The intestinal module in3.4 recapitulates microbial suppression of <i>angptl4</i>.

<p>(A) Semi-quantitative whole mount <i>in situ</i> hybridization of <i>angptl4</i> mRNA in 6 dpf germ-free (GF) and conventionalized (CONVD) animals. Arrowheads mark intestinal expression. Note that the background staining in the gills (arrows) is similar in GF and CONVD fish. Transverse sections show that microbial suppression of <i>angptl4</i> mRNA is specific to the intestinal epithelium. (B) Quantitative RT-PCR of <i>angptl4</i> and <i>GFP</i> mRNA levels in 6 dpf GF and CONVD <i>Tg(in3.4-Mmu.Fos:GFP)</i> animals. GF and CONVD animals were derived from the same <i>Tg(in3.4-Mmu.Fos:GFP)</i> stable line. <i>GFP</i> and <i>angptl4</i> mRNA were normalized to <i>18S</i> rRNA levels and are shown as fold difference compared to GF controls averaged across 3 experimental replicates ± SEM (2 biological replicate groups of 10 larvae per condition per experiment). Similar results were attained when normalized to <i>ribosomal protein L32</i> (<i>rpl32</i>) rRNA levels. Asterisks denote P-value<.01 from unpaired T-test between GF and CONVD conditions for each gene. See also Figure S8.</p

Functional evolution of the islet and intestinal regulatory modules in 12 fish species.

<p>(A) Unscaled phylogram based on information from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585-Hedges1" target="_blank">[58]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585-Peng1" target="_blank">[59]</a> showing images and relative relationships of 12 fish for which intronic sequences were analyzed. <i>Danio rerio</i> (<i>Dr</i>, zebrafish), <i>Danio nigrofasciatus</i> (<i>Dn</i>), <i>Danio albolineatus</i> (<i>Dalb</i>), <i>Danio choprae</i> (<i>Dc</i>), <i>Danio feegradei</i> (<i>Df</i>), <i>Devario aequipinnatus</i> (<i>Daeq</i>, giant danio), <i>Carassius auratus</i> (<i>Ca</i>, goldfish), <i>Cyprinus carpio</i> (<i>Cc</i>, carp), <i>Puntius conchonius</i> (<i>Pc</i>, rosy barb), <i>Chromobotia macracanthus</i> (<i>Cm</i>, clown loach), <i>Ictalurus punctatus</i> (<i>Ip</i>, channel catfish), <i>Oryzias latipes</i> (<i>Ol</i>, medaka). (B) VISTA plot displaying the global pairwise alignment of orthologous in3.2 regions from each species anchored to zebrafish (<i>Dr</i>) in3.2. Orange peaks correspond to regions in the alignment that correspond to <i>Dr</i> in3.3 (islet module). Blue peaks correspond to regions in the alignment that correspond to <i>Dr</i> in3.4 (intestine module). Percent identity is calculated from pairwise alignments of each module with zebrafish (VISTA parameters: 25 bp sliding window, LAGAN alignment). (C) Representative islet and intestinal images from injections of each orthologous in3.2 module. Orange or blue arrowheads mark positive islet or intestine expression, respectively. The absence of arrowheads denotes negative expression in each tissue. (D) Summary of mosaic expression for each species. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown. Orange or blue (+) denotes that the construct was sufficient to confer expression in the islet or intestine, respectively. Black (−) denotes insufficiency. Note that <i>Dalb</i> and <i>Cm</i> sequences were not tested (nt) in this heterologous functional assay. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s005" target="_blank">Figures S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s006" target="_blank">S6</a>.</p

Tissue-specific expression of zebrafish <i>angptl4</i> mRNA.

<p>(A) Distance phylogram of Angptl4 protein from zebrafish (<i>Dr</i>, <i>Danio rerio</i>), catfish (<i>Ip</i>, <i>Ictalurus punctatus</i>), medaka (<i>Ol</i>, <i>Oryzias latipes</i>), tetraodon (<i>Tn</i>, <i>Tetraodoan nigroviridis</i>), fugu (<i>Tr</i>, <i>Takifugu rubipres</i>), xenopus (<i>Xt</i>, <i>Xenopus tropicalis</i>), chicken (<i>Gg</i>, <i>Gallus gallus</i>), mouse (<i>Mm</i>, Mus <i>musculus</i>), human (<i>Hs</i>, <i>Homo sapiens</i>), dog (<i>Cf</i>, <i>Canis familiaris</i>), pig (<i>Ss</i>, <i>Sus scrofa</i>), cow (<i>Bt</i>, <i>Bos taurus</i>). All nodes are significant (>700/1000 bootstrap replicates) except those marked with an asterisk (*). Scale bar indicates phylogenetic distance, in number of amino acid substitutions per site. We found that the genomes of zebrafish, channel catfish (<i>Ictaluris punctatus</i>), and medaka (<i>Oryzias latipes</i>) encode a single ortholog of mammalian Angptl4, whereas two pufferfish species (<i>Takifugu rubripes</i> and <i>Tetraodon nigroviridis</i>) encode two Angptl4 paralogs. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s001" target="_blank">Figure S1</a>. (B–G) Whole-mount <i>in situ</i> hybridization (WISH) using a riboprobe targeting <i>angptl4</i> mRNA during various stages in zebrafish development reveals dynamic spatiotemporal gene expression patterns. (B) At 1 day post fertilization (dpf) embryos exhibit ubiquitous expression of <i>angptl4</i>. (C–D) By 4 dpf, marked expression is observed in the intestinal epithelium (in, black arrowhead), but by 6 dpf, robust expression becomes largely localized to the intestine (black arrowhead) and pancreatic islet (not shown). The black arrow marks the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). Scale bars = 500 µm. (E–F) Transverse sections of 6 dpf and 8 dpf animals confirm expression in the intestinal epithelium (E, in, black arrowhead) and pancreatic islet (F, is, black triangle). Scale bars = 50 µm. (G–H) At 17 dpf, strong expression is observed in the liver (li, white arrowhead, dotted line outlines the liver). G, Scale bar = 250 µm; H, Scale bar = 50 µm.</p

Summary of functional conservation and mapping of islet and intestinal regulatory information.

<p>(A) Conservation plots, module truncations, and predicted transcription factor binding sites (TFBS) in islet CRM in3.3 are overlayed and annotated to scale. The grey shaded box represents the region that is present in all positive truncations and has strong conservation in islet-positive species. (B) Conservation plots, module truncations, SDM data, and predicted transcription factor binding sites (TFBS) in intestinal CRM in3.4 are overlayed and annotated to scale. Two grey shaded boxes represent regions that are present in all positive truncations, are required for intestinal expression, and have strong conservation in intestine-positive species. Dotted boxes in panels A and B represent highly conserved regions from each (A) islet-positive or (B) intestine-positive species used to predict common TFBS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s005" target="_blank">Figures S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#pgen.1002585.s006" target="_blank">S6</a>, and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#s4" target="_blank">Materials and Methods</a>).</p

Multiple-species alignments reveal conservation in <i>angptl4</i> gene structure and location of conserved non-coding regions.

<p>(A) VISTA plot displaying the global pairwise alignment of the zebrafish <i>angptl4</i> locus with the orthologous medaka, tetraodon, and fugu regions and (B) human <i>ANGPTL4</i> locus with the orthologous mouse and dog regions. Purple conservation peaks correspond to exonic sequences, and green conservation peaks represent non-coding sequences. The zebrafish and human gene structure are denoted by purple boxes above the corresponding VISTA plot (VISTA parameters: 100 bp sliding window, LAGAN alignment). Note that the concentration of conservation peaks within intron 3 of both teleost and mammalian <i>angptl4</i> genes.</p

Truncation mapping of the islet and intestinal regulatory module.

<p>(A) Scaled schematic of the zebrafish <i>angptl4</i> locus showing annotations of truncations assayed for regulatory potential. Orange lines indicate sufficiency to confer islet expression, blue lines indicate sufficiency to confer intestinal expression, and black lines indicate insufficiency in intestine and islet. Dashed blue lines indicate reduced intestinal expression compared to in3.4. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (B) Representative images of islet views from mosaic injected fish of each truncation construct. Orange arrows mark islet expression (is). Scale bars = 100 µm. (C) Representative images of intestinal views from mosaic fish injected with each truncation construct. Blue arrows mark intestinal expression (in). Scale bars = 100 µm. (D) Relative mean intestinal fluorescence within the intestine was quantified in mosaic animals (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#s4" target="_blank">Materials and Methods</a>) and plotted per injected fish. Circles represent mean fluorescence averaged for three mosaic patches within one fish, and are colored blue or black to designate truncations that are sufficient or insufficient to confer intestinal expression, respectively. Statistical significance was tested using Kruskal-Wallis one-way analysis of variance (labels: a = P<.001, b = P<.05 vs. <i>Fos</i>; c = P<.001, d = P<.01 vs. in3.4). Scale bars = 100 µm.</p

Site-directed mutagenesis defines DNA motifs required for intestinal expression.

<p>(A) Scaled schematic showing 10 bp substitution blocks tiled across the zebrafish <i>angptl4</i> in3.11 region within the context of the entire in3.4 intestinal module. Black or blue blocks represent mutations that do or do not significantly alter intestinal expression compared to wild type in3.4, respectively (see below). Ratios of intestine positive fish versus total fish expressing GFP are shown in parentheses above or below substitution block labels. (B) Relative mean intestinal fluorescence was quantified in mosaic animals (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002585#s4" target="_blank">Materials and Methods</a>) and plotted per injected fish. Circles represent mean fluorescence averaged for three mosaic patches within a single fish and are colored blue or black to designate mutations that do or do not confer intestinal expression, respectively. Statistical significance was tested using Kruskal-Wallis one-way analysis of variance (labels: a = P<.01 vs. in3.4, P>.05 vs. <i>Fos</i>; b = P>.05 vs. <i>Fos</i>; unlabeled = P>.05 vs. in3.4, P<.01 vs. <i>Fos</i>). (C) Images from animals with mosaic expression of five representative mutant constructs are shown. Blue arrows indicate intestinal expression (in). Scale bars = 100 µm.</p

Non-overlapping regulatory modules within <i>angptl4</i> intron 3 confer liver, islet, and enterocyte-specific reporter expression.

<p>(A) Depiction of the 6 dpf zebrafish showing liver (li, green), intestine (in, blue), swim bladder (sb, grey), and muscle (mu, grey), with the fish oriented anterior (a) to the left and posterior (p) to the right. The opposite orientation reveals the exocrine pancreas (pa, yellow) and islet (is, orange). (B) Scaled schematic of the zebrafish <i>angptl4</i> locus and non-coding DNA assayed for regulatory potential. Modules are color coded according to the tissues in which they confer expression. Ratios of islet or intestine positive fish versus total fish expressing gfp are shown in parentheses next to truncation labels. (C–N) Representative images of GFP reporter expression in mosaic (column 1) and F₁ stable (column 2) animals driven by each non-coding DNA region (rows). Scale bars = 100 µm; li = liver, is = islet, in = intestine, sb = swim bladder. Colored arrowheads indicate tissue with specific reporter expression. (C–D) Full-length intron 3 (in3; 2,136 bp) is sufficient to promote expression of the reporter in the liver, islet (D, inset, scale bar = 50 µm), and intestine. (E–F) Truncation in3.1 (1,219 bp) confers expression in the liver. (G–H) Truncation in3.2 (701 bp) confers expression in both the intestine and islet (H, inset). Inset scale bar = 50 µm. (I–J) Truncation in3.3 (387 bp) confers islet expression. A transverse section (inset, J) reveals islet expression (nuclei stained with DAPI). Inset scale bar = 50 µm. (K–L) Truncation in3.4 (316 bp) confers intestinal expression. Insets in panels K and L contain transverse sections showing expression localized to the intestinal epithelium (nuclei stained with DAPI). Inset scale bar = 25 µm. The dotted lines in panels D, G, H, and I outline the pancreas. The white arrows in panels H, K, and L mark the boundary between the anterior intestine (segment 1) and mid-intestine (segment 2). (M–N) Cells expressing GFP driven by the in3.4 regulatory module colocalize with a marker (4E8, red, white arrow) of the brush border of absorptive enterocytes, but fail to co-localize with marker for secretory cells (2F11, red, asterisk). Nuclei stained with DAPI. Scale bars = 5 µm. (O) Intercross of <i>Tg(in3.2-Mmu.Fos:tdTomato)</i> with β-cell specific reporter line (<i>Tg(ins:CFP-NTR)^s892</i>) show colocalization of tdTomato and CFP in the islet. Scale bars = 10 µm. (P) Quantitative PCR shows that the in3.4 module and the <i>angptl4</i> promoter (TATA box), but not the in3.3 module, are hypersensitive to DNase I cleavage in intestinal epithelial cells isolated from adult zebrafish. Asterisks denote P-value<.01 from unpaired T-tests between TATA box or in3.4 and in3.3 regions. Error bars represent standard deviation from four biological replicates using cells pooled from 3 wild-type adult zebrafish per replicate.</p