentation and might have evolved from a state of uniform Toll activity associated with protecting insect eggs against pathogens.
The transforming growth factor beta (TGF)-β signaling pathway and its modulators are involved in many aspects of cellular growth and differentiation in all metazoa. Although most of the core components of the pathway are highly conserved, many lineage-specific adaptations have been observed including changes regarding paralog number, presence and absence of modulators, and functional relevance for particular processes. In the parasitic jewel wasp Nasonia vitripennis, the bone morphogenetic proteins (BMPs), one of the major subgroups of the TGF-β superfamily, play a more fundamental role in dorsoventral (DV) patterning than in all other insects studied so far. However, Nasonia lacks the BMP antagonist Short gastrulation (Sog)/chordin, which is essential for polarizing the BMP gradient along the DV axis in most bilaterian animals. Here, we present a broad survey of TGF-β signaling in Nasonia with the aim to detect other lineage-specific peculiarities and to identify potential mechanisms, which explain how BMP-dependent DV pattering occurs in the early Nasonia embryo in the absence of Sog.
Ancient and diverged TGF-β signaling components in Nasonia vitripennis
Özüak O., Buchta T., Roth S., Lynch J.A., Development Genes and Evolution Dev Genes Evol (2014) 224: 223. https://doi.org/10.1007/s00427-014-0481-0 2014 Oct 11.
The transforming growth factor beta (TGF)-β signaling pathway and its modulators are involved in many aspects of cellular growth and differentiation in all metazoa. Although most of the core components of the pathway are highly conserved, many lineage-specific adaptations have been observed including changes regarding paralog number, presence and absence of modulators, and functional relevance for particular processes. In the parasitic jewel wasp Nasonia vitripennis, the bone morphogenetic proteins (BMPs), one of the major subgroups of the TGF-β superfamily, play a more fundamental role in dorsoventral (DV) patterning than in all other insects studied so far. However, Nasonia lacks the BMP antagonist Short gastrulation (Sog)/chordin, which is essential for polarizing the BMP gradient along the DV axis in most bilaterian animals. Here, we present a broad survey of TGF-β signaling in Nasonia with the aim to detect other lineage-specific peculiarities and to identify potential mechanisms, which explain how BMP-dependent DV pattering occurs in the early Nasonia embryo in the absence of Sog.
Dorsoventral polarity of the Nasonia embryo primarily relies on a BMP gradient formed without input from Toll.
Özüak O, Buchta T, Roth S, Lynch JA, Curr Biol. 2014 Oct 20;24(20):2393-8. doi: 10.1016/j.cub.2014.08.035. Epub 2014 Oct 9.
In Drosophila, Toll signaling leads to a gradient of nuclear uptake of Dorsal with a peak at the ventral egg pole and is the source for dorsoventral (DV) patterning and polarity of the embryo. In contrast, Toll signaling plays no role in embryonic patterning in most animals, while BMP signaling plays the major role. In order to understand the origin of the novelty of the Drosophila system, we have examined DV patterning in Nasonia vitripennis (Nv), a representative of the Hymenoptera and thus the most ancient branch points within the Holometabola. We have previously shown that while the expression of several conserved DV patterning genes is almost identical in Nasonia and Drosophila embryos at the onset of gastrulation, the ways these patterns evolve in early embryogenesis are very different from what is seen in Drosophila or the beetle Tribolium. In contrast to Drosophila or Tribolium, we find that wasp Toll has a very limited ventral role, whereas BMP is required for almost all DV polarity of the embryo, and these two signaling systems act independently of each other to generate DV polarity. This result gives insights into how the Toll pathway could have usurped a BMP-based DV patterning system in insects. In addition, our work strongly suggests that a novel system for BMP activity gradient formation must be employed in the wasp, since orthologs of crucial components of the fly system are either missing entirely or lack function in the embryo.
Patterning the dorsal-ventral axis of the wasp Nasonia vitripennis: Novelty and Convergence
Buchta T, Özüak O, Stappert D, Roth S, Lynch JA, Dev Biol, Epub ahead of print (2013)
Regulatory networks composed of interacting genes are responsible for pattern formation and cell type specification in a wide variety of developmental contexts. Evolution must act on these regulatory networks in order to change the proportions, distribution, and characteristics of specified cells. Thus, understanding how these networks operate in homologous systems across multiple levels of phylogenetic divergence is critical for understanding the evolution of developmental systems. Among the most thoroughly characterized regulatory networks is the dorsal-ventral patterning system of the fly Drosophila melanogaster. Due to the thorough understanding of this system, it is an ideal starting point for comparative analyses. Here we report an analysis of the DV patterning system of the wasp, Nasonia vitripennis. This wasp undergoes a mode of long germ embryogenesis that is superficially nearly identical to that of Drosophila, but one that was likely independently derived. We have found that while the expression of genes just prior to the onset of gastrulation is almost identical in Nasonia and Drosophila, both the upstream network responsible for generating this pattern, and the downstream morphogenetic movements that it sets in motion, are significantly diverged. From this we conclude that many network structures are available to evolution to achieve particular developmental ends.
Developmental gene discovery in a hemimetabolous insect: de novo assembly and annotation of a transcriptome for the cricket Gryllus bimaculatus
Zeng V, Ewen-Campen B, Horch HW, Roth S, Mito T, Extavour CG, PLoS ONE 8(5): e61479 (2013)
Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus.
Co-option of a coordinate system defined by the EGFr and Dpp pathways in the evolution of a morphological novelty
Vreede BM, Lynch JA, Roth S, Sucena E, Evodevo 4:7 (2013)
Background
Morphological innovation is an elusive and fascinating concept in evolutionary biology. A novel structure may open up an array of possibilities for adaptation, and thus is fundamental to the evolution of complex multicellular life. We use the respiratory appendages on the dorsal-anterior side of the Drosophila eggshell as a model system for morphological novelty. To study the co-option of genetic pathways in the evolution of this novelty we have compared oogenesis and eggshell patterning in Drosophila melanogaster with Ceratitis capitata, a dipteran whose eggs do not bear dorsal appendages.
Results
During the final stages of oogenesis, the appendages are formed by specific groups of cells in the follicular epithelium of the egg chamber. These cells are defined via signaling activity of the Dpp and EGFr pathways, and we find that both pathways are active in C. capitata oogenesis. The transcription factor gene mirror is expressed downstream of EGFr activation in a dorsolateral domain in the D. melanogaster egg chamber, but could not be detected during C. capitata oogenesis. In D. melanogaster, mirror regulates the expression of two important genes: broad, which defines the appendage primordia, and pipe, involved in embryonic dorsoventral polarity. In C. capitata, broad remains expressed ubiquitously throughout the follicular epithelium, and is not restricted to the appendage primordia. Interestingly pipe expression did not differ between the two species.
Conclusions
Our analysis identifies both broad and mirror as important nodes that have been redeployed in the Drosophila egg chamber patterning network in the evolution of a morphologically novel feature. Further, our results show how pre-existing signals can provide an epithelium with a spatial coordinate system, which can be co-opted for novel patterns.
Does the Bicoid gradient matter?
Roth S, Lynch J, Cell 149, 511-512 (2012)
The generation and interpretation of positional information are key processes in developmental systems. In this issue, Chen et al. report discoveries made in the Drosophila embryo that give new insights into how positional information can be produced by patterning gradients.
Development. Making waves for segments.
Roth S, Panfilio KA, Science (New York, N.Y.) 336, 306-307 (2012)
Comparisons of the embryonic development of Drosophila, Nasonia, and Tribolium
Lynch JA, El-Sherif E, Brown SJ, Wiley Interdisciplinary Reviews: Developmental Biology, 1: 16–39 (2012)
Studying the embryogenesis of diverse insect species is crucial to understanding insect evolution. Here, we review current advances in understanding the development of two emerging model organisms: the wasp Nasonia vitripennis and the beetle Tribolium castaneum in comparison with the well-studied fruit fly Drosophila melanogaster. Although Nasonia represents the most basally branching order of holometabolous insects, it employs a derived long germband mode of embryogenesis, more like that of Drosophila, whereas Tribolium undergoes an intermediate germband mode of embryogenesis, which is more similar to the ancestral mechanism. Comparing the embryonic development and genetic regulation of early patterning events in these three insects has given invaluable insights into insect evolution. The similar mode of embryogenesis of Drosophila and Nasonia is reflected in their reliance on maternal morphogenetic gradients. However, they employ different genes as maternal factors, reflecting the evolutionary distance separating them. Tribolium, on the other hand, relies heavily on self-regulatory mechanisms other than maternal cues, reflecting its sequential nature of segmentation and the need for reiterated patterning.
Mathematics and biology: a Kantian view on the history of pattern formation theory.
Roth S, Development Genes and Evolution 221, 255-279 (2011)
Driesch's statement, made around 1900, that the physics and chemistry of his day were unable to explain self-regulation during embryogenesis was correct and could be extended until the year 1972. The emergence of theories of self-organisation required progress in several areas including chemistry, physics, computing and cybernetics. Two parallel lines of development can be distinguished which both culminated in the early 1970s. Firstly, physicochemical theories of self-organisation arose from theoretical (Lotka 1910-1920) and experimental work (Bray 1920; Belousov 1951) on chemical oscillations. However, this research area gained broader acceptance only after thermodynamics was extended to systems far from equilibrium (1922-1967) and the mechanism of the prime example for a chemical oscillator, the Belousov-Zhabotinski reaction, was deciphered in the early 1970s. Secondly, biological theories of self-organisation were rooted in the intellectual environment of artificial intelligence and cybernetics. Turing wrote his The chemical basis of morphogenesis (1952) after working on the construction of one of the first electronic computers. Likewise, Gierer and Meinhardt's theory of local activation and lateral inhibition (1972) was influenced by ideas from cybernetics. The Gierer-Meinhardt theory provided an explanation for the first time of both spontaneous formation of spatial order and of self-regulation that proved to be extremely successful in elucidating a wide range of patterning processes. With the advent of developmental genetics in the 1980s, detailed molecular and functional data became available for complex developmental processes, allowing a new generation of data-driven theoretical approaches. Three examples of such approaches will be discussed. The successes and limitations of mathematical pattern formation theory throughout its history suggest a picture of the organism, which has structural similarity to views of the organic world held by the philosopher Immanuel Kant at the end of the eighteenth century.
The phylogenetic origin of oskar coincided with the origin of maternally provisioned germ plasm and pole cells at the base of the Holometabola.
Lynch JA, Ozüak O, Khila A, Abouheif E, Desplan C, Roth S, PLoS Genetics 7, e1002029 (2011)
The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, we used comparative genomics, parental RNAi, and gene expression analyses in multiple insect species. We have found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. Our results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. We propose that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program.
The evolution of dorsal-ventral patterning mechanisms in insects.
Lynch JA, Roth S, Genes & Development 25, 107-118 (2011)
The gene regulatory network (GRN) underpinning dorsal-ventral (DV) patterning of the Drosophila embryo is among the most thoroughly understood GRNs, making it an ideal system for comparative studies seeking to understand the evolution of development. With the emergence of widely applicable techniques for testing gene function, species with sequenced genomes, and multiple tractable species with diverse developmental modes, a phylogenetically broad and molecularly deep understanding of the evolution of DV axis formation in insects is feasible. Here, we review recent progress made in this field, compare our emerging molecular understanding to classical embryological experiments, and suggest future directions of inquiry.
The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus.
Ewen-Campen B, Shaner N, Panfilio KA, Suzuki Y, Roth S, Extavour CG, BMC Genomics 12, 61 (2011)
BACKGROUND:Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.RESULTS:We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.CONCLUSIONS:Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (http://www.ncbi.nlm.nih.gov/sra?term=SRP002610). Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html. Seven Additional files are available.].
Novel modes of localization and function of nanos in the wasp Nasonia.
Lynch JA, Desplan C, Development (Cambridge, England) 137, 3813-3821 (2010)
Abdominal patterning in Drosophila requires the function of nanos (nos) to prevent translation of hunchback (hb) mRNA in the posterior of the embryo. nos function is restricted to the posterior by the translational repression of mRNA that is not incorporated into the posteriorly localized germ plasm during oogenesis. The wasp Nasonia vitripennis (Nv) undergoes a long germ mode of development very similar to Drosophila, although the molecular patterning mechanisms employed in these two organisms have diverged significantly, reflecting the independent evolution of this mode of development. Here, we report that although Nv nanos (Nv-nos) has a conserved function in embryonic patterning through translational repression of hb, the timing and mechanisms of this repression are significantly delayed in the wasp compared with the fly. This delay in Nv-nos function appears to be related to the dynamic behavior of the germ plasm in Nasonia, as well as to the maternal provision of Nv-Hb protein during oogenesis. Unlike in flies, there appears to be two functional populations of Nv-nos mRNA: one that is concentrated in the oosome and is taken up into the pole cells before evidence of Nv-hb repression is observed; another that forms a gradient at the posterior and plays a role in Nv-hb translational repression. Altogether, our results show that, although the embryonic patterning function of nos orthologs is broadly conserved, the mechanisms employed to achieve this function are distinct.
Generation of distinct signaling modes via diversification of the Egfr ligand-processing cassette.
Rousso T, Lynch J, Yogev S, Roth S, Schejter ED, Shilo B, Development (Cambridge, England) 137, 3427-3437 (2010)
Egfr ligand processing in Drosophila involves trafficking of the ligand precursor by the chaperone Star from the endoplasmic reticulum (ER) to a secretory compartment, where the precursor is cleaved by the intramembrane protease Rhomboid. Some of the Drosophila Rhomboids also reside in the ER, where they attenuate signaling by premature cleavage of Star. The genome of the flour beetle Tribolium castaneum contains a single gene for each of the ligand-processing components, providing an opportunity to assess the regulation and impact of a simplified ligand-processing cassette. We find that the central features of ligand retention, trafficking by the chaperone and cleavage by Rhomboid have been conserved. The single Rhomboid is localized to both ER and secretory compartments. However, we show that Tribolium Star is refractive to Rhomboid cleavage. Consequently, this ligand-processing system effectively mediates long-range Egfr activation in the Tribolium embryonic ventral ectoderm, despite ER localization of Rhomboid. Diversification of the Egfr signaling pathway appears to have coupled gene duplication events with modulation of the biochemical properties and subcellular localization patterns of Rhomboid proteases and their substrates.
Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo.
Nunes da Fonseca R, van der Zee M, Roth S, Developmental Biology 345, 80-93 (2010)
The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.
EGF signaling and the origin of axial polarity among the insects.
Lynch JA, Peel AD, Drechsler A, Averof M, Roth S, Current Biology 20, 1042-1047 (2010)
The eggs of insects are unusual in that they often have bilateral symmetry when they are laid, indicating that both anterior-posterior (AP) and dorsal-ventral (DV) symmetries are broken during oogenesis. The molecular basis of this process is well understood in Drosophila melanogaster, in which symmetry breaking events for both axes depend on the asymmetric position of the oocyte nucleus and on germline-soma signaling mediated by the Tgf alpha-like epidermal growth factor (EGF) ligand Gurken. Germline-soma signaling interactions centered around the oocyte nucleus have been proposed in other insect species, but the molecular nature of these interactions has not been elucidated. We have examined the behavior of the oocyte nucleus and the function of EGF signaling components in the ovaries of the wasp Nasonia vitripennis, the beetle Tribolium castaneum, and the cricket Gryllus bimaculatus. We have found that EGF signaling has broadly conserved roles in mediating the encapsulation of oocytes by the somatic follicle cell layer, in establishing polarity of the egg chambers, and in setting up the DV axis of the embryo. These results provide insights into the evolutionary origins of the unique strategy employed by insects to establish embryonic axial polarity during oogenesis.
Epithelial reorganization events during late extraembryonic development in a hemimetabolous insect.
Panfilio KA, Roth S, Developmental Biology 340, 100-115 (2010)
As extra-embryonic tissues, the amnion and serosa are not considered to contribute materially to the insect embryo, yet they must execute an array of morphogenetic movements before they are dispensable. In hemimetabolous insects, these movements have been known for over a century, but they have remained virtually unexamined. This study addresses late extraembryonic morphogenesis in the milkweed bug, Oncopeltus fasciatus. Cell shape changes and apoptosis profiles are used to characterize the membranes as they undergo a large repertoire of final reorganizational events that reposition the embryo (katatrepsis), and eliminate the membranes themselves in an ordered fashion (dorsal closure). A number of key features were identified. First, amnion-serosa "fusion" involves localized apoptosis in the amnion and the formation of a supracellular actin purse string at the amnion-serosa border. During katatrepsis, a 'focus' of serosal cells undergoes precocious columnarization and may serve as an anchor for contraction. Lastly, dorsal closure involves novel modifications of the amnion and embryonic flank that are without counterpart during the well-known process of dorsal closure in the fruit fly Drosophila melanogaster. These data also address the long-standing question of the final fate of the amnion: it undergoes apoptosis during dorsal closure and thus is likely to be solely extraembryonic.
Evolution of axis formation: mRNA localization, regulatory circuits and posterior specification in non-model arthropods.
Nunes da Fonseca R, Lynch JA, Roth S, Current Opinion In Genetics & Development 19, 404-411 (2009)
Drosophila melanogaster has been used as an arthropod model to understand the establishment of embryonic axes. However, the long germ type of embryogenesis in Drosophila is highly divergent from the ancestral, short germ mode seen in most other arthropods. Therefore, broader sampling of other arthropod taxa is required to understand the evolution of axial patterning mechanisms within this phylum. Here we describe recent progress toward this end. First, we describe differing strategies for providing patterning information along the anterior-posterior axis of arthropod embryos. Second, we discuss the recent findings on dorso-ventral axis of beetles and spiders that highlight the importance of regulatory interactions at the zygotic level. Third, recent discoveries of the roles of Wnt signalling, the Notch/Delta pathway and caudal in establishing and patterning the posterior embryonic region in spiders and beetles are described. In the end, we try to integrate these new findings with non-model arthropods in a common framework and discuss the major gaps of knowledge that should be addressed by future studies.
The genome of the model beetle and pest Tribolium castaneum.
Tribolium Genome Sequencing Consortium, Richards S, Gibbs RA, Weinstock GM, Brown SJ, Denell R, Beeman RW, Gibbs R, Beeman RW, Brown SJ, Bucher G et al., Nature 452, 949-955 (2008)
Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.
Self-regulatory circuits in dorsoventral axis formation of the short-germ beetle Tribolium castaneum.
Nunes da Fonseca R, von Levetzow C, Kalscheuer P, Basal A, van der Zee M, Roth S, Developmental cell 14, 605-615 (2008)
The rel/NF-kappaB transcription factor Dorsal controls dorsoventral (DV) axis formation in Drosophila. A stable nuclear gradient of Dorsal directly regulates approximately 50 target genes. In Tribolium castaneum (Tc), a beetle with an ancestral type of embryogenesis, the Dorsal nuclear gradient is not stable, but rapidly shrinks and disappears. We find that negative feedback accounts for this dynamic behavior: Tc-Dorsal and one of its target genes activate transcription of the IkB homolog Tc-cactus, terminating Dorsal function. Despite its transient role, Tc-Dorsal is strictly required to initiate DV polarity, as in Drosophila. However, unlike in Drosophila, embryos lacking Tc-Dorsal display a periodic pattern of DV cell fates along the AP axis, indicating that a self-organizing ectodermal patterning system operates independently of mesoderm or maternal DV polarity cues. Our results also elucidate how extraembryonic tissues are organized in short-germ embryos, and how patterning information is transmitted from the early embryo to the growth zone.
TGFbeta signaling in Tribolium: vertebrate-like components in a beetle.
van der Zee M, da Fonseca RN, Roth S, Development Genes and Evolution 218, 203-213 (2008)
The cytokines of the TGFbeta superfamily are highly conserved in evolution and elicit a diverse range of cellular responses in all metazoa. In Drosophila, the signaling pathways of the two TGFbeta subfamilies, Activins and Bone Morphogenetic Proteins (BMPs), have been well studied. To address the question of whether the findings from Drosophila are representative of insects in general, we analyzed the components of TGFbeta-signaling present in the genome of the beetle Tribolium castaneum. We were able to identify orthologs of the BMPs Decapentaplegic and Glass bottom boat, of the Activins Activinbeta and Dawdle, as well as orthologs of the less well-known ligands Myoglianin and Maverick, together with orthologs of all TGFbeta receptors and cytoplasmic signal transducers present in Drosophila. This indicates that the diversity of TGFbeta signaling components is generally well conserved between Drosophila and Tribolium. However, the genome of the beetle-and of the bee Apis mellifera-lacks an ortholog of the Drosophila BMP Screw but does contain a vertebrate-like BMP10 homolog which is not found in Drosophila. Concerning BMP inhibitors, Tribolium displays an even more vertebrate-like ensemble of components. We found two orthologs of the vertebrate DAN family, Dan and Gremlin, and show embryonic expression of a vertebrate-like BAMBI ortholog, all of which are absent in Drosophila. This suggests that Tribolium might have retained a more ancestral composition of TGFbeta signaling components and that TGFbeta signaling underwent considerable change in the Drosophila lineage. Tribolium is an excellent model to study the function of these ancestral signaling components in insects.
Sog/Chordin is required for ventral-to-dorsal Dpp/BMP transport and head formation in a short germ insect.
van der Zee M, Stockhammer O, von Levetzow C, Nunes da Fonseca R, Roth S, Proceedings Of The National Academy Of Sciences Of The United States Of America 103, 16307-16312 (2006)
Bone morphogenetic protein (BMP) signaling plays a major role in dorsoventral patterning in vertebrates and in Drosophila. Remarkably, in Tribolium, a beetle with an ancestral type of insect development, early BMP/dpp exhibits differential expression along the anteroposterior axis. However, the BMP/Dpp inhibitor Sog/chordin is expressed ventrally and establishes a dorsal domain of BMP/Dpp activity by transporting BMPs toward the dorsal side, like in Drosophila. Loss of Tribolium Sog not only abolishes dorsoventral polarity in the ectoderm, but also leads to the complete absence of the CNS. This phenotype suggests that sog is the main BMP antagonist in Tribolium, in contrast to vertebrates and Drosophila, which possess redundant antagonists. Surprisingly, Sog also is required for head formation in Tribolium, as are the BMP antagonists in vertebrates. Thus, in Tribolium, the system of BMP and its antagonists is less complex than in Drosophila or vertebrates and combines features from both, suggesting that it might represent an ancestral state.
Distinct functions of the Tribolium zerknüllt genes in serosa specification and dorsal closure.
van der Zee M, Berns N, Roth S, Current Biology 15, 624-636 (2005)
BACKGROUND:In the long-germ insect Drosophila, a single extraembryonic membrane, the amnioserosa, covers the embryo at the dorsal side. In ancestral short-germ insects, an inner membrane, the amnion, covers the embryo ventrally, and an outer membrane, the serosa, completely surrounds the embryo. An early differentiation step partitions the uniform blastoderm into the anterior-dorsal serosa and the posterior-ventral germ rudiment giving rise to amnion and embryo proper. In Drosophila, amnioserosa formation depends on the dorsoventral patterning gene zerknüllt (zen), a derived Hox3 gene.RESULTS:The short-germ beetle Tribolium castaneum possesses two zen homologs, Tc-zen1 and Tc-zen2. Tc-zen1 acts early and specifies the serosa. The loss of the serosa after Tc-zen1 RNAi is compensated by an expansion of the entire germ rudiment toward the anterior. Instead of the serosa, the amnion covers the embryo at the dorsal side, and later size regulation normalizes the early fate shifts, revealing a high degree of plasticity of short-germ development. Tc-zen2 acts later and initiates the amnion and serosa fusion required for dorsal closure. After Tc-zen2 RNAi, the amnion and serosa stay apart, and the embryo closes ventrally, assuming a completely everted (inside-out) topology.CONCLUSIONS:In Tribolium, the duplication of the zen genes was accompanied by subfunctionalization. One of the paralogues, Tc-zen1, acts as an early anterior-posterior patterning gene by specifying the serosa. In absence of the serosa, Tribolium embryogenesis acquires features of long-germ development with a single extraembryonic membrane. We discuss implications for the evolution of insect development including the origin of the zen-derived anterior determinant bicoid.
back to project area A