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

Abstract

<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