A Potential Method for Rapid Screening of Amphioxus Founder Harboring Germline Mutation and Transgene

Abstract

Amphioxus is a promising model organism for understanding the origin and evolution of vertebrates due to its basal phylogenetic position among chordates. We here compared the mutation efficacy and mutation type of tail tips and gametes of amphioxus founders injected with Cas9 protein and six different sgRNAs targeting five distinct genes, and revealed a strong correlation for mutation efficacy and a mild correlation for mutation type among the two tissues. In addition, we also observed a positive relationship between gene insertions observed in tail tips and gametes of amphioxus founders injected with Tol2 transposase and two different transgenic constructs. Finally, we showed that amphioxus larvae which had their tail tips cut at the 3–4 gill-slit stage were able to recover within 6 days and developed a normal number of gonads at the adult stage, and that F0 larvae carry similar mutation efficacy and type in the posterior end to that in the tail tips after their metamorphosis. Together, these findings suggest a great potential for obtaining valid amphioxus founders with desired mutations and transgenes at as early as the early larval stage, which will certainly speed up the generation of amphioxus mutants and transgenes and make it more cost- and labor-effective.

Investigating the developmental onset of regenerative potential in the annelid Capitella teleta

“…Specifically, ventral regions in larvae of P. flava regenerate better than dorsal regions and posterior segments regenerate better than anterior segments (Luttrell et al, 2018). The larval tail of the cephalochordate Branchiostoma floridae regenerates multiple tissues faster than adults following similar amputations (Zou et al, 2021). Unlike larvae of other clades, echinoderm larvae have been widely surveyed for regenerative potential in all classes with documented adult whole‐body regeneration abilities (Cary et al, 2019; Vickery et al, 2001).…”

Amphioxus as a model to study the evolution of development in chordates

“…Classical embryo micromanipulation techniques, including grafting, have also been developed ( Le Petillon et al, 2020 ). Importantly, through the use of the TALEN and CRISPR-Cas9 gene-editing approaches, and Tol2-based transgenesis, it has been possible to obtain knock out and transgenic lines in B. floridae and B. belcheri for different genes, which has lifted an important brake on functional studies using amphioxus and has boosted the research in the evolutionary developmental biology field (EvoDevo) ( Holland and Li, 2021 ; Li et al, 2014 ; Li et al, 2017 ; Hu et al, 2017 ; Zhong et al, 2020 ; Ren et al, 2020 ; Zhu et al, 2020 ; Zou et al, 2021 ; Su et al, 2020 ; Kozmikova and Kozmik, 2015 ).…”