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

Abstract

Background: The 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.

Results: hand2-2A-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.

Conclusions: hand2 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.

Keywords: CRISPR-Cas9; GeneWeld; Hand2 lineages; early and late emerging venous progenitors; germ layers; knock-in; neural crest; organogenesis; zebrafish.

Figure 1.. hand2 2A-cre and 2A-creERT2 knock-in lines generated with GeneWeld CRISPR/Cas9 targeted integration.

A hand2 diagram showing sequence of exon 2 gRNA in red relative to the hand2 translation stop codon TGA. Space, Cas9 cut site. PAM in bold and underlined. Primers for generating PCR amplicon to test hand2 gRNA mutagenesis efficiency are labeled hand2F and hand2R. Results of ICE analysis of Sanger sequenced PCR amplicon from showing 96% mutagenesis efficiency and range of indel mutations at the hand2 gRNA target site. B Diagrams of recovered hand2-2A-cre, gcry1:mRFPis78 and hand2-2A-creERT2, gcry1:eGFPis79 lines with primers used for 5’ and 3’ junction PCR analysis. C Sanger sequence results of 5’ and 3’ genomic DNA - cassette knock-in junction PCR amplicons from Founders and F2 adults. 48 bp homology arms are shown in green. 5‘ homology arm contained an additional 17 nucleotides to complete the hand2 coding sequence, scrambled, shown in red.

Figure 2.. hand2 lineage tracing live confocal imaging at 22 hpf and 2 dpf in hand2-2A-cre, gcry1:mRFP; ubi:Hulk-lox-Stop-Lox-eGFP embryos.

A, A’ GFP expression in 22 hpf embryo in mesothelium (large arrow), mesoderm-derived vessels (small arrows), and yolk periderm (small arrowheads). B, B’ Higher magnification dorsal view at 22 hpf shows GFP expression in the heart (arrows) and fin buds (arrowheads). C, C’ Higher magnification lateral view of posterior yolk extension show GFP is absent from cell layers of the presumptive gut (small arrows). D Lateral and E dorsal views of 2 dpf embryos show strong GFP expression in the heart (arrows) and fin buds (arrowheads). F, F’ Ventral view of the heart at 2 dpf shows GFP in the atrium (A) and ventricle (V). G, G’ Higher magnification dorsal views of GFP in the fin buds (arrowheads). H, H’ Higher magnification lateral views of 2 dpf mid trunk region show GFP in sprouting vessels from the posterior cardinal vein (small arrows), in presumptive cell layer of the developing gut (arrowheads) and pectoral fin fold (small arrowheads). Scale bars 50 μm.

Figure 3.. hand2 lineage tracing in 3 and 5 dpf hand2-2A-cre, gcry1:mRFP; ubi:Hulk-lox-Stop-Lox-eGFP larvae.

A-D Live confocal imaging of GFP expression in 3 dpf hand2-2a-cre; ubi:Hulk larvae. A Strong GFP expression is detected in heart (arrow), fin (arrowhead) and vasculature (small arrows), and pectoral fin fold (small arrowheads). B Higher magnification of the trunk shows GFP expression in intersegmental vessels and posterior cardinal vein (arrows), the developing intestine (arrowheads), and pectoral fin fold (small arrowheads). C, C’ Ventral view of GFP expression in the branchial arches (ba) and heart ventricle (V) and atrium (A) chambers. D Dorsal view of GFP expression in the pectoral fins (arrowheads). D’ More dorsal focal plane showing GFP expression in pectoral fins (large arrowheads) and in the developing gut (small arrowheads). E – J Live confocal imaging of GFP expression in 5 dpf hand2-2a-cre; ubi:Hulk larvae. E Strong GFP expression in the branchial arches (ba), heart ventricle (V) and along the lining of the developing gut (G). F Ventral view shows GFP expression in the branchial arches (PA) and strong expression in the heart ventricle (V) and atrium (A). G, G’ GFP expression is present in the heart bulbus arteriosus (BA), ventricle (V), atrium (A), and atrioventricular valve (arrowhead). H, H’ GFP expression in mesothelial tissue surrounding the liver (dashed line) and in hepatic stellate cells (asterisks). I, I’ GFP expression in the vasculature in the anterior trunk in intersegmental vessels and posterior cardinal vein (boxed region) and in the lining of the anterior gut (arrow). J, J’ Strong GFP expression extending along the length of the intestine (small arrows) and in the pectoral fin fold (small arrowheads). Scale bars: A, B, C, C’, D, D’ E, F, 50 μm; G, G’, H, H’, I, I’, J, J’, 20 μm.

Figure 4.. Vibratome transverse sections through the trunk of 24 hpf, 48 hpf and 72 hpf hand2-2A-cre, gcry1:mRFP; ubi:Switch lox-GFP-lox-mCherry embryos and larvae.

A – E 150 μm transverse sections labeled with DAPI (blue) and Phalloidin (White). A 24 hpf embryo shows mCherry (magenta) expression in the Lateral Plate Mesoderm (LPM) flanking the endoderm that will form the primitive gut. mCherry is present in a small number of cells in the neural tube. B 48 hpf embryo shows mCherry expression in the LPM surrounding the primitive gut (g). mCherry expression is present in the Posterior Cardinal Vein (PCV) and in a few cells in the neural tube. Nuclear mCherry present in the muscle may represent ectopic expression of the gcry1:nls-mRFP linked secondary marker in the hand2-2A-Cre knock-in allele. C – E Transverse sections along the anterior-posterior axis of a 72 hpf larva. C Anterior transverse section shows mCherry in the mesoderm-derived smooth muscle surrounding the Intestine, and in cells within the liver. D Mid trunk transverse section, and E Posterior trunk transverse section, shows mCherry expression in the smooth muscle surrounding the Intestine and in the wall of the PCV. n=8 embryos or larva per time point. Scale bars A, B, D, E 20 μm; C 30 μm.

Figure 5.. hand2-2A-creERT2, gcry1:eGFP; ubi:Switch-lox-GFP-STOP-lox-mCherry lineage tracing in 24 hpf and 2 dpf embryos after tamoxifen regulated switch at 6 hpf shield stage.

A – C Live confocal imaging of hand2-2A-creERT2 mCherry (magenta) labeled lineages in 24 hpf embryos. A, A’ Lateral view showing mCherry expression in the heart (large arrow), vasculature (small arrows) and fibroblasts of the pre-anal fin fold (PAFF) (small arrowheads). B, B’ Dorsal view showing mCherry expression in the developing heart (arrow). C, C’ Absence of GFP to mCherry switching in the cell layers of the primitive gut (arrows). D – F Live confocal imaging of hand2-2A-creERT2 mCherry labeled lineages in 2 dpf embryos. D Lateral view showing mCherry expression in the pericardium and heart (arrow), fin bud (large arrowhead) and PAFF (small arrowheads). E, E’ Higher magnification lateral view of trunk shows mCherry in posterior cardinal vein and intersegmental vessels (arrows), the primitive gut (arrowheads), and the PAFF (small arrowheads). F, F’ Ventral view embryo shows mCherry expression in the atrium (A) and ventricle (V) of the heart and a low level of expression in the presumptive heart outflow tract (arrow). G, G’ mCherry expression is not detected in 2 dpf control hand2-2A-creERT2; ubi:Switch embryo treated with EtOH vehicle alone. Scale bars: A, A’ 100 μm; B, B’, C, C’, D, D’ E, F, F’, G, G’ 50 μm.

Figure 6.. Tamoxifen and heat shock regulated lineage tracing in hand2-2A-creERT2; hsp70l:lox-STOP-lox-GFP embryos at 1.5 and 2.5 dpf.

A, A’ Live confocal imaging of hand2-2A-creERT2; hsp70l:lox-STOP-lox-GFP embryos after 4-OHT treatment at shield stage and heat shock at 1.5 dpf. GFP expression is present in the heart (large arrow), pectoral fin (large arrowhead), Posterior Cardinal Vein (small arrows), and PAFF (asterisks). B, B’, C, C’ Live confocal imaging of hand2-2A-creERT2; hsp70l:lox-STOP-lox-GFP embryos after 4-OHT treatment at shield stage and heat shock at 2.5 dpf. B, B’ Ventral view of 2.5 dpf embryo showing GFP expression in the cardiac atrium and ventricle (arrows) and branchial arches (arrowheads). C, C’ Lateral view of 2.5 dpf embryo showing GFP expression in the heart (large arrows), pectoral fin bud (large arrowheads), Posterior Cardinal Vein (small arrows), venous intersegmental vessels (small yellow arrows), yolk periderm (asterisks), PAFF (large yellow arrows), and primitive gut (small arrowheads). D, E Live confocal imaging of 1.5 dpf (D) and 2.5 dpf (E) hand2-2A-creERT2; hsp70l:lox-STOP-lox-GFP control embryos that were not treated with 4-OHT. Scale bars: A, A’, C, C’, D 500 μm; B, B’, E 200 μm.

Figure 7.. Comparison of hand2-2A-creERT2 lineage tracing in 5dpf larvae after induction at shield stage vs. 3 dpf.

A – E Live confocal imaging of 5 dpf hand2-2A-creERT2; ubi:Switch embryos treated with 4-OHT at 6 hpf. A, A’ Ventral view showing mCherry (magenta) expression in branchial arches (ba), heart (arrow) and pectoral fins (arrowheads). B, B’ mCherry expression in the cardiac bulbus arteriosus (BA), ventricle (V), atrium (A), atrioventricular valve (arrowhead), and pericardium (small arrows). C, C’ mCherry expression in the mesothelium surrounding the liver (dashed line) and hepatic stellate cells (asterisks). D, D’ Anterior trunk region showing mCherry expression in the intersegmental vessels and posterior cardinal vein (small arrows), intestinal bulb (arrow), mid intestine (arrowheads), and PAFF fibroblasts (asterisk). E, E’ Mid-trunk region showing mCherry expression in the intersegmental vessels and posterior cardinal vein (small arrows), intestine (arrowhead), and PAFF (asterisk). F – J Live confocal imaging of 5 dpf hand2-2A-creERT2; ubi:Switch embryos treated with 4-OHT at 3 dpf. F, F’ Ventral view showing mCherry expression in branchial arches (PA), heart (arrow) and pectoral fins (arrowheads). G, G’ mCherry expression in the heart bulbus arteriosus (BA), ventricle (V), atrioventricular valve (arrowhead), and atrium (A, dashed outline), absent from the pericardium (small arrows). H, H’ mCherry expression in liver hepatic stellate cells (asterisks). Dashed line outlines liver. I, I’ Anterior and (J, J’) mid trunk regions showing mCherry expression present in gut enteric neurons (arrowheads) and absent from the PAFF (asterisk). Scale bars: B, B’, E, E’. F, F’, G, G’ J, J’, K, K’, 50 μm; C, C’. D, D’, H, H’, I, I’, 20 μm.

Figure 8.. mCherry expression in hand2-2A-creERT2; ubi:Switch fixed 5 dpf larval liver and intestine after induction at 3 dpf.

A, A’ Confocal 3D projection of z-stack sections of mCherry (magenta) expression in liver hepatic stellate cells (arrows). B, B’ Confocal 3D projection of z-stack sections of mCherry expression in enteric neuron cell bodies and neurite extensions surrounding the intestine. Scale bars: A, A’, B, B’ 50 μm.

Figure 9.. hand2:creERT2 lineage tracing reveals contribution to the venous and intestinal vasculature.

A-D Live embryo confocal z-stack projections of vasculature in hand2:creERT2; actb2:loxP-BFP-loxP-dsRed; fli1:GFP 3 dpf larvae. A, B Images of 3 dpf larva treated with 4-OHT beginning at 6 dpf. A Lateral view of larval trunk showing eGFP expression throughout the vasculature and eGFP, dsRed double positive cells (B, arrows) in the posterior cardinal vein (PCV), venous Intersegmental Vessels (vISV), supraIntestinal Artery (SIA) and subIntestinal Vein (SIV). dsRed was not detected in vascular cells in the Dorsal Aorta (DA) or aortic Intersegmental Vessels (aISV). B Higher magnification of boxed region shown in A. C Images of 3 dpf larva treated with 4-OHT from 6 hpf to 1 dpf showing eGFP, dsRed double positive cells (arrows) in the PCV, vISV, SIA and SIV. D Lateral view of trunk vasculature in larva treated with 4-OHT beginning at 24 hpf through 3 dpd showing eGFP, dsRed double positive cells (arrows) in the SIA and SIV. E Quantification of eGFP, dsRed double positive vascular endothelial cells in hand2:creERT2; actb2:loxP-BFP-loxP-dsRed; fli1:GFP embryos treated with 4-OHT from 6 hpf – 3 dpf (n=9), from 6 hpf – 1 dpf (n=18), and from 1 dpf – 3 dpf (n=10). Data plots show mean ± s.e.m. Scale bars: A 100 μm; B, C, D 50 μm.