Collective Epithelial Migration Drives Kidney Repair after Acute Injury

Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney’s remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI

Asymmetric inheritance of the apical domain and self-renewal of retinal ganglion cell progenitors depend on Anillin function.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.journal articleresearch support, non-u.s. gov't2015 Mar 012015 02 05importe

Hemodynamic Forces Sculpt Developing Heart Valves through a KLF2-WNT9B Paracrine Signaling Axis.

Hemodynamic forces play an essential epigenetic role in heart valve development, but how they do so is not known. Here, we show that the shear-responsive transcription factor KLF2 is required in endocardial cells to regulate the mesenchymal cell responses that remodel cardiac cushions to mature valves. Endocardial Klf2 deficiency results in defective valve formation associated with loss of Wnt9b expression and reduced canonical WNT signaling in neighboring mesenchymal cells, a phenotype reproduced by endocardial-specific loss of Wnt9b. Studies in zebrafish embryos reveal that wnt9b expression is similarly restricted to the endocardial cells overlying the developing heart valves and is dependent upon both hemodynamic shear forces and klf2a expression. These studies identify KLF2-WNT9B signaling as a conserved molecular mechanism by which fluid forces sensed by endothelial cells direct the complex cellular process of heart valve development and suggest that congenital valve defects may arise due to subtle defects in this mechanotransduction pathway.journal articleresearch support, non-u.s. gov'tresearch support, n.i.h., extramural2017 11 062017 10 19importe

Mechanotransduction in cardiovascular morphogenesis and tissue engineering.

Cardiovascular morphogenesis involves cell behavior and cell identity changes that are activated by mechanical forces associated with heart function. Recently, advances in in vivo imaging, methods to alter blood flow, and computational modelling have greatly advanced our understanding of how forces produced by heart contraction and blood flow impact different morphogenetic processes. Meanwhile, traditional genetic approaches have helped to elucidate how endothelial cells respond to forces at the cellular and molecular level. Here we discuss the principles of endothelial mechanosensitity and their interplay with cellular processes during cardiovascular morphogenesis. We then discuss their implications in the field of cardiovascular tissue engineering.journal articlereview2019 Aug2019 10 03importe

Regenerating epithelia remain differentiated.

<p>Kidney epithelium is shown at 4–6 hours post injury (hpi), on the site of the laser ablation. (A, B): Anti-GFP (green), anti-acetylated-tubulin (red) and DAPI (magenta, A) staining of the injured tubule shows that cilia and cilia bundles (multiciliated cell in A) are present at the edge (white arrowheads, also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>) of surviving epithelium (white star in A and B). The aggregated cilia bundles in B (red star) are visible in the middle of the injured segment (between arrowheads). (C and D): Na/K ATPase (red) and Crumbs (green) expression in intact (C) and injured (D) epithelium (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>). AKI does not affect the expression of Crumbs on the apical surface of surviving cells (D, arrow), while its distribution is randomized in the injured segment (right of the arrowhead in D, which marks the border between the intact and the injured segment. (E, F): Vimentin is not expressed in intact kidney epithelium (E) and is not up-regulated after acute injury (F, also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>). Vimentin staining can be seen outside of the kidney (arrow in (F)). Bar lengths in (A-F) are 30 µm. (G) Electron microscopy of a longitudinal section at the edge of surviving epithelium (the edge itself is not shown, to the left). Apical junctional complexes (arrow) are preserved. Bar length is 1 µm. One degenerated mitochondrion indicates partial injury to the cell at the edge of the injury (arrowhead). Different colors are used to delineate individual epithelial cells. The virtual slice thickness in (A-F): A- 1.4 µm; B-5.6 µm (7 slices); C,D- 1.4 µm; E- 7.0 µm (8 slices); F- 11.2 µm (15 slices).</p

Modeling cell migration and cell proliferation during kidney repair.

<p>(A) Initial arrangement of cells in the simulation. Each square represents a single cell position. Our model is based on that published in¹⁹, with an addition of a ‘free edge’. (B, C) After 5 and 50 iterations of the algorithm, cells have migrated to mostly recover the epithelial gap. In the illustrated simulation, we introduced a delay in cell proliferative response to stretch. Simulations produce very similar results with or without delay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s012" target="_blank">Movie S8</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s013" target="_blank">S9</a>), but presence of a delay is more reflective of the experimental observations. (D, E, left, gray markers) show divided cells in response to cell stretch after ‘long segment ablation’. (A–E, right) demonstrate that after ‘short ablation’ no cell proliferation is induced. (F) Plots of the ‘rate of migration’ after long (left panel) vs. short (right panel) segment ablation. Arrows point to a rapid decrease of the rate of migration due to cell-cell interaction. This was similar to the experimentally observed migration rate decline (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone-0101304-g002" target="_blank">Figure 2</a>). A second small peak in migration (arrowhead) is due to cell division.</p

Regenerating epithelia remain differentiated.

<p>Kidney epithelium is shown at 4–6 hours post injury (hpi), on the site of the laser ablation. (A, B): Anti-GFP (green), anti-acetylated-tubulin (red) and DAPI (magenta, A) staining of the injured tubule shows that cilia and cilia bundles (multiciliated cell in A) are present at the edge (white arrowheads, also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>) of surviving epithelium (white star in A and B). The aggregated cilia bundles in B (red star) are visible in the middle of the injured segment (between arrowheads). (C and D): Na/K ATPase (red) and Crumbs (green) expression in intact (C) and injured (D) epithelium (also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>). AKI does not affect the expression of Crumbs on the apical surface of surviving cells (D, arrow), while its distribution is randomized in the injured segment (right of the arrowhead in D, which marks the border between the intact and the injured segment. (E, F): Vimentin is not expressed in intact kidney epithelium (E) and is not up-regulated after acute injury (F, also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101304#pone.0101304.s004" target="_blank">Figure S4</a>). Vimentin staining can be seen outside of the kidney (arrow in (F)). Bar lengths in (A-F) are 30 µm. (G) Electron microscopy of a longitudinal section at the edge of surviving epithelium (the edge itself is not shown, to the left). Apical junctional complexes (arrow) are preserved. Bar length is 1 µm. One degenerated mitochondrion indicates partial injury to the cell at the edge of the injury (arrowhead). Different colors are used to delineate individual epithelial cells. The virtual slice thickness in (A-F): A- 1.4 µm; B-5.6 µm (7 slices); C,D- 1.4 µm; E- 7.0 µm (8 slices); F- 11.2 µm (15 slices).</p

405 nm laser ablation induces kidney specific segmental epithelial injury in GFP transgenic zebrafish.

<p>(A) 12 dpf zebrafish was subjected to 405 nm laser treatment using a confocal microscope. The plane of maximal illumination was guided by kidney GFP fluorescence (here- E11-9 transgenic fish). The laser scan window is shown by the white rectangle. Efficient laser treatment was monitored by observing GFP bleaching in the ablated area (with a target of ∼50% initial reduction in GFP fluorescence). The ablated segment continued to lose GFP positivity with only an occasional cell surviving at 160 min post- laser treatment. The arrow points to a sharply defined edge of surviving epithelium. Scale bar = 70 µm. (B) Propidium iodide (PrI, red) staining confirmed that loss of GFP positivity was due to cell death, as opposed to other mechanisms. As cells in the injured segment lost GFP positivity, they became strongly PrI-positive. Every cell showed PrI positivity by 60 min post injury, and by 210 min PrI-positive material was seen exclusively in the distended lumen proximal to the obstructed segment (lower panel). PrI-positive material could also be seen transiting through the distal tubule (arrow in 20 min panel). Scale bar = 100 µm. (C) Electron microscopy of the injured epithelium 3 hours post-laser treatment shows compacted swollen degenerated mitochondria (arrowheads). The basement membrane (pink, arrows) and the adjacent, likely stromal or endothelial cell (green, star) is preserved. “n” – intact nucleus, “ly” – lysosome. Scale bar = 1000 nm.</p

Kidney epithelial proliferation after acute injury.

<p>(A,B) Two examples of BrdU staining after segmental ablation early (0–12 hpi, A) and late (36–48 hpi, B) in ET11-9 GFP transgenic fish. Arrows indicate the direction of injury. Confocal slice thickness is 1.5 µm in (A), and 1.6 µm in (B). (C) Number of BrdU+ cells at various intervals after segmental laser ablation (0–12 hpi, n = 4, 12–24 hpi, n = 3, 24–36 hpi, n = 4, 36–48 hpi, n = 4) and compared to non-injured control condition (n = 8). There was no significant increase in cell proliferation early in the repair process (up to 36 hpi) but a pronounced increase in cell proliferation first detectable between 36 and 48 hpi, compared to control condition (p<0.05, C). (D) Two possible mechanisms that could trigger cell proliferation (mechanical stretch due to cell migration vs. a secretory factor) lead to different predictions about the distribution of cell proliferation after segmental ablation. Red color indicates the predicted distribution of cell proliferation after short (left panel) vs. long (right panel) tubule segment ablation. Upper row: initial injury; middle row: stretch response scenario (mechanical model). Lower row: secretory factor scenario. (E) Long segmental ablation resulted in two distinct bands of BrdU incorporation both upstream (left and lower sub-panels) and downstream (right and lower sub-panels, each sub-panel represents a confocal projection image). Brackets in the lower sub-panel indicate bands of proliferation upstream and downstream of the injury. The right and left upper sub-panels show a higher magnification of the areas of proliferation marked by brackets in the lower sub-panel. Other BrdU+ nuclei are outside of the kidney. (F) Comparison of BrdU incorporation in the injured (rhombi) vs. contralateral non-injured tubule (squares). This pattern of BrdU incorporation is most consistent with the mechanical model of the cell proliferation trigger. Double-arrow bar indicates the approximate site of injury. (G) Total number of BrdU+ nuclei after long (80–100 µm) vs. short (20 µm) segmental ablation (24–48 hpi). Long segmental ablation resulted in significantly increased number of BrdU+ cells compared to a contralateral non-injured side (as well as kidney epithelium after short injury or in a non-injured control). Long injury vs. contralateral non-injured tubule: p = 0.039, long injury vs. short injury: p = 0.048, n = 3 per each condition. Scale bars in (A,B and E upper sub-panels) = 30 µm, and 60 µm in (E, lower sub-panel).</p

Surviving kidney epithelium responds by collective migration.

<p>Various kidney GFP transgenics were examined by time lapse confocal microscopy at different developmental stages. (A) Proximal tubule epithelium (ET33d10 transgenic, 2.5 dpf), after segmental ablation of the proximal tubule, shown in 30 min intervals. Scale bar –60 µm. (B) Proximal convoluted tubule epithelium (ET33d10 transgenic, 6 dpf), after segmental ablation of the proximal tubule, shown in 120 min intervals. Scale bar –60 µm. (C, D) Distal tubule epithelium (ET11-9 transgenic) after segmental photoablation shown in 120 min intervals in 7 dpf (C) and 5 dpf (D) fish. (D) shows marked obstructive dilatation of tubule epithelium, some of which is also seen in (C). This is a commonly observed phenomenon. Scale bars: (C) - 60 µm, (D) - 30 µm. (E) Migration rates as a function of time after the initiation of the migratory response. The rates are maximal right after the initiation of the migration and decrease over a few hours. The three early time points (green box) show statistically significant difference from the three late time points (red box, p<0.01, n = 10). The initial migration rates are similar in young (2–3 dpf) fish and older (> = 5 dpf) fish (p = 0.84), but the rates appear to persist longer in younger fish. Statistically significant differences between the rates can be seen at 50 min (p = 0.02, n = 5,5) and 110 min (p<0.01, n = 5,5) time points, as shown in (F).</p