Dynamic Expression of Notch Components in the IM and Pronephric Duct

<div><p>(A–D) <i>notch1a</i> (A) and <i>notch3</i> (C) are expressed in the IM at 10 ss. <i>notch1a</i> (B) is expressed in the distal duct region from somite 10 to 14 (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030018#pgen-0030018-sg003" target="_blank">Figure S3</a>B) at 18 ss, and <i>notch3</i> (D) is expressed in the whole duct from somite 3 to 20 at 24 hpf as indicated by the arrows.</p><p>(E–G) <i>jagged2a</i> expression in the IM appears gradually from anterior to posterior from 5 ss (E) (as indicated by the arrow) to 10 ss (F), and reaches the posterior by 15 ss (G).</p><p>(H–K) <i>jagged2a</i> expression is higher in some cells (arrows point to these cells in [I], which is magnified from [H]) than in neighboring cells in the distal duct at 17 ss (H and I), and transcription is limited to individual cells from 20 ss (J), to 24 hpf (K), to at least 36 hpf (unpublished data) in the demarcated region from somite 8 to 14 (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030018#pgen-0030018-sg003" target="_blank">Figure S3</a>D and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030018#pgen-0030018-sg003" target="_blank">S3</a>F) as indicated by arrows.</p><p>(L and M) <i>her9</i> is expressed in the distal pronephric duct at 18 ss (L) from somite 10 to 12 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030018#pgen-0030018-sg003" target="_blank">Figure S3</a>H) and at 21 hpf (M). The arrowhead marks the glomerulus, and arrows demarcate the <i>her9</i> expression region. Left and right inserts in (M) are the magnified images in the glomerulus and distal duct, respectively.</p><p>All embryos, anterior to the left. (A), (C), and (E–I) are dorsal views; the rest are lateral views. Bar scale: 200 μm (A, C, and E), 110 μm (B and D), 180 μm (F), 230 μm (G and H), 90 μm (I), 115 μm (J), 190 μm (K), and 100 μm (L and M).</p></div

Multi-Cilia Cells Start to Differentiate as a Result of Jagged2a-Notch Signaling from 17 ss Onwards

<div><p>(A–C) <i>rfx2</i> expression in (A) wt embryos, (B) <i>mib^ta52b</i> mutants, and (C) <i>jagged2a-sp</i> morphants at 15 ss.</p><p>(D–F) <i>rfx2</i> expression in (D) wt embryos, (E) <i>mib^ta52b</i> mutants, and (F) <i>jagged2a-sp</i> morphants at 18 ss.</p><p>Arrows point to the <i>rfx2</i> expression in the (A–C) IM and (D–F) pronephric duct. <i>rfx2</i> staining in the neural tube (arrowheads) indicates the neurogenic phenotype in (B and E) <i>mib^ta52b</i> mutants compared to that of (A and D) wt embryos.</p><p>All embryos, anterior to the left. (A–C) are dorsal views; (D–F) are lateral views. Bar scale: 200 μm (A–C) and 100 μm (D–F).</p></div

Model for the Role of Jagged2a/Notch Signaling during Differentiation of Multi-Cilia Cells and Principal Cells in the Zebrafish Distal Pronephric Duct

<p>Two adjacent cells of the developing pronephric duct are shown. The left cell, the winner in the lateral inhibition competition, differentiates as a multi-cilia cell (with cilia tuft and <i>rfx2</i> and <i>centrin2</i> expression, etc.), whereas the right cell is consequently inhibited and differentiates as a principal cell (with primary cilium and Na⁺, K⁺ ATPase expression). Activated components of the feedback regulatory system are highlighted in red, and inactive components are outlined in gray. Jagged2a is the sole ligand, Notch1a and Notch3 are two redundant receptors, and Her9 is one of the effectors that works downstream of the Notch receptors to prevent generating excessive multi-cilia cells at the expense of the principal cells. In this model, Mib affects Notch activity by interacting with Jagged2a and facilitating Jagged2a endocytosis in order to signal to neighboring cells. Procilia genes are hypothetical and have not been identified. In a manner similar to that of the proneural genes, procilia genes could encode bHLH transcription factors, stimulating expression of <i>jagged2a</i> and terminal differentiation (ciliogenesis) genes. Other components are mentioned in the text.</p

Multi-Cilia Cell Hyperplasia Is Due to Mib-Mediated Jagged2a Signaling Pathway via Notch1a and Notch3 Receptors

<div><p>(A) Effectiveness of splicing <i>jagged2a-sp</i> MO. RT-PCR of control embryos generates a 230-bp <i>jagged2a</i> fragment, bridging parts of exon 1 and exon 2 at 24 hpf (lane 1) and 48 hpf (lane 5). <i>jagged2a</i>-<i>sp</i> MO-injected embryos analyzed with the same primers at the same timepoints (lanes 3 and 7) show a larger amplicon of 708 bp caused by a nonsplicing intron 1, which encodes a premature stop codon. Lane 9 shows the amplicon from genomic DNA, and lane 10 shows the amplicon from <i>jagged2a</i> cDNA. No fragment can be amplified in the RT-PCR without reverse transcriptase in 24-hpf (lane 2) or 48-hpf (lane 6) wt embryos or in 24-hpf (lane 4) or 48-hpf (lane 8) <i>jagged2a-sp</i> MO-injected embryos. Lane L: 100-bp ladder.</p><p>(B) Pronephric duct (arrow) integrity is not affected in <i>jagged2a</i> morphants.</p><p>Panels C–L focus on the duct between somite 10 and 13.</p><p>(C–H) Multi-cilia cell number is increased in (D and F) <i>jagged2a-atg</i> morphants compared to (C and E) wt embryos as shown by (C and D) <i>rfx2</i> and (E and F) <i>centrin2</i> expression at 24 hpf, but principal cell number is decreased in (H) <i>jagged2a-atg</i> morphants compared to (G) wt embryos as revealed by <i>Na⁺, K⁺ ATPase β1a</i> expression at 24 hpf.</p><p>(I–L) Multi-cilia cell number is increased in (J) <i>mib^ta52b</i> embryos compared to (I) wt embryos as shown by <i>rfx2</i> expression at 24 hpf, but principal cell number is decreased in (L) <i>mib^ta52b</i> embryos compared to (K) wt embryos as revealed by <i>Na⁺, K⁺ ATPase α1a2</i> expression at 24 hpf.</p><p>Panels M–R focus on the duct around somite 11 to 13.</p><p>(M–O) Fluorescent double in situ hybridization of <i>rfx2</i> (green) and <i>Na⁺, K⁺ ATPase β1a</i> (red) in 36-hpf (M) wt embryos, (N) <i>jagged2a-sp</i> morphants, and (O) <i>mib^ta52b</i> mutants shows multi-cilia cell hyperplasia in <i>jagged2a</i> morphants and <i>mib^ta52b</i> mutants. Arrows point to the <i>rfx2</i>-expressing cells in the duct of (M) wt embryos; arrowheads point to the <i>Na⁺, K⁺ ATPase β1a</i>-expressing cells in the pronephric duct of (N) <i>jagged2a-sp</i> morphants.</p><p>(P–R) Double immunohistochemistry of α6F (green) and Pcm1 (red) in 36-hpf (P) wt embryos, (Q) <i>jagged2a-sp</i> morphants, and (R) <i>mib^ta52b</i> mutants shows multi-cilia cell hyperplasia in <i>jagged2a</i> morphants and <i>mib^ta52b</i> mutants. Arrows point to the Pcm1 staining in the pronephric duct of (P) wt embryos; arrowheads point to α6F staining in the pronephric duct of (Q) <i>jagged2a-sp</i> morphants.</p><p>(S) Immunoprecipitation of Myc-Jagged2a and Myc-Jagged2a^icd by Flag-Mib^ta52b. IP, immunoprecipitation; IB, immunoblotting.</p><p>(T–U) Expression of Myc-Jagged2a (T) and cotransfection of Myc-Jagged2a and Flag-Mib (U) in COS7 cells. </p><p>(V–Y) Compared to (V) wt embryos, mild cilia cell hyperplasia is observed in (W) <i>notch1a (des^th35b</i>) mutants and (X) <i>notch3-utr</i> morphants, while severe cilia cell hyperplasia is observed in (Y) <i>notch3-utr</i> MO-injected <i>notch1a (des^th35b)</i> mutants as shown by <i>rfx2</i> expression at 24 hpf.</p><p>All embryos, anterior to the left. Bar scale: 100 μm (B), 75 μm (C–L and V–Y), 50 μm (M–R), and 30 μm (T and U).</p></div

<i>jagged2a</i>-Expressing Cells in the Pronephric Duct are Multi-Cilia Cells

<div><p>(A–D) <i>rfx2</i> is expressed uniformly in the IM at (A) 15 ss, and expression is restricted to individual cells in the pronephric duct from (B) 17 ss onwards to (C) 20 ss and (D) 24 hpf. Arrows demarcate the distal duct region that contains <i>rfx2</i>-expressing cells.</p><p>(E–J) Fluorescent double in situ hybridization of (E and H) <i>jagged2a</i> and (F and I) <i>rfx2</i> revealed that they are (G and J) colocalized in the distal pronephric duct of (E–G) 17-ss and (H–J) 22-hpf embryos. Arrows point to the cells that express <i>jagged2a</i> and <i>rfx2,</i> and arrowhead points to the cells that express <i>jagged2a</i> only.</p><p>(K–M) Fluorescent double in situ hybridization of (K) <i>rfx2</i> and (L) <i>her9</i> revealed that they are expressed in the (M) alternate cells in the distal pronephric duct of 20-ss embryos. Arrowheads point to the <i>rfx2</i>-expressing cells and arrows point to the <i>her9</i>-expressing cells<i>.</i></p><p>All embryos, anterior to the left. (A) and (B) are dorsal views; (C–M) are lateral views. Bar scale: 135 μm (A), 150 μm (B), 100 μm (C), 120 μm (D), and 50 μm (E–M).</p></div

<i>her9</i> is a Downstream Target Gene of Jagged2a-Notch1a/Notch3 Signaling

<div><p>(A and B) Compared to (A) wt embryos, <i>her9</i> expression in the pronephric duct region at 18 ss is severely down-regulated in (B) <i>jagged2a-sp</i> morphants.</p><p>(C–F) Compared to (C) wt embryos, <i>her9</i> expression in the pronephric duct region at 17 ss is mildly down-regulated in (D) <i>notch1a</i> (<i>des^th35b</i>) mutants and (E) <i>notch3-sp</i> morphants, and is severely down-regulated in (F) <i>notch3-sp</i> MO-injected <i>notch1a</i> (<i>des^th35b</i>) mutants.</p><p>(G and H) Compared to (G) wt embryos, <i>her9</i> expression in the pronephric duct region at 18 ss is severely down-regulated in (H) <i>mib^ta52b</i> mutants.</p><p>(I and J) Coinjection of GFP mRNA (50 pg) and <i>notch1a^icd</i> mRNA (100 pg) into one blastomere at the two-cell stage leads to (I) somite boundary disruption in the right half of the embryo, while somites on the left side are segmented properly. (J) GFP expression demonstrates that mRNA is localized to the right half of the embryo.</p><p>(K) Compared to the left side of the embryo, <i>her9</i> expression in the duct (arrows) and glomerulus (arrowheads) is increased in the right side at 18 ss.</p><p>(L and M) Compared to (L) wt embryos, the multi-cilia cell number is increased in (M) <i>her9-utr</i> morphants as shown by <i>rfx2</i> expression at 24 hpf.</p><p>All embryos, anterior to the left. (A–K) are dorsal views; (L and M) are lateral views. Bar scale: 100 μm (A–J), 130 μm (K), and 50 μm (L and M).</p></div

Risk factors of adverse cardiac events.

<p>CI: confidence interval. NA: the odds ratio was not applicable due to the zero count.</p>*<p>Indicates a significant influence on the occurrence of an adverse cardiac event.</p

Characteristics of patients with and without adverse cardiac events.

<p>Data are presented as mean ± standard deviation or number (percentage).</p

cTnI levels in the various cardiac adverse event groups.

<p>Data were presented as count of positive cTnI and positive rate of cTnI.</p>*<p>Indicates a significant difference as compared to acute myocardial ischemia group.</p>†<p>Indicates a significant difference as compared to cardiac arrhythmia group.</p

Changes of cTnI, HIF-1α and HO-1 during the perioperative period.

<p>The cTnI positive rate (A) and serum HIF-1α (B) and HO-1 (C) levels at the 4 time points (baseline, 30 min after surgery, 48 h after surgery, and 72 h after surgery) from patients with or without adverse cardiac events. *Indicates a significant difference between those with and without an adverse cardiac event. (A) Data were presented as percentage and 95% confidence interval. (B, C) Data were presented as mean and standard deviation.</p