Embryo Microinjection and Electroporation in the Chordate <em>Ciona intestinalis</em>

Abstract

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Identifying adhesive components in a model tunicate

“…Adults of Ciona intestinalis and Pallusia mammillata were purchased from the Roscoff Marine Station of the Sorbonne University, France, and cultivated in an aquarium with circulating and oxygenated artificial seawater at 16°C until use. Fertilization and embryo culturing were performed as described [49], and embryos were developed on 10 cm Petri dishes coated with 1% agarose in filtered artificial seawater with HEPES (ASWH or ASW) to 18-24 hpf (hours post fertilization).…”

miR-7 Knockdown by Peptide Nucleic Acids in the Ascidian Ciona intestinalis

“…For each molecule, the maximum non-lethal concentration was chosen. Dechorionated eggs were microinjected with a solution of 0.7 mM PNAs (PNA-a7 or PNA-sc7) in distilled water or 0.3 mM AmiR-7 plus 5 μg/μL Fast Green as vital dye, as previously described [15]. Embryos were reared at 18 ± 1 °C until they reached late tailbud stage [46].…”

“…Particularly, the ascidian Ciona intestinalis is amenable to embryological manipulations. A variety of molecular tools were developed to perturb gene activity during its development, including microinjections of antisense molecules directly into unfertilized eggs [15,16].…”

Exploring miR-9 Involvement in Ciona intestinalis Neural Development Using Peptide Nucleic Acids

“…For PNA microinjections, only batches in which 90% or more of the eggs developed normally were used. Dechorionated eggs were microinjected with solutions of 0.7 mM and 1 mM PNAs (PNA-a9 and PNA-sc9) in distilled water, plus 5 µg/µL Fast Green as the vital dye, as described previously [59]. In vitro cross-fertilization was performed and embryos were reared at 16 • C in a 1% agarose-coated petri dish in ASW until they reached late tailbud and larva stages (16 and 22 h post fertilization) [60].…”