Urochordate Ciona spp.are ideal marine model organisms for studying embryogenesis and developmental and evolution-ary biology.However,the effective implementation of genetic labeling and CRISPR/Cas9-based editing tool...Urochordate Ciona spp.are ideal marine model organisms for studying embryogenesis and developmental and evolution-ary biology.However,the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging.This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp.The destination vector sets contained two Gateway cassettes flanked by Minos sites,allowing the N-or C-terminal tagging of a protein of interest with various fluorescent markers.In addition,we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry,a fluorescent indicator for Cas9 expression at cellular resolution.We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs.Furthermore,we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues.The genetic tools developed in this study offer a robust method for gene expression,cell tracking,and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.展开更多
Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ...Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ascidians,including those expressed in the larval tail muscle,the adult body-wall muscle,and adult heart muscle.In this study,a novel striated non-tail muscle actin gene was identified from the RNA-seq data of Ciona savignyi embryos.Phylogenetic analysis,alignment of the N-terminal amino acid sequences and comparation of diagnostic residues provided evidence that it had high similarity with vertebrate cardiac and skeletal muscle actin.In situ hybridization and promoter-driven GFP reporter assay revealed that it was specifically expressed in the primordia of the oral and atrial siphon.We hereby defined it as siphon-specific muscle actin coding gene(Cs-SMA).A 201 bp(−1350 bp to−1150 bp)sequence containing T-box and Six1/2 binding motif within the upstream region of Cs-SMA confined the expression of GFP in the siphons of electroporated embryos.Six1/2 binding motif was experimentally confirmed to play indispensable role in controlling the siphon-specific expression of Cs-SMA.The tissue-specific expression of Cs-SMA in the siphon primordia indicated its potential crucial roles in Ciona embryogenesis and organogenesis.展开更多
基金supported by the National Key Research and Development Program of China(2022YFC2601302)the Taishan Scholar Program of Shan-dong Province,China(B.D.).
文摘Urochordate Ciona spp.are ideal marine model organisms for studying embryogenesis and developmental and evolution-ary biology.However,the effective implementation of genetic labeling and CRISPR/Cas9-based editing tools at cellular resolution remains challenging.This study successfully developed and validated a collection of Gateway-based vectors for cell labeling in Ciona spp.The destination vector sets contained two Gateway cassettes flanked by Minos sites,allowing the N-or C-terminal tagging of a protein of interest with various fluorescent markers.In addition,we optimized the CRISPR/Cas9 and CRISPR/dCas9 systems by incorporating P2A-mCherry,a fluorescent indicator for Cas9 expression at cellular resolution.We demonstrated the effective destruction or inhibition of target genes when CRISPR constructs were introduced into fertilized eggs.Furthermore,we engineered a dual fluorescence sensor system that helps visualize successful gene knockouts at the cellular level in specific tissues.The genetic tools developed in this study offer a robust method for gene expression,cell tracking,and subcellular protein localization while also facilitating tissue-specific functional analysis in Ciona embryos and other model systems.
基金funded by the National Key Research and Development Program of China(Nos.2019YFE0190900,2018YFD0900705).
文摘Actin is a ubiquitous protein and plays essential roles on cellular structure maintenance and cellular motility in both muscle and non-muscle tissues.Multiple genes encoding muscle actin have been identified from the ascidians,including those expressed in the larval tail muscle,the adult body-wall muscle,and adult heart muscle.In this study,a novel striated non-tail muscle actin gene was identified from the RNA-seq data of Ciona savignyi embryos.Phylogenetic analysis,alignment of the N-terminal amino acid sequences and comparation of diagnostic residues provided evidence that it had high similarity with vertebrate cardiac and skeletal muscle actin.In situ hybridization and promoter-driven GFP reporter assay revealed that it was specifically expressed in the primordia of the oral and atrial siphon.We hereby defined it as siphon-specific muscle actin coding gene(Cs-SMA).A 201 bp(−1350 bp to−1150 bp)sequence containing T-box and Six1/2 binding motif within the upstream region of Cs-SMA confined the expression of GFP in the siphons of electroporated embryos.Six1/2 binding motif was experimentally confirmed to play indispensable role in controlling the siphon-specific expression of Cs-SMA.The tissue-specific expression of Cs-SMA in the siphon primordia indicated its potential crucial roles in Ciona embryogenesis and organogenesis.
