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. 2003 Nov;203(5):483-503.
doi: 10.1046/j.1469-7580.2003.00238.x.

Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor-induced epithelial tubulogenesis

Affiliations

Microscopic analysis of the cellular events during scatter factor/hepatocyte growth factor-induced epithelial tubulogenesis

M J Williams et al. J Anat. 2003 Nov.

Abstract

Scatter factor/hepatocyte growth factor (SF/HGF), a large multifunctional polypeptide growth and motility factor, is known to play important roles during embryonic development, adult tissue growth and repair. In an established three-dimensional type I collagen model, SF/HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching tubules (tubulogenesis). In addition, the composition of the surrounding extracellular matrix (ECM) has been shown to modulate SF/HGF-induced morphogenesis, where tubulogenesis was completely abrogated in Matrigel basement membrane. Many cellular events that occur during SF/HGF-mediated remodelling, and its modulation by the ECM, remain unclear. We have investigated these mechanisms through microscopic examination of the time-course of SF/HGF-induced responses in MDCK cysts cultured in type I collagen or Matrigel. We found that early responses to SF/HGF were matrix-independent. Changes included increased paracellular spacing between normally closely apposed lateral membranes, and the formation of filopodial processes, indicating a partial motile response. Cell-cell contact was maintained, with the persistence of cell junctions. Therefore, while one or a number of ECM components are preventing SF/HGF-primed cells from undergoing an invasive and/or migratory programme, non-permissive matrices are not preventing SF/HGF signalling to the cell. Later matrix-dependent responses, which occurred in type I collagen but not Matrigel, included the formation of basal protrusions that comprise two or more neighbouring cells, which extend to form nascent tubules. Modified polarity of cells comprising the basal protrusions was evident, with a marker for the apical membrane being found in the same region as adherens junctions and desmosomes, typically localized at lateral membranes. We propose a model for SF/HGF-induced tubulogenesis in which tubules form from basal protrusions of adjacent cells. This mechanism of in vitro tubule formation has many similarities to reported in vivo epithelial tubulogenesis.

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Figures

Fig. 1
Fig. 1
Phase-contrast photomicrographs of MDCK cysts cultured in type I collagen (a–e) or Matrigel (f–h) during SF/HGF treatment. Cysts cultured in type I collagen: untreated (a), or treated with SF/HGF for 6 h (b), 12 h (c), 24 h (d) or 72 h (e). Cysts cultured in Matrigel: untreated (f), or treated with SF/HGF for 6 h (g) or 24 h (h). Scale bar = 50 µm.
Fig. 2
Fig. 2
Transmission electron micrograph montages of MDCK cyst cultured in type I collagen. (a) Untreated, (b) 24-h SF/HGF, (c) 72-h SF/HGF. Scale bar = 5 µm.
Fig. 3
Fig. 3
Transmission electron micrographs of MDCK cysts cultured in type I collagen. Untreated (a,I–iii), SF/HGF treatment for 3 h (b,I–iii), 6 h (c,I–iii), 12 h (d,I–iii), 24 h (e,I–vi) or 72 h (f,I–iii); (i) cyst wall (a–e) or tubule (f), (ii) apical region, (iii) basal region, (iv–vi) protrusions. Lateral membranes are indicated by solid arrows; cell–cell contacts are indicated by open arrows. Scale bar = 10 µm (i), 2 µm (ii–vi).
Fig. 3
Fig. 3
Transmission electron micrographs of MDCK cysts cultured in type I collagen. Untreated (a,I–iii), SF/HGF treatment for 3 h (b,I–iii), 6 h (c,I–iii), 12 h (d,I–iii), 24 h (e,I–vi) or 72 h (f,I–iii); (i) cyst wall (a–e) or tubule (f), (ii) apical region, (iii) basal region, (iv–vi) protrusions. Lateral membranes are indicated by solid arrows; cell–cell contacts are indicated by open arrows. Scale bar = 10 µm (i), 2 µm (ii–vi).
Fig. 4
Fig. 4
Scanning electron micrographs of MDCK cysts cultured in type I collagen. Untreated (a) or SF/HGF treatment for 24 h (b–d). Solid arrows indicate basal protrusions; open arrow indicates secondary cell extension. Scale bar = 20 µm (i,ii), 2 µm (iii,iv).
Fig. 5
Fig. 5
Single-section confocal images of MDCK cysts cultured in type I collagen, stained for (a,i–iii) E-cadherin, (b,I–iii) β-catenin, (c,I–iii) desmoplakin, (d,i–iii) F-actin, (e–g) gp114. c(e) gp114 in untreated cyst, (f,g) gp114 in 24-h SF/HGF-treated cysts. Untreated (i), SF/HGF treatment for 12 h (ii) or 24 h (iii). Arrow, luminal F-actin. Scale bar = 30 µm.
Fig. 5
Fig. 5
Single-section confocal images of MDCK cysts cultured in type I collagen, stained for (a,i–iii) E-cadherin, (b,I–iii) β-catenin, (c,I–iii) desmoplakin, (d,i–iii) F-actin, (e–g) gp114. c(e) gp114 in untreated cyst, (f,g) gp114 in 24-h SF/HGF-treated cysts. Untreated (i), SF/HGF treatment for 12 h (ii) or 24 h (iii). Arrow, luminal F-actin. Scale bar = 30 µm.
Fig. 6
Fig. 6
Transmission electron micrographs of basal regions of MDCK cells, and confocal images of F-actin staining, in MDCK cysts cultured in type I collagen. TEM (a,c), confocal microscopy (b,d). Untreated (a,b) or SF/HGF treatment for 24 h (c,d). Solid arrows indicate dense basal actin microfilament distribution; open arrow indicates sparse basal actin microfilament distribution. Scale bar = 1.5 µm (a,c), 30 µm (b,d).
Fig. 7
Fig. 7
Transmission electron micrographs of MDCK cysts cultured in Matrigel. Untreated (a,i–iii), SF/HGF treatment for 6 h (b,i–iii) or 24 h (c,i–iii); (i) cyst wall, (ii) apical region, (iii) basal region. Lateral membranes are indicated by solid arrows; cell–cell contacts are indicated by open arrows. Scale bar = 10 µm (i), 2 µm (ii,iii).
Fig. 8
Fig. 8
Single-section confocal images of MDCK cysts cultured in Matrigel, stained for (a,i&ii) β-catenin, (b,i&ii) E-cadherin, (c,i&ii) desmoplakin, (d,i&ii) F-actin. Untreated (i), or SF/HGF-treated for 24 h (ii). Scale bar = 30 µm.
Fig. 9
Fig. 9
Proposed model of matrix-independent and matrix-dependent SF/HGF-induced morphological responses in MDCK cysts. Schematic representation of the cyst wall during SF/HGF treatment. (a) Untreated cysts, (b) 3-h SF/HGF-treated cysts, and (c) 6-h SF/HGF-treated cysts cultured in type I collagen and Matrigel. (d) SF/HGF treatment for 12 h (e), 24 h (f) or 72 h in type I collagen cultures only. Matrix-independent responses (both type I collagen and Matrigel cultures): increased paracellular spacing during first 6 h of SF/HGF treatment; filopodial processes at lateral membranes; responses persist throughout treatment. Matrix-dependent responses (type I collagen cultures only): formation of basal protrusions, comprising extensions of two or more cells, after 12 h of SF/HGF treatment, which enlargened and extended to form nascent tubules during prolonged treatment; progressive reduction in apical microvilli density. Cell–cell adhesion maintained throughout SF/HGF treatment

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