Biology subjects

Lalonde, R. L.

Publications and source records attributed to Lalonde, R. L..

5 recordsLinked to original sources

bioRxiv2025

Genetic context of transgene insertion can influence neurodevelopment in zebrafish

The Gal4/UAS system is used across model organisms to overexpress target genes in precise cell types and relies on generating transgenic Gal4 driver lines. In zebrafish, the Tg(elavl3:KalTA4) (HuC) Gal4 line drives robust expression in neurons. We observed an increased prevalence of swim bladder defects in Tg(elavl3:KalTA4) zebrafish larvae compared to wildtype siblings, which prompted us to investigate whether transgenic larvae display additional neurobehavioral phenotypes. Tg(elavl3:KalTA4) larvae showed alterations in brain activity, brain morphology, and behavior, including increased hindbrain size and reduced activity of the cerebellum. Bulk RNA-seq analysis revealed dysregulation of the transcriptome and suggested an increased ratio of neuronal progenitor cells compared to differentiated neurons. To understand whether these phenotypes derive from Gal4 toxicity or from positional effects related to transgenesis, we used economical low-pass whole genome sequencing to map the Tol2-mediated insertion site to chromosome eight. Reduced expression of the neighboring gene gadd45ga, a known cell cycle regulator, is consistent with increased proliferation and suggests a role for positional effects. Challenges with creating alternative pan-neuronal lines include the length of the elavl3 promoter (over 8 kb) and random insertion using traditional transgenesis methods. To facilitate the generation of alternative lines, we cloned five neuronal promoters (atp6v0cb, smaller elavl3, rtn1a, sncb, and stmn1b) ranging from 1.7 kb to 4.3 kb and created KalTA4 lines using Tol2 and the phiC31 integrase-based pIGLET system. Our study highlights the importance of using appropriate genetic controls and interrogating potential positional effects in new transgenic lines. ARTICLE SUMMARYThe Gal4/UAS system is a genetic tool that allows researchers to overexpress specific genes in specific cell types by pairing a Gal4 "driver" line with a UAS "reporter" line. In zebrafish, more than 500 transgenic Gal4 lines have been generated using transgenesis methods that insert DNA into random sites in the genome. Here, the authors report that a pan-neuronal Gal4 zebrafish line, Tg(elavl3:KalTA4), has neurobehavioral phenotypes, including changes in brain activity, brain structure, and behavior. These findings emphasize the importance of using appropriate controls and highlight the utility of methods for targeted transgenesis.

genetics↗

bioRxiv2024

Lineage labeling with zebrafish hand2 Cre and CreERT2 recombinase CRISPR knock-ins

BackgroundThe ability to generate endogenous Cre recombinase drivers using CRISPR- Cas9 knock-in technology allows lineage tracing, cell type specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration. Resultshand2-2A-cre and hand2-2A-creERT2 knock-ins crossed with ubiquitous loxP-based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in cardiac, pectoral fins, pharyngeal arch, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24 hours reveals late emerging hand2 progenitors in the 24 - 48 hour embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larva restricts hand2 lineage labeling to mesoderm-derived components of the branchial arches, heart, liver and enteric neurons. Conclusionshand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. Later emerging hand2 progenitors become restricted to a subset of lineages in the larva. The hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/626907v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@12f58cborg.highwire.dtl.DTLVardef@16e95c8org.highwire.dtl.DTLVardef@12b8e9dorg.highwire.dtl.DTLVardef@a01848_HPS_FORMAT_FIGEXP M_FIG C_FIG Zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR/Cas9 precision targeted integration label numerous lineages from the mesoderm and ectoderm. Temporal CreERT2 Tamoxifen regulated switching re{-} veals late emerging hand2 progenitors contribute to the embryonic venous vasculature and larval organs.

developmental biology↗

bioRxiv2023

pIGLET: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish

Standard methods for transgenesis in zebrafish depend on random transgene integration into the genome followed by resource-intensive screening and validation. Targeted vector integration into validated genomic loci using phiC31 integrase-based attP/attB recombination has transformed mouse and Drosophila transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying attP-based landing or safe harbor sites suitable for universal transgenesis applications in zebrafish have not been established. Here, using CRISPR-Cas9, we converted two well-validated single insertion Tol2-based zebrafish transgenes with long-standing genetic stability into two attP landing sites, called phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). Generating fluorescent reporters, loxP-based Switch lines, CreERT2 drivers, and gene-regulatory variant reporters in the pIGLET14a and pIGLET24b landing site alleles, we document their suitability for transgenesis applications across cell types and developmental stages. For both landing sites, we routinely achieve 25-50% germline transmission of targeted transgene integrations, drastically reducing the number of required animals and necessary resources to generate individual transgenic lines. We document that phiC31 integrase-based transgenesis into pIGLET14a and pIGLET24b reproducibly results in representative reporter expression patterns in injected F0 zebrafish embryos suitable for enhancer discovery and qualitative and quantitative comparison of gene-regulatory element variants. Taken together, our new phiC31 integrase-based transgene landing sites establish reproducible, targeted zebrafish transgenesis for numerous applications while greatly reducing the workload of generating new transgenic zebrafish lines. SHORT ABSTRACTTargeted transgenesis into pre-established, "safe harbor," landing sites remains a missing technique in zebrafish. Here, we established phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET) by CRISPR-Cas9-based conversion of two previously validated Tol2 transgenes into attP sites. phiC31-mediated transgenesis into our pIGLET14a and pIGLET24b attP landing sites results in 25-50% germline transmission efficiency and quantifiable transgene activity. Our landing sites are suitable for reproducible, routine zebrafish transgenesis for diverse applications.

genetics↗

bioRxiv2023

Multiple embryonic sources converge to form the pectoral girdle skeleton in zebrafish

The morphological transformation of the pectoral/shoulder girdle is fundamental to the water-to-land transition in vertebrate evolution. Although previous studies have resolved the embryonic origins of the tetrapod shoulder girdle, those of the fish pectoral girdle remain uncharacterized, creating a gap in the understanding of girdle transformation mechanisms from fish to modern tetrapods. Here, we identified the embryonic origins of the pectoral girdle of zebrafish (Danio rerio), including the cleithrum as an ancestral pectoral girdle element lost in extant tetrapods. Our combinatorial approach of photoconversion and genetic cell lineage tracing mapped that cleithrum development combines three adjoining embryonic populations: cranial neural crest cells and lateral plate mesoderm-derivatives (trunk lateral plate mesoderm and cardiopharyngeal mesoderm-associated cells). The topographical position of the cleithrum at the head/trunk interface is a shared characteristic among cleithrum-bearing fish, thus its multiple embryonic origins are likely a conserved feature. Moreover, a comparison of the pectoral girdle progenitors between aquatic fish and extant amniotes suggests that cleithrum loss is associated with the disappearance of its unique developmental environment by the insertion of the neck lateral plate mesoderm into the head/trunk interface. Overall, our study establishes an embryological framework for pectoral/shoulder girdle formation and their evolutionary trajectories from their origin in water to diversification on land.

developmental biology↗

bioRxiv2021

Heterogeneity and genomic loci of ubiquitous Cre reporter transgenes in zebrafish

The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of two additional Switch reporters due to favorable transgene integration sites. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. Our data documents the heterogeneity among lox-based Switch transgenes towards informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

genetics↗