Optochemical Dissection of T‑box Gene-Dependent Medial Floor Plate Development

In addition to their cell-autonomous roles in mesoderm development, the zebrafish T-box transcription factors <i>no tail a</i> (<i>ntla</i>) and <i>spadetail</i> (<i>spt</i>/<i>tbx16</i>) are required for medial floor plate (MFP) formation. Posterior MFP cells are completely absent in zebrafish embryos lacking both Ntla and Spt function, and genetic mosaic analyses have shown that the two T-box genes promote MFP development in a non-cell-autonomous manner. On the basis of these observations, it has been proposed that Ntla/Spt-dependent mesoderm-derived signals are required for the induction of posterior but not anterior MFP cells. To investigate the mechanisms by which Ntla and Spt regulate MFP development, we have used photoactivatable caged morpholinos (cMOs) to silence these T-box genes with spatiotemporal control. We find that posterior MFP formation requires Ntla or Spt activity during early gastrulation, specifically in lateral margin-derived cells that converge toward the midline during epiboly and somitogenesis. Nodal signaling-dependent MFP specification is maintained in the absence of Ntla and Spt function; however, midline cells in <i>ntla</i>;<i>spt</i> morphants exhibit aberrant morphogenetic movements, resulting in their anterior mislocalization. Our findings indicate that Ntla and Spt do not differentially regulate MFP induction along the anterior–posterior axis; rather, the T-box genes act redundantly within margin-derived cells to promote the posterior extension of MFP progenitors

Functional imaging of NO in pre-contracted arteries.

<p>3D reconstruction and luminal diameter measured from explanted murine carotid arteries <i>ex </i><i>vivo</i> using Cu ₂FL2E (20 µM) (<b>a</b>) before precontraction (<b>b</b>) after precontraction with NA, (<b>c</b>) in post NA and ACh stimulation (2.5min), error bars indicate s.d. (n=3), (<b>d</b>) luminal diameter measured from arteries with conditions mentioned in a, b and c, error bars indicate s.d. (n=3).</p

Detection of NO produced in explanted murine carotid arteries <i>ex</i><i>vivo</i> using Cu ₂FL2E (20 µM) after precontraction.

<p>(<b>a</b>) Detection of NO in response to NA (ECs and SMCs are not apparent), (<b>b</b>) Detection of NO in post NA and ACh stimulation (2.5min) (ECs and SMCs are apparent), (<b>c</b>) Syto 41 staining of nucleus of ECs and SMCs, (<b>d</b>) plot of fluorescence intensities of the ECs and SMCs (from carotid artery) measured with NA and ACh stimulation for 15min.</p

Detection of NO produced in explanted murine carotid arteries <i>ex</i><i>vivo</i> using Cu ₂FL2E (20 µM).

<p>(<b>a</b>) & (<b>b</b>) Magnified images of vessel showing basal NO signal detected after 5 min incubation of Cu ₂FL2E without any stimulus at medial and intimal focal planes, respectively. (<b>c</b>) NO signal detected in smooth muscle cells (SMCs) and (<b>d</b>) endothelial cells (ECs) of the tissue with 5 min incubation of Cu ₂FL2Eand, subsequently 45min incubation of H₂O₂ (150 µM). Scale bar is 50 µm, (<b>e</b>) & (<b>f</b>) Magnified images of vessel showing NO signal detected after 5 min incubation of Cu ₂FL2E and subsequently, 45 min incubation of H₂O₂ (150 µM) in SMCs at medial plane and in ECs at intimal plane respectively, (<b>g</b>) Quantification of spatial distribution of fluorescence intensity as measure of NO in cells of vessel wall stimulated with H₂O₂ (n = 5). (<b>h</b>) Quantification of spatial distribution of fluorescence intensity as measure of NO in cells of vessel wall stimulated with flow (flow rate= 2.1 Pa, time=45min), (n = 5).</p

Sensitivity and specificity of Cu ₂FL2E.

<p>(<b>a</b>) Fluorescence response of Cu ₂FL2E (2 µM) to various concentrations of NO after 1 min of SNAP administration. n = 5 for each concentration, (<b>b</b>) Linear regression curve plotted from (a), (<b>c</b>) Fluorescence response of Cu ₂FL2E to NO (50 µM SNAP in PBS at 37°C, pH 7.4) and H₂O₂ (150 µM). The spectra were obtained 1 min after SNAP addition n = 5. Error bars indicate s.d., (<b>d</b>) Cytotoxicity assay with different concentrations Cu ₂FL2E.</p

Scheme of NO detection by Cu ₂FL2E in endothelial cells, showing the probe is trapped in the cell via hydrolysis of the pendant ester groups by intracellular esterases to give Cu ₂FL2A since the ester (E) is hydrolyzed to the acid (A).

<p>Scheme of NO detection by Cu ₂FL2E in endothelial cells, showing the probe is trapped in the cell via hydrolysis of the pendant ester groups by intracellular esterases to give Cu ₂FL2A since the ester (E) is hydrolyzed to the acid (A).</p

Detection of NO with Cu ₂FL2E produced by endothelial cells <i>in</i><i>vitro</i>.

<p>(<b>a</b>) NO detection in porcine aortic endothelial cells (PAECs); Left: 45 min incubation of Cu ₂FL2E (20 µM). Right: 45 min incubation of Cu ₂FL2E (20 µM) and H₂O₂ (150 µM). Top: bright-field images of cells. Bottom: fluorescence images of cells. Scale bar is50 µm. (<b>b</b>) Quantification of fluorescence intensity plotted against incubation time. (<b>c</b>) Detection of NO with Cu ₂FL2E in HCAECs cells, with or without NO-inhibitor (L-NAME). Shown are the fluorescence images after 45min co-incubation of the probe (Cu ₂FL2E =2 µM) with H₂O₂ (150 µM), L-NAME (100 µM), and/or ACh (10 µM) according to scheme. Scale bar is 75 µm. (<b>d</b>) Quantification of fluorescence intensity from (c) plotted against each condition mentioned in (c) (n = 5). Error bars indicate s.d.</p

Functional imaging of NO.

<p>(<b>a</b>) 3D reconstruction of vessels with Cu ₂FL2E (20 µM) without/ with stimulus (here ACh), (<b>b</b>) luminal diameter measured from arteries with conditions mentioned in (a), (<b>c</b>) normalized fluorescence intensities of the arteries with conditions mentioned in (a), (<b>d</b>) 3D reconstruction of vessels with Cu ₂FL2E without/ with stimulus (here ACh) and also in combination with L-NAME, (<b>e</b>) luminal diameter measured from arteries with conditions mentioned in (d), (<b>f</b>) normalized fluorescence intensities of the arteries with conditions mentioned in (c), error bars indicate s.d. (n=5).</p