EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel

AbstractDuring vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell–cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation

Selective inhibition of serine protease proteolytic activity by SFTI-FCQR Asn₁₄.

<p>Examination of fibrinogen proteolysis by trypsin and kallikreins by SDS-PAGE. Bands were visualised with Coomassie blue staining after resolving on 10% polyacrylamide gels. Images are representative of three separate experiments. Inhibition of KLK4 proteolytic activity by (A) SFTI-FCQR Asp₁₄ and (B) SFTI-FCQR Asn₁₄. Inhibition of trypsin proteolytic activity by (C) SFTI-1 and (D) SFTI-FCQR Asn₁₄. Inhibition of proteolytic activity of (F) KLK12 and (F) KLK14 by SFTI-FCQR Asn₁₄.</p

Relationship between <i>K</i>_i and number of internal hydrogen bonds.

<p>Plot of the average number of internal (circles), intermolecular (squares) and total (triangles) hydrogen bonds of SFTI-FCQR variants (Asn₁₄, Tyr₁₄, Lys₁₄, Asp₁₄, Gly₁₄, Ala₁₄ and Ser₁₄) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019302#pone-0019302-t001" target="_blank">Table 1</a> versus Morrison <i>K</i>_i values from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019302#pone-0019302-t002" target="_blank">Table 2</a>.</p

Representation of a trypsin/SFTI-1 complex and internal hydrogen bonding within SFTI variants during MD.

<p>Ribbon plot of SFTI-1 in complex with trypsin (A) with β-sheets and α-helices coloured in yellow and blue respectively, excluding SFTI-1 which is displayed in magenta. The residues of the catalytic triad of trypsin and the P1 Lys of SFTI-1 are shown in stick models with carbon in green, nitrogen in blue and oxygen in red. The structure of SFTI variants are shown in ball and stick 2D model with intramolecular hydrogen bond networks for (B) SFTI-1, (C) SFTI-FCQR Asp₁₄ and (D) SFTI-FCQR Asn₁₄. Amino acids are labelled with one letter code and residue number in subscript while the frequency of hydrogen bonds per residue is in brackets (rounded to nearest tenth). Carbons, oxygen, nitrogen and sulphur are represented by gray, red, blue and yellow respectively while hydrogens are excluded for clarity. Bond lengths and angles are intentionally unrealistic to enable easy viewing of hydrogen bonds, represented by dotted green line. Only hydrogen bonds occurring in more than 50% of trajectory frames are shown. Data is represented as mean from three independent 5 ns MD trajectories.</p

<i>In silico</i> Internal Hydrogen Bond Analysis of SFTI-FCQR Residue 14 Variants.

<p><i>In silico</i> Internal Hydrogen Bond Analysis of SFTI-FCQR Residue 14 Variants.</p

Inhibitory Properties of SFTI-1, SFTI-FCQR and SFTI-FCQR Residue 14 Variants.

<p>Amino acids are represented by the one letter code.</p

Bioavailability of SFTI-FCQR Asn₁₄ in mice.

<p>Serum levels of SFTI-FCQR Asn₁₄ administered at 3 mg/kg via the intravenous (IV), intraperitoneal (IP) routes in mice. Serum half life was 25-28 minutes with 10.0±0.8 nM inhibitor serum levels at 4 hours. The data is expressed as mean ± SEM (IV, n = 3; IP, n = 2).</p

RMSD analysis for SFTI variants during MD.

<p>RMSD values between Cα of SFTI-1, SFTI-FCQR Asp₁₄ and SFTI-FCQR Asn₁₄ during MD and the (A) SFTI-1 starting structure or (B) calculated average simulation structures. (C) Ribbon plot showing the average simulation structures coloured according to Cα RMSD from low to high as blue, purple, magenta, orange, and red, labelled with odd residue numbers. Data is represented as mean from three independent 5 ns MD trajectories.</p

Stability of SFTI variants in contact with prostate cancer cells <i>in vitro.</i>

<p>Residual activity of (A) SFTI-FCQR Asn₁₄ and (B) SFTI-FCQR Lys₁₄ in cell culture media from prostate cancer cells treated with a single dose of inhibitor. Endogenous inhibitors were removed by boiling and centrifugation. Stability was assessed against LNCaP (closed circles), 22Rv1 (triangles), and PC3 cells (open circles). Data are mean ± SEM from three experiments in triplicate.</p