Figures and data
podxl knockdown decreases HSC number.
Tg(wt1b:egfp) zebrafish injected with CRISPR/Cas9 targeting either tyr or podxl, examined at 6 dpf. Representative confocal projections of uninjected (A), tyr KD (B), and podxl sgRNA #1 KD (C) (livers outlined in white) and quantified HSC counts (D). (E) HSC counts of 6-dpf podxl sgRNA #2 KD larvae and uninjected controls. Bars show mean +/- SD. Brown-Forsythe and Welch ANOVA tests (D) and Welch’s t test (E). NS, not significant; *, p<0.05; ***, p<0.001.
Decrease in HSC number seen with podxl KD is not due to developmental delay or an artifact of using the Tg(wt1b:eGFP) zebrafish line.
Liver area (A) and HSC count (B) of podxl KD zebrafish at 6 dpf compared to uninjected siblings. HSC number of podxl KD zebrafish at 5 dpf and 7 dpf (C). (D) HSC count of podxl KD in Tg(hand2:EGFP) zebrafish compared to uninjected siblings at 6 dpf, and representative images (E,F) (livers outlined in white). Bars show mean +/- SD. Welch’s t test (A, B, D) and ANOVA (C). NS, not significant; *,p<0.05; **, p<0.01; ***, p<0.001.
Analysis of HSC number in podxl mutant lines shows no change in HSC number for some mutants but an increase in promoter mutants.
Schematic representation of the wildtype podxl gene (A) and podxl mutants (B-F). Deleted regions of podxl are depicted by a black arrow (B) or dotted lines (C-F). Premature stop codons are represented by black hexagons (B,C). (G-K) HSC counts of podxl mutants compared to wildtype siblings. (L) HSC number in podxlEx1(p)_Ex7Δmutant combined with podxl sgRNA #1. Welch’s t-test (G-K). Ordinary one-way ANOVA (L). Bars show mean +/- SD. NS, not significant; **, p<0.01;****, p<0.0001.
Expression of podxl mRNA in podxl mutants.
(A) Wildtype podxl gene schematic with brackets indicating the regions of mRNA amplified by qPCR; 5’UTR - exon 1(left), exon 7 - exon 8 (center), and partial exon 8 (right). (B-F) Normalized expression levels of each region from the livers of 3-mpf podxl Ex1,-5bpΔ(B), podxlEx1(p)_Ex7Δ (C), podxlEx5_Ex7Δ (D), podxl-194_Ex7Δ (E), and podxl-319_Ex1(p)Δ (F) mutants and wildtype siblings. Primers lacking binding sites in the mutant allele are indicated by bold blue graph titles. Welch’s t-test. NS, not significant; *, p<0.05; **, p<0.01; ND, not detected.
Expression of podxl pre-mRNA in podxl mutants.
(A) Wildtype podxl gene schematic with brackets indicating the regions of pre-mRNA amplified by qPCR; exon 2 - intron 2 (left), intron 2 - exon 3 (center), and intron 7 - exon 8 (right). (B-F) Normalized expression levels of each region from the livers of 3mpf podxl Ex1,-5bpΔ (B), podxlEx1(p)_Ex7Δ (C), podxlEx5_Ex7Δ (D), podxl- 194_Ex7Δ (E), podxl-319_Ex1(p)Δ (F) mutants and wildtype siblings. Primers lacking binding sites in the mutant allele are indicated by bold blue graph titles. Welch’s t test. NS, not significant; *, p<0.05; ND, not detected.
Endoglycan does not compensate for podxl loss.
(A) HSC count of 6-dpf podxlEx1(p)_Ex7Δmutants with CRISPR knockdown of endoglycan. (B-F) qPCR of endoglycan in podxl Ex1,-5bpΔ (B), podxlEx1(p)_Ex7Δ (C), podxlEx5_Ex7Δ (D), podxl-194_Ex7Δ (E), and podxl-319_Ex1(p)Δ (F). Brown-Forsythe and Welch ANOVA test (A), Welch’s t test (B-F). Bars show mean +/- SD. ns, not significant; ND, not detected.
