# Novel Phenotypic Elements of Type IV Collagenopathy Revealed by the _Drosophila_ Model
## 2 Myopathic Defects in _Drosophila col4a1_ Mutants  
Under restrictive temperatures, the female heterozygotes become progressively more female-sterile [47]. We therefore chose the single-layer muscle fibers that encircle the common oviduct as a model. The system allowed the direct study of single muscle fibers and their sarcoplasmic membrane, without sectioning [24]. Muscle fibers in the _col4a1C552D2_ mutant detached laterally from each other and a thickened collagen IV-associated label appeared to fill the gap. The basement membrane covering the myofib appeared distorted, thinned or absent, demonstrating that the mutant COL4A1 protein induced severe oviductal myopathy [24; 26]. Integrin deposition in muscle fibers was recorded by punctate staining at the level of Z-discs, overlapping with the signals provided by the Z-disc protein kettin and bordering each sarcomeric unit in immunohistochemical preparation [29]. High expressivity of the trait occurred at low penetrance, i.e., shifting Z-disc integrin-binding sites toward the middle of sarcomeres in _col4a1C552D1_ mutants at the restrictive condition (Figure 1). The normal integrin-kettin staining still persisted at the level of the Z-discs and duplication of the Z-discs occurred at the level of M-discs (Figure 1, B4, B5, B6, white arrows). In some of the replications this phenomenon occurred at nearly full expressivity; integrin staining was shifted quantitatively up to the level of M-discs, into the middle of the anisotropic band (Figure 1, C1, C2, C3, white arrows).  
Next, we quantitatively analyzed the phenomenon of Z-disc shifting in the wild-type control (_Oregon_) and _col4a1_[5552D1] mutants and we labeled the level of expressivity by the Z-disc marker, kettin (Figure 2). The expressivity of Z-disc shifting occurred at over 8% in the wild-type control and over 12% in _col4a1_[5552D2] mutants (Figure 2). We obtained the results by counting 6796 kettin-staining structures within the single-layer striated muscle of the common oviduct. Statistical analysis with the aid of the Welch two-sample t-test revealed significance at a value of \(p=0.003973\), indicating higher expressivity of the Z-disc shifting in the mutants (Figure 2).  
Figure 1: Shifting of Z-discs into the middle of sarcomeres, to the level of M-discs in striated muscle fibers of oviducts. The scaffold protein kettin is deposited at the level of the Z-discs (**A1**, **A4**, **B1**, **B4**) and overlaps with the integrin label (**A2**, **A5**, **B2**, **B5**) providing yellow-orange complementary colors in the overlays (**A3**, **A6**, **B3**, **B6**). Note the irregular integrin expression in the mutant at 29 \({}^{\circ}\)C, zig-zag pattern and streaming of Z-discs (**B5**). Additional kettin-integrin expression appears in the middle of sarcomeres at the level of M-discs (white arrows, **B4**, **B5**, **B6**). Higher magnification of areas in **B4**, **B5** and **B6** are marked by arrows (**B7**, **B8**, **B9**). The shifting of the integrin expression toward the middle of the sarcomeres is nearly at full expressivity (**C4**, **C5**, **C6**, white arrows). The area marked by arrows in C6 are at higher magnification (**C7**). Integrin expression remains at the sides of sarcomeres in Z-discs in the wild-type control (**C1**, **C2**, **C3**). Bars, lower left: 10 micrometers. Experiments were carried out as described [24; 25; 26; 28; 29].