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. 2019 Apr 8;132(7):jcs226860.
doi: 10.1242/jcs.226860.

DSS-induced damage to basement membranes is repaired by matrix replacement and crosslinking

Angela M Howard  1   2   3 , Kimberly S LaFever  1 , Aidan M Fenix  1 , Cherie' R Scurrah  1   3   4 , Ken S Lau  1   3   4 , Dylan T Burnette  1   3 , Gautam Bhave  1   2   5 , Nicholas Ferrell  5   6 , Andrea Page-McCaw  7   2   3   8
Affiliations

DSS-induced damage to basement membranes is repaired by matrix replacement and crosslinking

Angela M Howard et al. J Cell Sci. .

Abstract

Basement membranes are an ancient form of animal extracellular matrix. As important structural and functional components of tissues, basement membranes are subject to environmental damage and must be repaired while maintaining functions. Little is known about how basement membranes get repaired. This paucity stems from a lack of suitable in vivo models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult Drosophila DSS becomes incorporated into the basement membrane, promoting its expansion while decreasing its stiffness, which causes morphological changes to the underlying muscles. Remarkably, two days after withdrawal of DSS, the basement membrane is repaired by all measures of analysis. We used this new damage model to determine that repair requires collagen crosslinking and replacement of damaged components. Genetic and biochemical evidence indicates that crosslinking is required to stabilize the newly incorporated repaired Collagen IV rather than to stabilize the damaged Collagen IV. These results suggest that basement membranes are surprisingly dynamic.

Keywords: Basement membrane; Collagen IV; Dextran sodium sulfate; Drosophila; Enterocytes; Matrix stiffness; Midgut.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
DSS alters gut muscle morphology, similar to loss of Pxn. (A) Schematic representation of the Drosophila gut based on imaging in this study. See also Figs S1, S3. (B–D′) A functional Vkg–GFP (Collagen IV α2) protein allows visualization of the basement membrane under the enterocytes and surrounding the muscles. (B) Basement membrane under control conditions. (C) Morphology is disrupted in DSS-fed adult wild-type flies. (D) Morphology is similarly disrupted in flies with adult-onset knockdown of Pxn (Pxn-KD), a basement membrane cross-linking enzyme, knocked down ubiquitously using TubP-Gal4. Muscle aspect ratio measurements are illustrated in D′. (E–F′) Basement membrane labeled with Vkg–GFP (E,F) surrounds muscles stained with phalloidin (E′,F′). After DSS feeding, apparent displacement of basement membrane represents changes in muscle morphology (F,F′). (G) Muscle aspect ratio, measured as in D′, changes in response to either DSS feeding or Pxn knockdown. Five to six flies were analyzed for each condition. Data shows mean±s.e.m. P-values calculated by ANOVA to determine significance followed by unpaired t-tests with Bonferroni correction. (H,I) A functional LanB1–GFP labels basement membrane under the enterocytes and surrounding the muscles. LanB1–GFP-expressing flies fed DSS recapitulate changes in muscle morphology visualized with Vkg–GFP. (J,K) A functional Trol–GFP labels basement membrane under the enterocytes and surrounding the muscles. Trol–GFP-expressing flies fed DSS recapitulate changes in muscle morphology visualized with Vkg–GFP and LanB1–GFP. B–F,H–K are optical cross sections, as illustrated in A. Scale bar: 5 µm.
Fig. 2.
Fig. 2.
DSS-induced muscle damage is similar to muscle damage from loss of basement membrane. (A) Schematic representation of the linkage between the actomyosin contractile machinery and the basement membrane. The linkage between the basement membrane and the muscles can occur through integrins or dystroglycans. (B,C,E,F) Phalloidin staining revealed sarcomeres in longitudinal gut muscles. Yellow brackets indicate sarcomeres. Scale bar: 5 µm. (D) Knockdown of LanB1 (LanB1-KD) ubiquitously using TubP-Gal4 reduced sarcomere size ∼10% compared to control. Sarcomeres were measured in five flies for each condition. (G) Feeding flies DSS reduced sarcomere size ∼30% compared to control. Sarcomeres were measured in four control and six DSS-fed flies. See also Fig. S2 for sarcomere length in relaxing buffer. Data in D,G show mean±s.e.m. P-values calculated by unpaired t-test.
Fig. 3.
Fig. 3.
DSS expands gut basement membrane without changing matrix protein levels. (A,B) TEM images showing the peristalsis muscles and basement membrane of the midgut of a control or DSS-fed fly. (A′,B′) Same images as in A,B with labels. Basement membrane is pseudo-colored green. Orange lines illustrate how basement membrane thickness under the enterocytes (as shown in C) was measured at regular intervals; actual measurements extended across the entire micrograph but orange lines are confined to one region for illustration. Yellow arrows in B′ indicate shredded muscle. Scale bar: 1 µm. (C) Quantification shows a significant increase in the thickness of the basement membrane after DSS feeding. (D,E) Structured illumination microscopy (SIM, super-resolution) images of Vkg–GFP in the midgut basement membrane in control (D) or DSS-fed (E) flies. Scale bar: 5 µm. (F) Quantification shows a significant increase in the thickness of the Vkg–GFP-labeled basement membrane after DSS feeding, measured in a blinded fashion as in insets below x-axis, corresponding to boxes in D,E. Measurements were made on four control and five DSS-fed flies. (G–I) Basement membrane protein levels are not significantly different in control versus DSS-fed fly midguts, as indicated by fluorescence levels of Vkg–GFP (G), LanB1–GFP (H) or Trol–GFP (I). Each dot represents one midgut. Data in C,F,G–I show mean±s.e.m. P-values calculated by unpaired t-test. NS, not significant.
Fig. 4.
Fig. 4.
DSS accumulates in basement membranes where it irreversibly decreases basement membrane stiffness. (A–C) Feeding conditions for testing DSS localization. (D–F″) After feeding, FITC–DSS specifically localizes to the basement membrane, labeled with Trol–RFP, in the midgut. Feeding regimens in D–F″ match the corresponding schematics above in A–C. (G–I) FITC–DSS specifically localizes to the basement membrane (arrows in I) of the Malpighian tubules after soaking ex vivo. Soaking regimens in G–I match the corresponding schematics above in A–C. (J) Assay for measuring tubule stress-strain response. The tubule is stretched between a cantilever and a holding pipette. Stress and strain are calculated from the bending of the cantilever and the changes in the tubule length. (K) Normalized stiffness for intact and detergent-decellularized tubules. There was no significant difference between cellularized and decellularized tubules, indicating that resistance to strain is imparted by the basement membrane. Five cellularized and three decellularized tubules were analyzed. (L) Stress-strain curves for control, DSS-treated, and DSS-treated and washed tubules showing a downward shift in the stress-strain curves for DSS-treated tubules. Five flies were analyzed for each condition. (M) Elastic modulus for control and DSS treated tubules, calculated from the data in L. DSS treatment significantly reduced basement membrane stiffness. No significant difference was detected between DSS-treated tubules following removal of DSS (wash). Scale bars: 5 µm in F″, I; 200 µm in J. Data in K–M show mean±s.d. P-values calculated by unpaired t-test (K) or ANOVA followed by unpaired t-tests with Bonferroni correction (M). NS, not significant.
Fig. 5.
Fig. 5.
Basement membrane damage precedes loss of epithelial barrier integrity. (A) In control-fed fly (left), blue dye remained in the gut. In DSS-fed fly (right), the gut lost barrier integrity and blue dye escaped to the body. (B,C) Percentage of living control (B) and DSS-fed (C) flies that lost barrier integrity. Green box highlights 2-day timepoint when basement membrane damage was observed by EM, SIM and muscle morphology. (D,E) Survival of the same flies as in B,C over the course of 6 days on the liquid feeding regimen. 60 flies per condition.
Fig. 6.
Fig. 6.
Basement membrane is repaired 48 h after termination of DSS feeding. (A) Muscle morphology was analyzed at the indicated times after withdrawing animals from DSS food to normal food. The muscle aspect ratio was restored at 48 h after terminating DSS feeding. Four to eight flies were analyzed for each time point. (B–D) TEM image of muscles and basement membrane of the midguts in a control-fed fly (A), DSS-fed fly (B), and a fly that recovered on normal food for 48 h after DSS feeding (C). Basement membranes are indicated with yellow arrows. Both muscle morphology and basement membrane thickness have been repaired by 48 h after termination of DSS feeding. (E) Quantification showing repair of basement membrane thickness 48 h after termination of DSS feeding, measured on TEM micrographs. Scale bar: 1 µm. Data in A,E show mean±s.e.m. P-values calculated by ANOVA to determine significance followed by unpaired t-tests with Bonferroni correction. NS, not significant.
Fig. 7.
Fig. 7.
Peroxidasin is required for basement membrane repair. (A–F) SIM images of Vkg–GFP in the midgut basement membrane of control flies (A–C) or flies with Pxn knocked down (Pxn-KD) in adults using TubP-Gal4, Gal80ts. Basement membrane was thicker upon DSS feeding (B,E) but 48 h after termination of DSS treatment, basement membrane returned to its undamaged thickness in control flies (C) but not Pxn-KD flies (F). Repair was also deficient with a second Pxn RNAi line (not shown). Scale bar: 5 µm. (G) Quantification of the basement membrane thickness in SIM micrographs. Five to seven flies were analyzed for each condition. (H–M) Epifluorescence images of Vkg–GFP outlining muscles in midguts of no-drug control flies (H–J) or flies fed the Pxn inhibitor PHG (K–M). Muscles become dysmorphic with DSS feeding (I,L), but 48 h after DSS withdrawal muscles return to their undamaged state in control flies (J) but not Pxn-inhibited flies (M). Scale bar: 10 µm. (N) Quantification of the muscle aspect ratio. Four to six flies were analyzed for each condition. Data in G,N show mean±s.e.m. For statistical analysis of significance, see Tables S1 and S2.
Fig. 8.
Fig. 8.
Basement membrane repair requires new Collagen IV and Laminin. (A–I) Epifluorescence microscopy images of Vkg–GFP (A–C,G–I) or LanB1–GFP (D–F) showing the basement membrane and muscle morphology before, during or after DSS feeding. When vkg (vkg-KD) or LanB1 (LanB1-KD) is knocked down in adults using TubP-Gal4, Gal80ts, basement membranes do not repair as inferred from the muscle morphology (F,I). Scale bar: 10 µm. (J) Quantification of the muscle aspect ratio. Four to six flies were analyzed for all conditions, except three flies for the LanB1-KD 48 h sucrose control. Data shows mean±s.e.m. For statistical analysis of significance, see Table S3. The repair-deficient phenotype was observed with second RNAi lines targeting vkg or LanB1 (not shown).
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