Biology subjects

McGrail, M.

Publications and source records attributed to McGrail, M..

10 recordsLinked to original sources

bioRxiv2026

Collagen4 fluorescent protein knock-in lines reveal remodeling of cardiovascular basement membranes

Basement membranes (BMs) are specialized extracellular matrix structures that provide structural support and regulate cellular behavior. Although BM composition and organization are dynamically remodeled during development and tissue regeneration, the remodeling of individual BM components in the cardiovascular system (CVS) remain incompletely characterized. Here, we focused on Collagen4 (Col4), a major structural component of BMs, to investigate its remodeling during cardiovascular development and regeneration. To monitor the remodeling of Col4 in vivo, the Venus fluorescent tag was integrated in frame into the zebrafish endogenous col4a1 and col4a2 genes, enabling live confocal imaging of expression. Our data reveal dynamic patterns of Col4a1 and Col4a2 within the developing CVS, with deposition progressively increasing as vascular and cardiac structures develop and mature during both development and regeneration. In the regenerating heart, Col4a2 accumulated at the injury site during early stages of regeneration and became increasingly associated with the newly regenerated myocardium. Together, these findings demonstrate that Col4 deposition is dynamically reorganized across developmental and regenerative stages of CVS and describe new tools for following Col4 dynamics in vivo.

developmental biology↗

bioRxiv2026

Tracing developmental and adult hematopoiesis with an endogenous zebrafish runx1-2A-CreERT2 CRISPR knock-in

Zebrafish combines the power of genetics and unparalleled in vivo imaging to investigate the dynamics of vertebrate hematopoietic development. The conserved transcription factor Runx1 is essential for definitive hematopoiesis. We generated an endogenous zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Runx1 lineage tracing and characterized its activity using recombinase-dependent fluorescence reporters, live imaging and flow cytometry. Tamoxifen induction of Cre in the early embryo labeled all expected Runx1 lineages, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and emerging definitive hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Short windows of tamoxifen treatment allowed separation of Runx1 primitive from definitive hematopoiesis and visualization of HSPC colonization of the larval caudal hematopoietic tissue, thymus and kidney. Runx1 HSPCs labeled during development persisted into adulthood and contributed to hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The runx1-2A-creERT2 knock-in line provides a novel genetic tool for inducible spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis.

developmental biology↗

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↗

bioRxiv2024

Stat3 mediates Fyn kinase driven dopaminergic neurodegeneration and microglia activation

The Alzheimers Disease and Parkinsons Disease risk locus Fyn kinase is implicated in neurodegeneration and inflammatory signaling. To investigate in vivo mechanisms of Fyn driven neurodegeneration, we built a zebrafish neural specific Gal4:UAS model of constitutively active FynY531F signaling. Using in vivo live imaging we demonstrate neural FynY531F expression lead to dopaminergic neuron loss and mitochondrial aggregation in 5 day larval brain. Dopaminergic loss coincided with microglia activation and induction of tnfa, il1b, and il12a inflammatory cytokine expression. Transcriptome analysis revealed Stat3 signaling as a potential Fyn target. Chemical inhibition experiments confirmed Fyn driven dopaminergic neuron loss and the inflammatory response were dependent upon activation of Stat3 and NF-{kappa}B pathways. Dual chemical inhibition demonstrated Stat3 acts synergistically with NF-{kappa}B in dopaminergic neuron degeneration. These results identify Stat3 as a novel downstream effector of Fyn signaling in neurodegeneration and inflammation. Summary StatementThis study describes a novel in vivo model of neural Fyn Kinase activation and identifies Stat3 signaling as a downstream Fyn effector in dopaminergic neuron degeneration and neuroinflammation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/602238v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1d783a4org.highwire.dtl.DTLVardef@3b838org.highwire.dtl.DTLVardef@33790eorg.highwire.dtl.DTLVardef@1508c64_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO - Neural Fyn signaling drives dopaminergic neurodegeneration, mitochondria accumulation, and microglia activation - Fyn driven neurodegeneration and cytokine expression are dependent on Stat3 - Stat3 and NF-kB pathways synergize in dopaminergic neuron degeneration C_FIG

developmental biology↗

bioRxiv2024

A zebrafish system for identifying genetic dependencies in melanocytes and melanoma

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

genetics↗

bioRxiv2021

Zebrafish Cre/lox regulated UFlip alleles generated by CRISPR/Cas targeted integration provide cell-type specific conditional gene inactivation

The ability to regulate gene activity spatially and temporally is essential to investigate cell type specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish, but simple and efficient methods for isolation of stable, Cre/lox regulated alleles are lacking. Here we applied our GeneWeld CRISPR/Cas9 short homology-directed targeted integration strategy to generate floxed conditional alleles that provide robust gene knockdown and strong loss of function phenotypes. A universal targeting vector, UFlip, with sites for cloning short 24-48 bp homology arms flanking a floxed mRFP gene trap plus secondary reporter cassette, was integrated into an intron in hdac1, rbbp4, and rb1. Active, gene off orientation hdac1-UFlip-Off and rb1-UFlip-Off integration alleles result in >99% reduction of gene expression in homozygotes and recapitulate known indel loss of function phenotypes. Passive, gene on orientation rbbp4-UFlip-On and rb1-UFlip-On integration alleles do not cause phenotypes in trans-heterozygous combination with an indel mutation. Cre recombinase injection leads to recombination at alternating pairs of loxP and lox2272 sites, inverting and locking the cassette into the active, gene off orientation, and the expected mutant phenotypes. In combination with our endogenous neural progenitor Cre drivers we demonstrate rbbp4-UFlip-On and rb1-UFlip-On gene inactivation phenotypes can be restricted to specific neural cell populations. Replacement of the UFlip mRFP primary reporter gene trap with a 2A-RFP in rbbp4-UFlip-Off, or 2A-KalTA4 in rb1-UFlip-Off, shows strong RFP expression in wild type or UAS:RFP injected embryos, respectively. Together these results validate a simplified approach for efficient isolation of highly mutagenic Cre/lox responsive conditional gene alleles to advance zebrafish Cre recombinase genetics.

genetics↗

bioRxiv2020

Endogenous zebrafish neural Cre drivers generated by CRISPR/Cas9 short homology directed targeted integration

The Cre/lox recombinase system has been widely used for spatiotemporal control of gene expression in animal model systems, however, efficient methods to isolate zebrafish Cre drivers that reliably recapitulate endogenous gene expression patterns are needed. Here, we apply CRISPR/Cas9 targeting to integrate a 2A-Cre recombinase transgene with 48bp homology arms into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10%-100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre)is75, Tg(olig2-2A-Cre)is76, and Tg(neurod1-2A-Cre)is77 expressed functional Cre recombinase in the expected proneural cell populations. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre drivers whose spatial and temporal restricted expression mimics endogenous genes, surmounting the challenges associated with promoter BAC cloning and transposon mediated transgenesis.

genetics↗

bioRxiv2019

The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease.

genomics↗

bioRxiv2018

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

genetics↗

bioRxiv2018

Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

Retinoblastoma-binding protein 4 (Rbbp4) is a WDR adaptor protein for multiple chromatin remodelers implicated in human oncogenesis. Here we show Rbbp4 is overexpressed in zebrafish rb1-embryonal brain tumors and is upregulated across the spectrum of human embryonal and glial brain cancers. We demonstrate in vivo Rbbp4 is essential for zebrafish neurogenesis and has distinct roles in neural stem and progenitor cells. rbbp4 mutant neural stem cells show delayed cell cycle progression and become hypertrophic. In contrast, rbbp4 mutant neural precursors accumulate extensive DNA damage and undergo programmed cell death that is dependent on Tp53 signaling. Loss of Rbbp4 and disruption of genome integrity correlates with failure of neural precursors to initiate quiescence and transition to differentiation. rbbp4; rb1 double mutants show that survival of neural precursors after disruption of Rb1 is dependent on Rbbp4. Elevated Rbbp4 in Rb1-deficient brain tumors might drive proliferation and circumvent DNA damage and Tp53-dependent apoptosis, lending support to current interest in Rbbp4 as a potential druggable target.\n\nAuthor SummaryExamining the developmental mechanisms controlling neural stem and progenitor cell behavior is critical to our understanding of the processes driving brain tumor oncogenesis. Chromatin remodelers and their associated adaptor proteins are thought to be key drivers of brain development and disease through epigenetic regulation of gene expression and maintenance of genome integrity, but knowledge of their in vivo roles in vertebrate neurogenesis is limited. The chromatin remodeler adaptor protein Rbbp4 has recently been shown to function in a mouse model of neuroblastoma and in glioblastoma multiforme cell resistance to the chemotherapeutic temozolomide. However, an in vivo requirement for Rbbp4 in neurogenesis has only just been shown by isolation of a recessive lethal mutation in zebrafish rbbp4. Here we provide conclusive genetic evidence that zebrafish rbbp4 is essential in neural stem and progenitor cell function during development. Our data reveal for the first time in vivo that Rbbp4 prevents DNA damage and activation of Tp53 signaling pathway that leads to programmed cell death. Importantly, neural progenitors that are mutant for the tumor suppressor Rb1 also depend on Rbbp4 for survival. Finally, we show that neural stem cells that have lost Rbbp4 cease dividing, and may enter a senescent like state. Together, these observations provide novel evidence that elevated expression of Rbbp4 in rb1-mutant tumors may contribute to cancer cell survival by blocking senescence and/or DNA damage-induced cell death.

genetics↗