6 research outputs found
A model for MpT cell intercalation.
<p>(A) Schematic showing a stage 13 MpT with distal to proximal (D–P) and circumferential (C) coordinates indicated. An asymmetric source of EGF ligand from the distally placed TC (TC) establishes a gradient of EGF pathway activity (red shading) in the distal tubule extending to the kink (k). A small cluster of tubule cells is highlighted. (B) Basal view of this cluster of cells during elongation. Individual cells read differential EGF activity across their D-P axis (higher distal relative to proximal; dashed lines in i), to produce an asymmetric accumulation of Myosin II (green, ii) at the basal, proximal cortex (buff cell). This leads to contraction along the circumferential axis (dashed arrows, ii). The resulting change in cell shape facilitates progressive, small movements (red arrows) between circumferential neighbours (pink and orange cells, iii). Multiple cycles of Myosin II pulses lead to cell intercalation (iv). Asynchronous pulses in a neighbouring cell (cyan), contracts its circumferential axis (black arrows, iii) facilitating intercalation of the buff cell, and producing a change in cell shape to initiate another cell intercalation event between the orange and grey cells (red arrows).</p
Convergent-extension movements drive MpT elongation.
<p>(A) Cartoon depicting embryonic MpT morphogenesis. At stage 13, MpTs are short, thick single-layered epithelial tubes with up to 12 cells surrounding the central lumen. Over 5–6 hours, the tubules elongate by cell rearrangement until just two cells surround the lumen. (B) Diagram showing the distal (red arrow) and proximal (blue arrow) aMpT regions, with the TC (pink) at the distal end. Arrowhead indicates the “kink” region. A-P, anterior-posterior; D-V, dorso-ventral embryonic axes. (C) Time-lapse sequence showing the morphogenesis of a <i>ctB>Stinger::dsRed</i> aMpT (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s008" target="_blank">Movie S1</a>). (D) Transverse sections of embryonic aMpTs stained with anti-FasII (stages 13–16). Sections taken from the distal region show that the number of cells surrounding the lumen decreases from 12 to 2. (E) MpTs increase in length from 82.1±0.8 µm at stage 13 to 330.6±24.2 µm 5 hours later (stage 16) (<i>n</i> = 4 aMpTs from four different embryos; ± standard error of the mean [SEM]). (F) Left panels, stills from live imaged <i>ctB>Stinger::dsRed</i> aMpT (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s009" target="_blank">Movie S2</a>) with 4-D reconstructions of distal region (right panels). Positions of tubule nuclei are represented as spheres. Circumferential rings of cells (black/white) rearrange into proximo-distally adjacent groups of cells by the end of tubule elongation. (G–G″) rotation of the tubule through 90° demonstrates the circumferential arrangement (two rings of cells, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s010" target="_blank">Movie S3</a>). Star, TC. (H) 4-D reconstruction of a few distal cells in the top plane of an aMpT. Arrows indicate a cell (green) that intercalates between yellow and red neighbours. (I). Cartoon of MpT epithelium as a 2-D sheet. As the tubule elongates in the distal-proximal axis, circumferential neighbours intercalate.</p
Polarised basal Myosin II pulses drive circumferential movement of MpT cells.
<p>(A–C) Stage 16 MpTs stained for Cut (green) and Baz (red) in control (A), <i>ctB>UAS-YFP-Zip<sup>DN</sup></i> (B), or <i>ctB>UAS-Sqh<sup>E20E21</sup></i> (C) embryos. Insets in (A–C) show cross-sections through tubules. (D) Basal view of ∼ten cells in the distal tubule region (dashed lines), showing Myosin II/Sqh::GFP (white) from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s016" target="_blank">Movie S9</a>. Distal is to the right. (D′) Time-lapse images of the single cell arrowed in (D) (blue dashed lines) showing Myosin II (arrowhead) accumulating in dynamic pulses localised asymmetrically to the proximal cortex. Inter-pulse periods lack discernable Sqh::GFP accumulation (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s017" target="_blank">Movie S10</a>). (E, F) Still images from control (E, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s018" target="_blank">Movie S11</a>) or <i>EGFR<sup>DN</sup></i> (F) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s019" target="_blank">Movie S12</a>) tubules (Myosin II/Sqh::mCherry, green; cell membranes, magenta). Proximal Myosin II pulses are absent in <i>EGFR<sup>DN</sup></i> tubules, only static puncta of Myosin II are seen (F, dashed arrow). (G) Basal area of a control (green), <i>EGFR<sup>DN</sup></i> (red) and YFP-<i>Zip<sup>DN</sup></i> (blue) MpT cell over time. Basal area and circumferential length fluctuate widely in control compared to <i>EGFR<sup>DN</sup></i> and YFP-<i>Zip<sup>DN</sup></i> cells. 1.0 represents normalised average area. (H) Histogram of the coordinates of MpT cell shape in relation to Myosin II pulses. Normalised length of D-P and circumferential cell axes (<i>n</i> = 15 pulses and 25 interpulses in 10 aMpT cells). While D-P length shows no significant difference between pulse (green) and interpulse (red) periods (<i>p</i> = 0.12>0.05), circumferential length decreases significantly during pulses (<i>p</i> = 0.02<0.05). 1.0 (dashed line) is the normalised average D-P or circumferential length; error bars ± standard deviation (SD). (I) Polar plot showing displacement of single cell centroids in control (green circles, <i>n</i> = 10 cells) and <i>EGFR<sup>DN</sup></i> (orange circles, <i>n</i> = 6 cells) MpTs. Tubule D-P axis 0°–180°, circumferential axis 90°–270°. Radial rings indicate speed of cell movement (µm min<sup>−1</sup>) and central open circle marks the starting position for all cells. Control cell movement is biased along the circumferential axis (shaded green). <i>EGFR<sup>DN</sup></i> cells move very little and their final position remains aligned with the D-P axis. (J) Cartoon of cell-shape changes and movement caused by the proximal Myosin II pulse (green shading). Oscillating circumferential cell length (red and black arrows) correlates with myosin pulses, allowing cells to inch around the tubule circumference. Tubule outline and cell position over time is shown. Panels on the right show basal views of a cell labelled for membrane (GAP43::GFP, magenta) and Myosin II (Sqh::mCherry, green). Change in circumferential cell length (C, double headed arrow) during proximal Myosin II accumulation (white, J′–J′″) can be seen. Scale bar = 3 µm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s004" target="_blank">Figure S4B</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s021" target="_blank">Movie S14</a>.</p
Normal tubule elongation is important for renal function.
<p>(<b>A</b>) Representative traces of wild-type (upper) and <i>ctB>EGFR<sup>act</sup></i> (lower) 3rd instar tubules. Asterisk, distal tubule tip. (<b>B</b>, <b>C</b>) 3rd instar distal tubule regions (DAPI, nuclei; stellate cells, smaller nuclei coloured orange). The regular spacing of stellate cells (arrows) is disturbed when EGF signalling is activated in all tubule cells. (<b>D</b>) Secretory rates (nl min<sup>−1</sup>) of control (blue) and <i>EGFR<sup>act</sup></i> (green) MpTs. Cyclic adenosine monophosphate (cAMP) and Leucokinin (LK) were added at 30 and 60 minutes, respectively (arrows). <i>EGFR<sup>act</sup></i> tubules have strongly reduced basal secretory rates and are refractory to diuretic stimulation. (<b>E</b>) Adult flies aged for ∼24 hours after eclosion. <i>EGFR<sup>act</sup></i> animals are grossly bloated compared with controls. Wet and dry weight measurements from <i>ctB-Gal4</i> control (orange, <i>n</i> = 5), <i>ctB>EGFR<sup>act</sup></i> (green, <i>n</i> = 11) and <i>UAS-EGFR<sup>act</sup></i> control (blue, <i>n</i> = 13) animals. Weight (mg) for the weight of three flies is given on y-axis. Error bars = standard deviation (SD). The difference in wet weight between control and experimental animals is highly significant, <i>p</i> = 2.82×10<sup>−6</sup> (orange asterisk) and <i>p</i> = 8.66×10<sup>−10</sup> (blue asterisk). Dry weights are approximately equal.</p
Polarised EGF signalling is required for MpT elongation.
<p>(A–C, E) Stage 16 embryos stained for MpTs (Cut). (A) Wild type embryo. (B) <i>EGFR<sup>ts</sup></i> embryo raised at restrictive temperature during period of elongation. (C) <i>ctB>EGFR<sup>DN</sup></i>, (E) <i>ctB>EGFR<sup>act</sup></i>. Perturbation of EGF signalling disrupts tubule elongation. (D, D′, F, F′) Beginning and end point still images from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s013" target="_blank">Movies S6</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013.s015" target="_blank">S8</a> in <i>ctB>EGFR<sup>DN</sup></i> (D) and <i>ctB>EGFR<sup>act</sup></i> (F) embryos, right panels show 4-D reconstruction, stars in (D, D′) indicate TC. Circumferential cell rows (coloured) achieve no (D,D′) or highly reduced (F,F′) cell intercalation (arrowheads). (G) Graph to show tubule elongation in wild type embryos and in those with perturbed EGFR signalling. Tubule length is significantly reduced in <i>ctB>EGFR<sup>DN</sup></i> (<i>p</i> = 4×10<sup>−6</sup><0.05) and <i>ctB>EGFR<sup>act</sup></i> (<i>p</i> = 1×10<sup>−4</sup><0.05) compared to WT. (H, I) Stage 16 <i>ctB>sSpi</i> (H) and <i>ase>sSpi</i> (I) embryos stained for Cut. (H′, I′) Enlarged views showing aMpTs.</p
The tip cell lineage is required for tubule elongation.
<p>(A) Stage 14 aMpT stained with anti-Cut (red) with TC (arrowhead) and SC (arrow) highlighted with Neuromusculin-LacZ (A37, green). (B) Stage 14 aMpT stained with anti-Cut (green) and anti-Rhomboid (red) showing Rhomboid in tip (arrowhead) and sibling (arrow) cells (inset, higher magnification). (C) A late stage 14 aMpT (Cut, magenta) stained for dpERK (green). dpERK is detected in the distal-most tubule cells, declining proximally towards the kink. High dpERK associated with the proximal tubule probably corresponds to hemocytes (asterisk) clustered around the MpTs <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002013#pbio.1002013-Bunt2" target="_blank">[64]</a>. Arrowhead, TC. A heat map showing dpERK is shown in (C′). (D) Stage 15 aMpT (Cut, magenta) in a <i>Capicua::Venus</i> (GFP, green) embryo. (D′) A heat map reveals high levels of nuclear Capicua in proximal tubule cells, while Capicua is absent or present at low levels in the cytoplasm of distal cells. Arrowhead, position of TC. (E) Quantification of dpERK activation along the D-P axis for ∼60 µm of the distal tubule (spanning ∼ten cell diameters) from the TC from stage 13 embryos. Average intensity (blue curve) of dpERK staining (<i>n</i> = 7 tubules) is plotted against tubule length (µm). Error bars (grey area) = standard error of the mean (SEM). Red line; best fit curve using fourth-order regression analysis. (F, F′) Stage 13 tubule stained for Cut (F) or dpERK (F′) showing tubule region measured in (E). D-P distance from TC in µm (red numbers), TC (arrowhead). (G, G′) Stage 16 <i>Df(os)1</i> embryo stained for Cut (white) showing that both anterior (aMpT) and posterior (pMpT) tubules lack TCs and SCs and fail to undergo normal elongation. (H, I) Embryos cultured to stage 16 following laser ablation of the TC and SC at late stage 12. Tubule cells, Cut (red); TCs, 22c10 (anti-Futsch, green). (H′, I′) Higher magnification views. TC ablated tubules fail to undergo elongation, whereas control tubules elongate normally. Distal (d) and proximal (p) tubule ends; arrowheads TC (I, I′) or ablated site (H, H′).</p