Cyp26 Enzymes Facilitate Second Heart Field Progenitor Addition and Maintenance of Ventricular Integrity

<div><p>Although retinoic acid (RA) teratogenicity has been investigated for decades, the mechanisms underlying RA-induced outflow tract (OFT) malformations are not understood. Here, we show zebrafish embryos deficient for Cyp26a1 and Cyp26c1 enzymes, which promote RA degradation, have OFT defects resulting from two mechanisms: first, a failure of second heart field (SHF) progenitors to join the OFT, instead contributing to the pharyngeal arch arteries (PAAs), and second, a loss of first heart field (FHF) ventricular cardiomyocytes due to disrupted cell polarity and extrusion from the heart tube. Molecularly, excess RA signaling negatively regulates <i>fibroblast growth factor 8a</i> (<i>fgf8a</i>) expression and positively regulates <i>matrix metalloproteinase 9</i> (<i>mmp9</i>) expression. Although restoring Fibroblast growth factor (FGF) signaling can partially rescue SHF addition in Cyp26 deficient embryos, attenuating matrix metalloproteinase (MMP) function can rescue both ventricular SHF addition and FHF integrity. These novel findings indicate a primary effect of RA-induced OFT defects is disruption of the extracellular environment, which compromises both SHF recruitment and FHF ventricular integrity.</p></div

Restoring FGF signaling rescues SHF addition but not ventricular integrity.

<p>(A,B) Graphs indicating fold difference of mRNA relative to <i>β-actin</i> assayed with RT-qPCR of <i>fgf8a</i> expression in whole embryos and isolated hearts at 48 hpf. (C) Graph depicting quantification of ventricular addition to the OFT (control <i>n</i> = 8, CA-Fgfr <i>n</i> = 6, Cyp26 deficient <i>n</i> = 10, Cyp26 deficient+CA-Fgfr <i>n</i> = 6). (D) Graph of ventricular cardiomyocyte counts at 48 and 72 hpf (<i>n</i> = 10 per group). (E–H) Confocal images of optical slices from hearts of <i>Tg(myl7</i>:<i>Kaede)</i> and <i>Tg(myl7</i>:<i>Kaede);Tg(hsp70</i>:<i>ca-fgfr1)</i> embryos at 48 hpf after heat-shock at 24 hpf and photoconversion at 36 hpf. Brackets indicate added ventricular cells (green only). (I–L) IHC for hearts from <i>Tg</i>(<i>myl7</i>:<i>DsRed-NLS</i>) and <i>Tg</i>(<i>myl7</i>:<i>DsRed-NLS</i>);<i>Tg(hsp70</i>:<i>ca-fgfr1)</i> at 48 hpf after heat-shock at 24 hpf. Arrows indicate ectopic cardiomyocytes. Error bars are SEM, asterisk denotes <i>p</i> < 0.05 by Student’s <i>t</i> test. Frontal views with anterior up (E–L); <i>n</i> > 20 embryos per group (E–L). Scale bar: 50 μm.</p

Ventricular cardiomyocytes can exit the heart tube in Cyp26-deficient embryos.

<p>(A,B) 30-min interval frames from confocal time-lapse movies of control and Cyp26-deficient <i>Tg(myl7</i>:<i>EGFP)</i> hearts. Images of hearts were depth-coded with the spectrum ranging from pink at 120 μm to blue at 0 μm. (C,D) Hematoxylin–eosin (HE) stained frontal sections of the hearts from control and Cyp26-deficient embryos. Endocardium (white arrowheads) and myocardium (black arrowheads). Control hearts <i>n</i> = 4. Cyp26-deficient hearts <i>n</i> = 5. (E,F) Control and Cyp26-deficient <i>Tg(myl7</i>:<i>EGFP);Tg</i>(<i>kdrl</i>:<i>mCherry</i>) embryos at 48 hpf. Endocardium (red) and myocardium (green). Arrowheads indicate the inner border of endocardial and outer border of myocardial cells. (G) Graph showing the distance between the endocardial and myocardial layers (control <i>n</i> = 5, Cyp26 deficient <i>n</i> = 7). (H–I”‘) Confocal images of control and Cyp26-deficient <i>Tg(myl7</i>:<i>EGFP)</i> hearts stained for zonula occludens 1 (ZO1) (red) and green fluorescent protein (GFP) (green), with schematized outlines of cell boundaries and ZO1 staining. Arrow denotes cardiomyocyte protruding into the pericardial space. (J) Graph depicting the percentage of ZO1 expression along the height of cardiomyocytes (control <i>n</i> = 15, Cyp26 deficient <i>n</i> = 15). (K) Graph depicting circularity measurement of ventricular cells (control <i>n</i> = 15, Cyp26 deficient <i>n</i> = 30). (L–M”‘) Confocal images of control and Cyp26-deficient <i>Tg(myl7</i>:<i>EGFP)</i> hearts stained for β-catenin (red) and GFP (green), with schematized outlines of cell boundaries and β-catenin staining. Error bars are SEM, asterisk denotes <i>p</i> < 0.05 by Student’s <i>t</i> test. Frontal views, anterior up (A–F,H,I,L,M); <i>n</i> > 20 embryos per group (E,F,H,I,L,M). A, apical; B, basal. Scale bar for C–M’: 50 μm. Scale bar for H”–M”‘: 25 μm.</p

SHF progenitors do not add to the heart tube in Cyp26-deficient embryos.

<p>(A–H) ISH of control and Cyp26-deficient <i>Tg</i>(<i>nkx2</i>.<i>5</i>:<i>ZsYellow</i>) embryos at 24 (A,E), 30 (B,F), 36 (C,G), and 48 (D,H) hpf. <i>Nkx2</i>.<i>5</i>:<i>zsyellow</i> (red) and <i>myl7</i> (purple). (A–C,E–G) Brackets indicate <i>nkx2</i>.<i>5</i>:<i>ZsYellow+</i> cells in the first <i>nkx2</i>.<i>5+</i> pharyngeal arch. (F,G) Arrows indicate accumulation of <i>nkx2</i>.<i>5+</i> cells adjacent to the arterial pole of the heart. (I,I’,K,K’) <i>Tg(nkx2</i>.<i>5</i>:<i>Kaede)</i> before and after photoconversion of the anterior lateral population of <i>nkx2</i>.<i>5+</i> cells (bracket) in control and Cyp26-deficient embryos at 24 hpf. (J–J”,L–L”) Position of photoconverted cells (red, arrows) relative to the heart tube (green) in control and Cyp26-deficient embryos at 48 hpf. Images in I–J” and K–L”, respectively, are of the same control and Cyp26-deficient embryos. (M) Schematic depicting the three regions where photoconverted <i>nkx2</i>.<i>5</i>:<i>Kaede+</i> cells contributed at 48 hpf. (N) Graph depicting percentage of contribution to the third and fourth PAAs, non-ventricular OFT, and ventricular cardiomyocytes (control-lateral <i>n</i> = 39, control-adjacent <i>n</i> = 8, Cyp26-deficient–lateral <i>n</i> = 52, Cyp26-deficient–adjacent <i>n</i> = 5). (O,P) Confocal images of arch arteries in <i>Tg(nkx2</i>.<i>5</i>:<i>Kaede; kdrl</i>:<i>nlsEGFP)</i> control and Cyp26-deficient embryos. (Q) Graph depicting quantification of the endothelial cell number in the third and fourth PAAs in control (<i>n</i> = 18) and Cyp26-deficient (<i>n</i> = 24) embryos. Ventral view, anterior up (A–H); dorsal view, anterior up (I,I’,K,K’); lateral view, anterior up (J,J’,J”,L,L’,L”); lateral view, anterior right (O,P). <i>n</i> > 20 per group for (A–H). Asterisk denote <i>p</i> < 0.05 by Chi Squared test. H, heart. Scale bar: 50 μm.</p

Attenuating MMP function restores SHF addition and ventricular integrity.

<p>(A,B) Graphs indicating fold difference of mRNA relative to <i>β-actin</i> assayed with RT-qPCR of <i>mmp9</i> expression in whole embryos and isolated hearts at 48 hpf. (C) Graph showing quantification of ventricular addition to the OFT (control <i>n</i> = 11, GM6001 treated <i>n</i> = 11, Cyp26 deficient <i>n</i> = 12, Cyp26 deficient + GM6001 <i>n</i> = 12). (D) Graph depicting ventricular cardiomyocyte counts at 48 and 72 hpf (<i>n</i> = 10 per group). (E–H) Confocal images of optical slices from hearts of <i>Tg(myl7</i>:<i>Kaede)</i> embryos at 48 hpf after photoconversion at 36 hpf. Brackets indicate ventricular addition (green only). (I–L) IHC of hearts from control and Cyp26-deficient <i>Tg</i>(<i>myl7</i>:<i>DsRed-NLS</i>) embryos after DMSO or GM6001 treatment. Arrows indicate ectopic cardiomyocytes. (M) Graph depicting the percentage of embryos with ectopic cardiomyocytes in Cyp26 deficient treated with DMSO or GM6001 at 48 hpf. (N) Graph depicting average number of ectopic cardiomyocytes (per embryo with ectopic cardiomyocytes) at 48 hpf. (O,P) Confocal images of optical slices through control (lineage tracer alone) or activated MMP9-injected <i>Tg(myl7</i>:<i>EGFP);Tg</i>(<i>kdrl</i>:<i>mCherry</i>) hearts at 48 hpf. Arrowheads denote the inner border of endocardial and outer border of myocardial cells. (Q) Graph depicting the quantification of the distance between the endocardium and myocardium (control <i>n</i> = 12, MMP9 injected <i>n</i> = 10). (R) Graph depicting the percentage of embryos with linearized, dysmoprhic hearts and cells outside the heart (<i>n</i> > 50 per group). (S) Graph indicating fold difference of mRNA relative to <i>β-actin</i> assayed with RT-qPCR of <i>fgf8a</i> expression at 48 hpf in control and Cyp26-deficient embryos treated with DMSO or GM6001. (T) Graph indicating fold difference of mRNA relative to <i>β-actin</i> assayed with RT-qPCR of <i>mmp9</i> expression at 48 hpf after heat-shock induction of CA-Fgfr1. (U) Model of Cyp26 enzyme function in the ventricular OFT development. Red indicates reagents used to inhibit function. Blue indicates reagents used to activate function. Controls in C–E, I, and S were DMSO treated. Controls in O, Q, R indicate Cascade blue-dextran injected alone. Error bars are SEM, asterisks denote <i>p</i> < 0.05 compared to controls by Student’s <i>t</i> test (A–D,N,Q–T), asterisk denotes p<0.05 by Chi Squared test (M). Frontal views, anterior up (E–L,M,N); <i>n</i> > 20 embryos per group (E–L,M,N). CMs, cardiomyocytes. Scale bar: 50 μm.</p

Rdh10a depletion enhances defects in <i>nls</i>/<i>aldh1a2</i> mutant embryos.

<p>(A, D) WT control and Rdh10a deficient embryos. (B,E) Control and Rdh10a deficient embryos heterozygous for the <i>nls</i> allele. (C,F) Control and Rdh10a deficient embryos homozygous for the <i>nls</i> allele. <i>Nls</i> mutant embryos (C) show anteriorization of nervous system (black arrowhead), increases in the size of the head (black bracket), and pericardial edema. Rdh10a deficient; <i>nls</i> mutant embryos (F) have an accentuated anteriorization of the nervous system (arrowhead), larger and dysmorphic head (red bracket), and enhanced pericardial edema compared to <i>nls</i> embryos. Dorsal is up and anterior is to the right.</p

Rdh10a Provides a Conserved Critical Step in the Synthesis of Retinoic Acid during Zebrafish Embryogenesis

<div><p>The first step in the conversion of vitamin A into retinoic acid (RA) in embryos requires retinol dehydrogenases (RDHs). Recent studies have demonstrated that RDH10 is a critical core component of the machinery that produces RA in mouse and <i>Xenopus</i> embryos. If the conservation of Rdh10 function in the production of RA extends to teleost embryos has not been investigated. Here, we report that zebrafish Rdh10a deficient embryos have defects consistent with loss of RA signaling, including anteriorization of the nervous system and enlarged hearts with increased cardiomyocyte number. While knockdown of Rdh10a alone produces relatively mild RA deficient phenotypes, Rdh10a can sensitize embryos to RA deficiency and enhance phenotypes observed when Aldh1a2 function is perturbed. Moreover, excess Rdh10a enhances embryonic sensitivity to retinol, which has relatively mild teratogenic effects compared to retinal and RA treatment. Performing Rdh10a regulatory expression analysis, we also demonstrate that a conserved teleost <i>rdh10a</i> enhancer requires Pax2 sites to drive expression in the eyes of transgenic embryos. Altogether, our results demonstrate that Rdh10a has a conserved requirement in the first step of RA production within vertebrate embryos.</p></div

Summary of enhancer analysis.

<p>Summary of enhancer analysis.</p

Rdh10a deficient embryos have increased CM number.

<p>(A, B) Hearts from control sibling (n = 10) and Rdh10a deficient (n = 10) <i>Tg(-5</i>.<i>1myl7</i>:<i>DsRed2-NLS)</i>^<i>f2</i> embryos. Images are frontal views. Red indicates ventricle. Green indicates atrium. (C) Mean CM number at 48 hpf. (D) RT-qPCR for CM differentiation marker gene expression at 48 hpf.</p

Differential abilities of ROL, RAL and RA to promote RA signaling.

<p>(A-D) EGFP fluorescence in <i>Tg(12XRARE-ef1a</i>:<i>EGFP)</i>^<i>sk72</i> embryos after treatment with 15 μM ROL, 1 μM RAL, and 0.5 μM RA beginning at 24 hpf. (E-H) ISH for <i>egfp</i> expression in <i>Tg(12XRARE-ef1a</i>:<i>EGFP)</i>^<i>sk72</i> embryos after treatment with 15 μM ROL, 1 μM RAL, and 0.5 μM RA beginning at 24 hpf. Arrows indicate the boundaries of expression. Views are lateral with dorsal right and anterior up.</p