Genetic analysis of zebrafish homologs of human FOXQ1, foxq1a and foxq1b, in innate immune cell development and bacterial host response

Abstract

FOXQ1 is a member of the forkhead-box transcription factor family that has important functions in development, cancer, aging, and many cellular processes. The role of FOXQ1 in cancer biology has raised intense interest, yet much remains poorly understood. We investigated the possible function of the two zebrafish orthologs (foxq1a and foxq1b) of human FOXQ1 in innate immune cell development and function. We employed CRISPR-Cas9 targeted mutagenesis to create null mutations of foxq1a and foxq1b in zebrafish. Using a combination of molecular, cellular, and embryological approaches, we characterized single and double foxq1a bcz11 and foxq1b bcz18 mutants. This study provides the first genetic mutant analyses of zebrafish foxq1a and foxq1b. Interestingly, we found that foxq1a, but not foxq1b, was transcriptionally regulated during a bacterial response, while the expression of foxq1a was detected in sorted macrophages and upregulated in foxq1a-deficient mutants. However, the transcriptional response to E. coli challenge of foxq1a and foxq1b mutants was not significantly different from that of their wildtype control siblings. Our data shows that foxq1a may have a role in modulating bacterial response, while both foxq1a and foxq1b are not required for the development of macrophages, neutrophils, and microglia. Considering the implicated role of FOXQ1 in a vast number of cancers and biological processes, the foxq1a and foxq1b null mutants from this study provide useful genetic models to further investigate FOXQ1 functions.

Fig 1. Sequence analysis of foxq1a^bcz11 and foxq1b^bcz18 mutations generated by CRISPR-Cas9 targeting.

a Top, DNA and amino acid sequences of the 5’ end of foxq1a coding region. DNA sequencing chromatograms show the expected foxq1a sequence in the wildtype sibling, and a large 95 base pair deletion (red box) in the homozygous bcz11 mutant, which causes a premature stop (black box with an asterisk) at the beginning of the gene. Bottom, DNA and amino acid sequences of the 5’ end of foxq1b coding region. DNA chromatograms show wildtype sequence in foxq1b wildtype sibling, while the homozygous bcz18 mutant carries a 4 bp deletion and 12 bp insertion (red box) causing a nonsense mutation at the beginning of the gene. gRNA, target sites of the guide RNAs used. b Whole mount live imaging of 5 dpf larvae show normal gross morphological development of foxq1a^bcz11 and foxq1b^bcz18 mutants as compared to wildtype siblings (at least 10 animals were analyzed per genotype group).

Fig 2. Gene expression analysis of foxq1a and foxq1b.

a Isolation of macrophages based on GFP expression by FACS in mpeg1:EGFP transgenic zebrafish embryos at 2.5 dpf. Non-fluorescent embryos were used as a negative control for gating. Top left, P4 shows the cell fraction sorted as GFP+ macrophages, also shown in the right panels in brightfield and green channel. b RT-PCR analysis of gene markers validated the different cell populations sorted by FACS, as denoted by E, erythrocytes; M, macrophages; A, all remaining non-fluorescent cells. The following genes were used: translation elongation factor 1 (ef1a) as a reference marker, hemoglobin beta embryonic-1.1 (hbbe1.1) as an erythrocyte marker, and macrophage expressed gene 1 (mpeg1) and interferon regulatory factor 8 (irf8) as well-established macrophage markers in zebrafish. As expected, the ‘A’ cells expressed all genes, while erythrocytes ‘E’ cells expressed hbbe1.1, ef1a, and DsRed genes and macrophage ‘M’ cells expressed macrophage markers, GFP, and ef1a. c Using the sorted cell populations, we found expression of foxq1a in macrophages and neither gene in erythrocytes. Both genes are expressed at 2 to 8 dpf of development as well as in the adult gut tissue.

Fig 3. Single and double foxq1a^bcz11 and foxq1b^bcz18 mutants have normal macrophage and neutrophil development.

a Whole mount in situ hybridization of 2.5 dpf single and double foxq1a^bcz11 and foxq1b^bcz18 mutants and their siblings using RNA probes for mfap4 (macrophage marker) and mpx (neutrophil marker). Arrows, macrophages or neutrophils in the caudal hematopoietic tissue in the embryo tail. b Scatter plots showing number of macrophages and neutrophils in the tail region for each embryo quantified in the different genotype categories. n, number of embryos analyzed beneath each scatter plot. Plots report average ± standard deviation (s.d.). Multiple unpaired t-tests comparing between control siblings and each of the mutants groups were conducted with correction for multiple comparisons using the Sidak-Bonferroni method and without assuming equal variance to determine statistical significance. n.s., no statistical significance as defined by p > 0.05 was found in all the comparisons.

Fig 4. Neutral red analysis shows normal microglia development in single and double foxq1a and foxq1b mutants at 4 dpf.

a Neutral red staining in 4 dpf wildtype (WT) sibling, foxq1a^bcz11, foxq1b^bcz18, and foxq1a^bcz11;foxq1b^bcz18 mutants shows a stereotypical pattern of microglia in the midbrain (arrow). b Bar graph shows all larvae analyzed in all genotype groups had normal numbers of microglia (>25). Number of larvae analyzed (n) shown to the right of the corresponding bar.

Fig 5. foxq1a^bcz11 and foxq1b^bcz18 mutants exhibit wildtype transcriptional response to E. coli challenge in the larval brain.

a Graphical representation of the brain challenge assay. E. coli was injected into right brain tectum, and at 6 hours post injection (hpi), injected larvae were individually processed for RNA extraction, genotyping, cDNA synthesis, and qPCR analysis. b Bar chart shows significant upregulation of immune activation genes il1β, irg1, mpx, and tnfα after bacteria challenge at 6 hpi in wildtype larvae compared with uninjected controls. Water injected wildtype larvae exhibit no induction of activation genes, whereas LPS injected wildtype animals have a significant upregulation of il1β and irg1. After microbial activation by E. coli or LPS, a significant downregulation of foxq1a was found by about a factor of 2. No significant change was found for foxq1b after immune challenge. Data from wildtype injections validate the efficacy of the assay to activate the innate immune system. Dotted line marks no fold difference at 1. At least 6 or more independent biological samples were measured per category. c Bar plot shows transcriptional changes of target genes after E. coli injection in the brain at 6 hpi for control siblings and foxq1a and foxq1b mutants. n = 9–14 independent biological samples were measured per genotype. All error bars show standard error of means. Statistical significance in b and c was determined by multiple unpaired t-tests comparing between uninjected or sibling controls and the experimental groups with correction for multiple comparisons using the Sidak-Bonferroni method and without assuming equal variance. n.s., no statistical significance as defined by p > 0.05. *, p < 0.05; **, p < 0.01; ***, p < 0.001.