I-SceI Meganuclease-mediated transgenesis in the acorn worm, Saccoglossus kowalevskii

Abstract

Hemichordates are a phylum of marine invertebrate deuterostomes that are closely related to chordates, and represent one of the most promising models to provide insights into early deuterostome evolution. The genome of the hemichordate, Saccoglossus kowalevskii, reveals an extensive set of non-coding elements conserved across all three deuterostome phyla. Functional characterization and cross-phyla comparisons of these putative regulatory elements will enable a better understanding of enhancer evolution, and subsequently how changes in gene regulation give rise to morphological innovation. Here, we describe an efficient method of transgenesis for the characterization of non-coding elements in S. kowalevskii. We first test the capacity of an I-SceI transgenesis system to drive ubiquitous or regionalized gene expression, and to label specific cell types. Finally, we identified a minimal promoter that can be used to test the capacity of putative enhancers in S. kowalevskii. This work demonstrates that this I-SceI transgenesis technique, when coupled with an understanding of chromatin accessibility, can be a powerful tool for studying how evolutionary changes in gene regulatory mechanisms contributed to the diversification of body plans in deuterostomes.

Transgenesis in the acoel worm Hofstenia miamia

“…We cloned regulatory regions of the candidate genes into a backbone plasmid with restriction sites for the I-SceI endonuclease flanking the transgenic cassette (Fig. 2A), a system that has been used successfully in multiple systems including cnidarians, protostomes, and vertebrates (Thermes et al, 2002;Deschet, Nakatani and Smith, 2003;Ogino, McConnell and Grainger, 2006;Renfer et al, 2010;Minor et al, 2019).…”

“…Numerous animal species have emerged as new systems for laboratory research, e.g., short-lived killifish for studying aging, hardy water bears for studying tolerance to extreme environments, or the anemone Aiptasia for studying coral bleaching (Lehnert, Burriesci and Pringle, 2012;Goldstein, 2018;Hu and Brunet, 2018). The lack of genetic tools in emerging systems can limit the depth of molecular and cellular insight, however, advances in genome-scale sequencing technology and tools for genetic manipulations (e.g., via CRISPR/CAS9) are releasing this constraint (Ikmi et al, 2014;Wudarski et al, 2017;Minor et al, 2019). Here, we developed a method for transgenesis in the acoel worm, Hofstenia miamia (Corrêa, 1960), which has emerged as a new research organism for studying regeneration and stem cell biology.…”

Beyond Adult Stem Cells: Dedifferentiation as a Unifying Mechanism Underlying Regeneration in Invertebrate Deuterostomes

“…Stable transgenic animals, or genetic labeling with inducible systems, permit by far the best control and resolution, and will be required to truly show changes in differentiation state and potency. Currently, transgenesis has been developed in some sea urchins (Buckley et al, 2018), amphioxus (Kozmikova and Kozmik, 2015), and the direct developing hemichordate Saccoglossus kowalevskii (Minor et al, 2019). A considerable molecular and genetic toolkit is also available for solitary ascidians, predominantly C. intestinalis but also Phallusia mammillata (Cota, 2018).…”

An atlas of anteriorhoxgene expression in the embryonic sea lamprey head:hox-code evolution in vertebrates

“…Characterization of lamprey Hox enhancer elements and comparison with those of gnathostomes will enable inference of common ancestral Hox regulatory mechanisms in vertebrates, and may elucidate how Hox functions have been differentially partitioned across Hox clusters in lamprey versus gnathostome lineages (Parker et al, 2014b). Looking deeper in chordate evolution, these studies will provide a platform for regulatory comparisons with non-vertebrate deuterostomes (Minor et al, 2018), to investigate how vertebrate segmental Hox regulation arose.…”