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Body Plan, Early Embryonic Patterning and Other Research

 

Evolution of Arthropod Body Plans

 

Although the insect body plan is well conserved, the developmental mechanisms of body patterning are surprisingly varied. The fruit fly Drosophila melanogaster has long served as a model organism to investigate the molecular mechanisms regulating embryonic axial patterning, long germ segmentation (in which all body segments are established near-simultaneously), and acquisition of specific body region and segment identities. However, basally branching insects and other arthropods develop their segments in a sequential fashion, similar to non-insect arthropods and to vertebrates. The arthropod body plan, including appendage development, has also undergone extensive evolutionary modifications in different clades.

behl

An adult Gryllus bimaculatus female perched on a human finger. Photo courtesy of David Behl.

We have studied the evolution of the underlying genetic mechanisms that result in the differences in arthropod body patterning using a number of model organisms and developmental processes. These include segmentation in the cricket Gryllus, appendage development in daddy longlegs, and Hox genes in scorpions. In the process, we have helped collaborator Prashant Sharma (American Museum of Natural History) develop the harvestman Phalangium opilio into the first daddy longlegs model laboratory system suitable for functional genetic analysis.

References

Kainz, F., Ewen-Campen, B., Akam, M. and Extavour, C.G. Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus. Development 138(22): 5015-5026 (2011) PDF PubMed

Sharma, P.P., Schwager, E.E., Extavour, C. G. and Giribet, G. Evolution of the chelicera: a dachshund domain is retained in the deutocerebral appendage of Opiliones (Arthropoda, Chelicerata). Evolution and Development 14(6): 522-533 (2012) PDF PubMed Recommended by the Faculty of 1000

Sharma, P.P., Schwager, E.E., Extavour, C. G. and Giribet, G. Hox gene expression in the harvestman Phalangium opilio reveals divergent patterning of the chelicerate opisthosoma. Evolution and Development 14(5): 450-463 (2012) PDF PubMed

Sharma, P.P., Schwager, E.E., Giribet, G.G., Jockusch, E. and Extavour, C.G. Distal-less and dachshund pattern both plesiomorphic and derived structures in chelicerates: RNA interference in the harvestman Phalangium opilio (Opiliones) Evolution and Development 15(4): 228-242 (2013) PDF PubMed Cover Article

Sharma, P.P., Gupta, T., Schwager, E.E., Wheeler, W., and Extavour C.G. Subdivision of arthropod cap-n-collar expression domains is restricted to Mandibulata. EvoDevo (in press) [PDF of accepted MS]

Sharma, P.P., Schwager, E.E., Extavour, C.G.. and Wheeler, W. Hox gene duplications correlate with posterior heteronomy in scorpions. (in review)

 


Early Cell Fates and Cell Movements in Holoblastic Arthropod Embryogenesis

Development from zygote to hatchling occurs through a complex and coordinated series of mitotic divisions, or cleavages. Many arthropods, particularly crustaceans, display a stereotyped pattern of early cleavage. Using time-lapse microscopy and semi-automated cell lineage analysis, we established a complete cell lineage for the early stages of the amphipod crustacean Parhyale hawaiensis up to the onset of gastrulation. We found that removal of any of the four micromeres at the eight-cell stage does not alter the cleavage or migration behavior of the remaining blastomeres through to gastrulation stages. We have documented our blastomere ablation technique in the form of a video protocol. This work provides the basis for continued research into the molecular genetic mechanisms underlying cell fate acquisition and early embryonic cell movements.

We are also investigating how Parhyale germ cell fate is established, and have found that a morphologically distinct cytoplasmic region in the one-cell embryo contains germ line-associated RNAs. Removal of this cytoplasmic region results in a loss of embryonic germ line cells, but also in changes in the behavior of other early cleavage blastomeres. We hypothesize that this cytoplasmic region acts as a putative germ plasm in Parhyale, and may also direct some aspect of cell fate acquisition in somatic cells. 

References

Alwes, F., Hinchen, B. and Extavour, C G. Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis. Developmental Biology 359(1): 110-123 (2011) PDF PubMed

Nast, A. R. and Extavour, C.G. Ablation of a single cell from eight-cell embryos of the amphipod crustacean Parhyale hawaiensis. Journal of Visualized Experiments doi:10.3791/51073 (2014) [PDF of accepted MS]

Gupta, T. and Extavour, C.G. Identification of a putative germ plasm in the crustacean Parhyale hawaiensis. EvoDevo 4:34 (2013) PDF PubMed



Developing Genomic Resources and Functional Genetic Tools for Emerging Model Organisms

 

Evolutionary developmental biology studies of non-model organisms are often limited by the absence of a sequenced genome, and thus tend to focus on small numbers of candidate genes cloned individually using degenerate PCR. To facilitate larger-scale gene discovery and move beyond the candidate gene approach, we have used next-generation sequencing to generate transcriptomes from the ovaries and embryos of multiple emerging model arthropod organisms, and de novo assemble and annotate them. In each of the transcriptomes from the milkweed bug Oncopeltus fasciatus, the cricket Gryllus bimaculatus, the amphipod Parhyale hawaiensis and the spider Parasteatoda tepidariorum, we identified over 10,000 genes, including members of all conserved metazoan signaling pathways and genes implicated in many important developmental processes. We have created a searchable database called ASGARD to provide public access to these annotated transcriptomes, their associated raw data, and bioinformatic tools for analyzing and annotating such de novo transcriptomes.

 

ASGARD

Origin and processing pipeline for transcriptomic data contained in ASGARD database as of November 2013.

We are also interested in improving the tools available for functional genetic analysis in non-traditional model organisms. Maternal RNA interference is frequently used in non-model insects to determine the function of genes in embryonic development. However, we find that while this technique is often effective at knocking down maternally supplied transcripts, the effects on zygotic transcripts can be highly variable. We have therefore optimized embryonic injection protocols that enable us to bypass maternal requirements by performing zygotic knockdowns, and to assess requirements for gene functions at different stages of embryogenesis.We have also collaborated with Prashant Sharma (American Museum of Natural History) to develop the first laboratory model for developmental studies in a daddy longlegs species, and have generated protocols for in situ hybridization and RNAi in this animal. Building on the recently reported success of transgenesis and targeted genome editing in Gryllus, we are currently working on CRISPR-based knockout and knock-in strategies, as well as tissue-specific reporter constructs, for use in the cricket. In collaboration with Quaid Morris (University of Toronto), we are working towards generating predictive bioinformatics tools to discover gene networks involved in developmental processes of interest. If successful, these techniques should be broadly applicable to a wide range of study organisms.

References

Abzhanov, A., Extavour, C.G., Groover, A., Hodges, S., Hoekstra, H., Kramer, E M., Monteiro, A. Are We There Yet? Tracking the Development of New Model Systems. Trends in Genetics 24(7): 353-360 (2008) PDF PubMed

Ewen-Campen, B., Shaner, N., Panfilio, K., Suzuki, Y., Roth, S. and Extavour, C.G. The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus. BMC Genomics 12(1): 61 (2011) PDF PubMed Highly Accessed

Zeng, V., Villanueva, K.E., Ewen-Campen, B., Alwes, F., Browne, W.E. and Extavour, C.G. De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis. BMC Genomics 12(1): 581 (2011) PDF PubMed

Zeng, V. and Extavour, C.G. ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species. Database doi: 10.1093/database/bas048 (2012) PDF PubMed

Zeng, V. Ewen-Campen, B., Horch, H.W., Roth, S., Mito, T., and Extavour, C.G. Developmental Gene Discovery in a Hemimetabolous Insect: De Novo Annotation and Assembly of a Transcriptome for the Cricket Gryllus bimaculatus. PLoS ONE 8(5): e61479 (2013) PDF PubMed

Sharma, P.P., Schwager, E.E., Giribet, G.G., Jockusch, E. and Extavour, C.G. Distal-less and dachshund pattern both plesiomorphic and derived structures in chelicerates: RNA interference in the harvestman Phalangium opilio (Opiliones) Evolution and Development 15(4): 228-242 (2013) PDF PubMed Cover Article

Posnien, N., Zeng, V., Schwager, E.E., Damen, W., Prpic, N., McGregor, A., and Extavour, C.G. A developmental transcriptome for the spider Parasteatoda tepidariorum. (in preparation)