Genetic dissection of endothelial transcriptional activity of zebrafish aryl hydrocarbon receptors (AHRs)

<div><p>The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor conserved across phyla from flies to humans. Activated by a number of endogenous ligands and environmental toxins, studies on AHR function and gene regulation have largely focused on a toxicological perspective relating to aromatic hydrocarbons generated by human activities and the often-deleterious effects of exposure on vertebrates mediated by AHR activation. A growing body of work has highlighted the importance of AHR in physiologic processes, including immune cell differentiation and vascular patterning. Here we dissect the contribution of the 3 zebrafish AHRs, <i>ahr1a</i>, <i>ahr1b</i> and <i>ahr2</i>, to endothelial <i>cyp1a1/b1</i> gene regulation under physiologic conditions and upon exposure to the AHR ligand Beta-naphthoflavone. We show that in fish multiple AHRs are functional in the vasculature, with vessel-specific differences in the ability of <i>ahr1b</i> to compensate for the loss of <i>ahr2</i> to maintain AHR signaling. We further provide evidence that AHR can regulate the expression of the chemokine receptor <i>cxcr4a</i> in endothelial cells, a regulatory mechanism that may provide insight into AHR function in the endothelium.</p></div

Transcriptional control of <i>cxcr4a</i> by AHR.

<p><b>(A)</b> Core DNA binding sequence of AHR::ARNT complex from JASPAR database. (<b>B)</b> Schematic of the zebrafish <i>cxcr4a</i> gene. Using strict search criteria (a relative profile score threshold of 90%), the promoter sequence spanning 2 kb from the start codon was analyzed for AHR binding sites in the JASPAR database. Two putative AHR binding sites lie close to the transcription start site (TSS). (<b>C-H)</b> Whole mount ISH for <i>cxcr4a</i> in WT and triple AHR mutant embryos treated from 48 hpf to 52 hpf with DMSO (<b>C, F</b>), 1 uM BNF (<b>D, G</b>) or 15 uM nifedipine (<b>E, H</b>). Arrows indicate subintestinal vasculature, while arrowheads point to DLAV. Scale bar is 250 um. (<b>I-K</b>) High magnification images of hindbrain capillaries in WTs and triple AHR mutants. Note similar levels of <i>cxcr4a</i>-positive CtAs (yellow arrowheads) in WTs and triple AHR mutants treated with DMSO (<b>I</b>), and markedly fewer <i>cxcr4a</i>-positive cells in triple mutants treated with BNF compared to WT (<b>J</b>). Both WTs and triple AHR mutants display marked increase in <i>cxcr4a</i> expression in all CtA endothelial cells after nifedipine treatment (<b>K</b>). Scale bar is 100 um. (<b>L)</b> Quantification of <i>cxcr4a</i>-positive endothelial cells in high magnification images of the hindbrain of WT, <i>ahr1a -/-</i>, <i>ahr1b</i>,<i>2 -/-</i> and triple AHR mutants treated from 48–52 hpf with DMSO or nifedipine. All genetic combinations of AHR mutations have similar numbers of <i>cxcr4a</i>-positive cells at basal levels. The BNF-induced increase of <i>cxcr4a</i> expression in WT still occurs in <i>ahr1a -/-</i> embryos, but does not show in <i>ahr1b</i>,<i>2 -/-</i> or triple AHR mutants. Analyzed by One-way ANOVA (DMSO treatment N = 4 WT, 12 <i>ahr1a -/-</i>, 13 <i>ahr1b</i>,<i>2 -/-</i> and 14 triple AHR fish. 1 uM BNF treatment N = 9 WT, 12 <i>ahr1a -/-</i>, 10 <i>ahr1b</i>,<i>2 -/-</i> and 6 triple AHR fish.) (<b>M)</b> Summary of transcriptional control of <i>cxcr4a</i> expression by flow (negative regulator) and BNF (positive regulator). Induction by BNF is in an AHR-dependent manner. **P<0.01, error bars indicate s.e.m. Abbreviations–AHR: aryl hydrocarbon receptor, ARNT: aryl hydrocarbon nuclear translocator, BNF: beta-naphthoflavone, CtA: central artery, CYP: cytochrome p450, DLAV: dorsal longitudinal anastomotic vessel, DMSO: dimethylsulfoxide, hpf: hours post fertilization, ISH: <i>in situ</i> hybridization, LDA: lateral dorsal aortae, PHBC: primordial hindbrain channel, TSS: transcription start site, WT: wildtype.</p

Regulation of <i>cyp1b1</i> by AHR.

<p><b>(A-E)</b> Overview pictures of <i>cyp1b1</i> expression at 52 hpf in DMSO or BNF-treated WT (<b>A</b>), <i>ahr1a -/-</i> (<b>B</b>), <i>ahr2 -/-</i> (<b>C</b>), <i>ahr1b</i>,<i>2 -/-</i> <b>(D</b>) and triple AHR mutants (<b>E</b>). No changes in expression pattern were evident in DMSO-treated embryos that lacked any AHR genes. Induction in the skin by BNF was dramatically reduced in <i>ahr2 -/-</i> (compare <b>A</b> to <b>C</b>, lower panels). Scale bar is 500 um. <b>F-T)</b> High magnification images of blood vessels in 3 different planes of the head vasculature: the LDA (<b>F-J</b>), the hindbrain blood vessels (<b>K-O</b>) and the hindbrain capillaries (<b>P-T</b>) in all genetic combinations with DMSO and BNF treatment. Black asterisks denote approximate location of liver. Yellow arrowheads label individual CtAs. Note maintenance of endogenous <i>cyp1b1</i> expression in the ear (<b>K-O</b>, upper panels) and head (<b>P-T</b>, upper panels) of all mutants. Expression of <i>cyp1b1</i> was not detected in the liver for either treatment in any genetic combination (<b>F-J</b>). Note lack of BNF-induced staining in LDA of <i>ahr2</i> mutants, but strong staining in PHBCs (<b>H</b> and <b>M,</b> lower panels). Numbers indicate embryos with indicated expression pattern/total embryos of that genotype analyzed. Scale bar is 100 um. Abbreviations–AHR: aryl hydrocarbon receptor, BNF: beta-naphthoflavone, CtA: central artery, CYP: cytochrome p450, DMSO: dimethylsulfoxide, hpf: hours post fertilization, LDA: lateral dorsal aortae, PHBC: primordial hindbrain channel, WT: wildtype.</p

Zebrafish AHR genes are differentially expressed during development.

<p><b>(A)</b> Schematic of genomic arrangement of zebrafish AHR genes. Note linkage of <i>ahr1b</i> and <i>ahr2</i> (~3 kb intergenic distance). (<b>B)</b> Experimental design. Embryos were exposed to DMSO or 1 uM BNF for 4 hrs starting at 48 hpf, then fixed in 4% PFA for ISH. (<b>C-H)</b> Stereomicroscope images of whole mount ISH in 52 hpf embryos exposed to DMSO or 1 uM BNF showing expression of <i>ahr1a</i> (<b>C, D</b>), <i>ahr1b</i> (<b>E, F</b>) and <i>ahr2</i> (<b>G, H</b>). Boxes indicate region of high magnification image in inset. Numbers indicate embryos with indicated expression pattern/total embryos analyzed. Scale bar is 500 um. (<b>I-N)</b> High magnification images of trunk in 52 hpf embryos exposed to DMSO or 1 uM BNF showing expression of <i>ahr1a</i> (<b>I, J</b>), <i>ahr1b</i> (<b>K, L</b>) and <i>ahr2</i> (<b>M, N</b>). No staining evident for <i>ahr1a</i> or <i>ahr1b</i> in either condition. <i>ahr2</i> expression is detected in DLAV, gut (white arrowhead) and region between DA and PCV (white bracket), and is increased by BNF-treatment. Scale bar is 100 um. Abbreviations—AHR: aryl hydrocarbon receptor, BNF: beta-naphthoflavone, DA: dorsal aorta, DLAV: dorsal longitudinal anastomotic vessel, DMSO: dimethylsulfoxide, hpf: hours post fertilization, ISH: <i>in situ</i> hybridization, kb: kilobase pair PCV: posterior cardinal vein, PFA: paraformaldehyde.</p

Regulation of <i>cyp1a1</i> by AHR.

<p><b>(A)</b> Overview pictures of <i>cyp1a1</i> expression at 52 hpf in DMSO or BNF-treated WT (<b>A</b>), <i>ahr1a -/-</i> (<b>B</b>), <i>ahr2 -/-</i> (<b>C</b>), <i>ahr1b</i>,<i>2 -/-</i> <b>(D</b>) and triple AHR mutants (<b>E</b>). <i>ahr1a -/-</i> are indistinguishable from WT. (*) indicates expression in the eye in DMSO and BNF-treated embryos, which is lost only in <i>ahr1b</i>,<i>2 -/-</i> and triple AHR mutants. Note reduction of endogenous vascular <i>cyp1a1</i> expression in <i>ahr2</i> mutants (<b>C,</b> upper panels) that is lost in <i>ahr1b</i>,<i>2 -/-</i> and triple AHR mutants (<b>D, E</b> upper panels). <i>ahr2 -/-</i> embryos treated with BNF do not induce <i>cyp1a1</i> expression in the skin, revealing staining in blood vessels underneath (<b>C</b>, lower panel), which is abolished in <i>ahr1b</i>,<i>2 -/-</i> and triple AHR mutants (<b>D, E</b>, lower panels). Scale bar is 500 um. (<b>F-T)</b> High magnification images of blood vessels of 3 different planes of the head vasculature: the LDA (<b>F-J</b>), the hindbrain blood vessels (<b>K-O</b>) and the hindbrain capillaries (<b>P-T</b>) in all genetic combinations with DMSO and BNF treatment. Note strong dependence of LDA (but not hindbrain arteries, PHBC or CtAs) on <i>ahr2</i> for full <i>cyp1a1</i> expression. White asterisk denotes liver. Yellow arrowheads label individual CtAs. Numbers indicate embryos with indicated expression pattern/total embryos of that genotype analyzed. Scale bar is 100 um. Abbreviations–AHR: aryl hydrocarbon receptor, BNF: beta-naphthoflavone, CtA: central artery, CYP: cytochrome p450, DMSO: dimethylsulfoxide, hpf: hours post fertilization, LDA: lateral dorsal aortae, PHBC: primordial hindbrain channel, WT: wildtype.</p

Generation of zebrafish <i>ahr1a</i> and <i>ahr1b</i> mutants.

<p><b>(A, B)</b> Depiction of genomic locus for <i>ahr1a</i> (<b>A</b>) and <i>ahr1b</i> (<b>B</b>). TALEN pairs were designed targeting the bHLH domain in exon 2 of both genes. Genomic lesions in the TALEN spacer indicated in alignment to WT sequence, and cartoons show predicted protein products. For <i>ahr1b</i>, alleles mu133-mu135 were generated in a WT genetic background, while mu136-mu145 were made in an <i>ahr2</i>^{<i>hu3335</i>} homozygous mutant background. (<b>C)</b> Brightfield images of WT, <i>ahr1a</i> -/-, <i>ahr1b</i>,<i>2 -/-</i> and triple AHR mutant embryos at 5 dpf. No gross morphological defects in AHR mutants are apparent. Boxes indicate region of imaging for different vascular beds in <b>D-I</b>. Numbers correspond to “number of embryos with inflated swim bladders/total number of embryos of that genotype”. Scale bar is 1000 um. (<b>D-I)</b> Maximum intensity projections of confocal z-stacks comparing the trunk (<b>D, E</b>), brain (<b>F, G</b>) and liver (<b>H, I</b>) vascular beds in <i>ahr1a</i> -/- and triple AHR mutant zebrafish embryos at 5 dpf. Vascular patterning appears normal in both mutants compared to WT. Yellow arrowheads point to forming intercostal vessels in the trunk. Orange brackets mark midbrain vessels, yellow brackets mark hindbrain vessels. Red arrows show the liver. Numbers indicate the “number of embryos of a particular genotype with vascular characteristics as in image/total numbers of that genotype analyzed.” Scale bar is 100 um. Abbreviations—AHR: aryl hydrocarbon receptor, bHLH: basic helix-loop-helix, hpf: hours post fertilization, TALEN: transcription activator-like effector nuclease, WT: wildtype.</p

Differential vascular expression of CYP genes under basal and BNF-induced conditions.

<p><b>(A)</b> Stereomicroscope images of whole mount ISH for <i>cyp1a1</i> and <i>cyp1b1</i> in 52 hpf WT embryos after 4 hrs exposure to DMSO (<b>A</b>) or 1 uM BNF (<b>B</b>). Strong upregulation of <i>cyp1a1</i> in skin can be detected. <i>cyp1b1</i> is also induced in the skin, but to a lesser extent. Numbers indicate embryos with indicated expression pattern/total embryos analyzed. Scale bar is 500 um. (<b>C)</b>. <i>Camera lucida</i> drawing of 48 hpf zebrafish embryo from Kimmel, et al 1995 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183433#pone.0183433.ref036" target="_blank">36</a>]. Dashed lines indicate plane of images to visualize central arteries (CtAs, red) and hindbrain vessels (blue). Confocal image shows dorsal view of hindbrain vasculature of live embryo at 48 hpf (scale bar is 200 um). Cartoon schematic depicts arrangement of hindbrain vessels. <b>(D-I)</b> High magnification images of basal <i>cyp1a1/b1</i> expression at 52 hpf in the hindbrain (<b>D, G</b>) and CtAs (<b>E, H</b>) of the head vasculature in embryos treated with DMSO. Schematics in <b>F,I</b> summarize hindbrain expression patterns of both genes. (<b>J-O)</b> High magnification images of BNF-induced <i>cyp1a1/b1</i> expression at 52 hpf in the hindbrain (<b>J, M</b>) and CtAs (<b>K, N</b>) of the head vasculature in embryos treated with 1 uM BNF. Schematics in <b>L,O</b> summarize hindbrain expression patterns of both genes. Analysis of <i>cyp1a1</i> expression was performed in <i>ahr2</i> mutant embryos to permit visualization of hindbrain vasculature. Yellow arrowheads mark CtAs. Scale bar is 100 um in all high magnification images. Abbreviations–AHR: aryl hydrocarbon receptor, BA: basilar artery, BCA: basal communicating artery, BNF: beta-naphthoflavone, CtA: central artery, CYP: cytochrome p450, DMSO: dimethylsulfoxide, hpf: hours post fertilization, ISH: <i>in situ</i> hybridization, PCS: posterior communicating segments, PHBC: primordial hindbrain channel, WT: wildtype.</p

Mitochondrial impairment and melatonin protection in parkinsonian mice do not depend of inducible or neuronal nitric oxide synthases

<div><p>MPTP-mouse model constitutes a well-known model of neuroinflammation and mitochondrial failure occurring in Parkinson’s disease (PD). Although it has been extensively reported that nitric oxide (NO^●) plays a key role in the pathogenesis of PD, the relative roles of nitric oxide synthase isoforms iNOS and nNOS in the nigrostriatal pathway remains, however, unclear. Here, the participation of iNOS/nNOS isoforms in the mitochondrial dysfunction was analyzed in iNOS and nNOS deficient mice. Our results showed that MPTP increased iNOS activity in substantia nigra and striatum, whereas it sharply reduced complex I activity and mitochondrial bioenergetics in all strains. In the presence of MPTP, mice lacking iNOS showed similar restricted mitochondrial function than wild type or mice lacking nNOS. These results suggest that iNOS-dependent elevated nitric oxide, a major pathological hallmark of neuroinflammation in PD, does not contribute to mitochondrial impairment. Therefore, neuroinflammation and mitochondrial dysregulation seem to act in parallel in the MPTP model of PD. Melatonin administration, with well-reported neuroprotective properties, counteracted these effects, preventing from the drastic changes in mitochondrial oxygen consumption, increased NOS activity and prevented reduced locomotor activity induced by MPTP. The protective effects of melatonin on mitochondria are also independent of its anti-inflammatory properties, but both effects are required for an effective anti-parkinsonian activity of the indoleamine as reported in this study.</p></div

The treatment with MPTP induced a drastic decrease in the activity of the complex I in SN and ST independently of the absence/presence of iNOS/nNOS.

<p>The administration of melatonin counteracted this effect. The graphs show the changes in the activity of complex I in SN and ST of iNOS^+/+ (above left) and iNOS^-/- (above right) and SN and ST of nNOS^+/+ (down left) and nNOS^-/- (down right). Mean ± SD of 6 animals per group, triplicated; *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ****<i>P</i> < 0.0001 <i>vs</i>. control; <i>P</i> < 0.05 ^## and ^####<i>P</i> < 0.0001 <i>vs</i>. MPTP; ^ΦΦ<i>P</i> < 0.05 and ^ΦΦΦΦ<i>P</i> < 0.0001 <i>vs</i>. basal complex I in SN.</p

Values of the ADP:O ratio in ST and SN mitochondria.

<p>Values of the ADP:O ratio in ST and SN mitochondria.</p