Novel Phenotypic Elements of Type IV Collagenopathy Revealed by the Drosophila Model

Featured Application: A great number of drugs can be screened with the aid of the high-throughput Drosophila system to combat type IV collagenopathy. Abstract: Type IV collagen is proposed to be a key molecule in the evolvement of multicellular animals by forming the architectural unit basement membrane, a specialized form of the extracellular matrix. Functions of the basement membrane include guiding organ regeneration, tissue repair, modulation of cell di ﬀ erentiation, apical–basal polarity identiﬁcation, cell migration and adhesion, regulation of growth factor signaling gradients, maintenance of tissue architecture and compartmentalization. collagenopathy is a devastating systemic disease a ﬀ ecting the circulatory, renal and visual systems and the skeletal muscles. It is observed in patients carrying mutations in the COL4A1 gene, which codes for the ubiquitous basement membrane component. Col4a1 mouse mutants display the human symptoms of type IV collagenopathy. We chose the Drosophila melanogaster model as we recorded dominant, temperature-sensitive mutations in the cognate col4a1 gene of the fruit ﬂy and demonstrated phenotypic elements which have not yet been explored in humans or in mouse models. In this paper we show a transition of the Z-discs, normally bordering each sarcomere, to the level of M-discs signiﬁcantly penetrant in the mutants, uneven distribution of fused mitochondria in the Malpighian tubules of the excretory organ and a loss of sarcomere structure in the visceral muscles in the gut of mutants. Our observations demonstrate the systemic nature of the col4a1 mutations in the fruit ﬂy. However, these traits are elements of the type IV collagen-associated pathology and may provide insights into approaches that can alleviate symptoms of the disease.


Introduction
The highly differentiated form of the extracellular matrix is the planar-organized basement membrane (BM) found at the basal side of epithelial and endothelial cells in neural, vascular and adipose tissues, surrounding each individual striated muscle fiber.
Major proteins of BMs are type IV collagen, laminin, nidogen or entactin, perlecan and integrins [1]. Nidogens and perlecan are required perinatally but not at earlier phases of development [2]. The BM is anchored to the cell-surface integrin and to the dystroglycan receptors and provides a BM-cytoskeleton linkage [3]. The family of laminins consists of five alpha, four beta and three gamma chains in mammals. They harbor three pairs of type IV collagen genes, COL4A1 through to COL4A6, with each gene pair with Walker-Warburg Syndrome or muscle-eye-brain disease, a distinct form of congenital muscular dystrophy [40].
Mouse Col4a1 mutants recapitulate human disease. An allelic series of induced dominant mouse mutants was identified and showed ocular anterior segment defects or Axenfeld-Rieger anomaly, vacuolar cataracts, corneal opacities, iris defects, iris or corneal adhesions, buphthalmos, optic nerve cupping, retinal detachment and developed renal glomerulopathy [41]. Similar ophthalmic defects were noted in the Col4a1 splice site of mutant C57BL6/J mice. When C57BL6/J mice were crossed with 129/SvEvTac and CAST/EiJ inbred strains, the F1 progeny were phenotypically almost indistinguishable from the wild-type. Genetic mapping identified a co-segregating dominant locus on chromosome 1, that likely contains the suppressor and modifier genes [42]. Col4a1 G498V/G498V homozygous mice showed severe muscular dystrophy, including muscle mass decrease, fiber atrophy, centronuclear fibers, fibrosis, focal perivascular inflammation and intramuscular hemorrhaging [43]. Variants closer to the C-terminus tended to result in increased intracellular levels of the Col4a1 protein, whereas those closer to the N-terminus tended to accumulate lower amounts of intracellular proteins with unchanged clinical severity [44]. The renal manifestation in mouse models of HANAC syndrome include albuminuria, hematuria, glomerular cysts and delays in glomerulogenesis and podocyte differentiation [45].
In col4a1 fly mutants, we recorded severe myopathy, reduced concentration of COL4A1 protein [24], chronic inflammation, intestinal dysfunction [25], detachment of the cells from the BM by electron microscopy [26], compromised excretory system [27], heavy membrane peroxidation in epithelial cells of the Malpighian tubules [28] and the onset of muscular dystrophy in all mutants [29]. Temperature-sensitive col4a1 alleles in Drosophila were generated by ethyl-methanesulfonate mutagenesis, resulting in G to A transitions in all alleles, in turn leading to glycine substitutions by Glu, Asp and Ser [29]. The phenotypic changes appeared in adult flies following 14 days of incubation at 29 • C and were not reversible, the flies did not recover at 20 • C following a shift in heat. We established the complementation map of the col4a1-col4a2 locus with the aid of 34 mutants in all combinations in 561 independent crosses. The map was sharply divided into two non-overlapping parts with the dominant, temperature-sensitive alleles on one side, suggesting that only one of the two genes carries a temperature-sensitive mutation. Two mutant alleles of the same gene may restore the wild-type function in interallelic complementation [46]. We observed interallelic complementation among the temperature-sensitive col4a1 alleles. This genetic observation indirectly suggests that at least two COL4A1 chains interact in forming the trimeric protomer, allowing the homotrimer [COL4A1] 3 , [COL4A2] 3 or heterotrimer [COL4A1] 2 COL4A2 composition of the triple helices [24]. In this paper, we present further phenotypic elements of the col4a1 mutations, including Z-disc shifting to the level of M-discs, aberrant mitochondria in the epithelial cells of the Malpighian tubules and loss of sarcomere structure in the visceral muscles, providing further data of a BM-linked systemic condition.

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 col4a1 G552D2 mutant detached laterally from each other and a thickened collagen IV-associated label appeared to fill the gap. The basement membrane covering the myofibers 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 col4a1 G552D1 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).
Appl. Sci. 2019, 9, x FOR PEER REVIEW 4 of 11 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 providing yellow-orange complementary colors in the overlays (A3, A6, B3, B6). Note the irregular integrin expression in the mutant at 29 °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 (C2, C3). Bars, lower left: 10 micrometers. Experiments were carried out as described [24,25,26,28,29].
Next, we quantitatively analyzed the phenomenon of Z-disc shifting in the wild-type control (Oregon) and col4a1 G552D1 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 G552D2 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 • 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].
Next, we quantitatively analyzed the phenomenon of Z-disc shifting in the wild-type control (Oregon) and col4a1 G552D1 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 G552D2 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 2.
Elevated expressivity of Z-disc shifting in mutants. Statistically significant expressivity of Z-disc shifting in the mutants at p = 0.003973 (**).

Compromised Excretory System in the Mutants
Excretion is accomplished by the kidneys in vertebrates and by the Malpighian tubules in insects. As part of the clinical spectrum amino acid substitutions in the ubiquitous human BM component, COL4A1 can cause nephropathy. Affected patients develop renal dysfunction and chronic kidney failure with or without hematuria. Mouse Col4a1 mutants present the same symptoms as humans. The Malpighian tubules are functionally similar to the mammalian kidneys and offer a versatile and tractable model [48]. The freely floating tubules are in continuous movement within the hemocoel, the blood-filled body cavity with open circulation. The mechanical load of periodic movements contributes to stress-induced cytoskeletal reorganization in col4a1 mutant animals. We therefore tested the effects of mechanical load in the col4a1 G552D2 mutant and recorded the development of actin stress fibers and irregular, uneven actin staining in epithelial cells of the Malpighian tubules [27]. In a separate study, we noted mitochondrial fusion and co-localization of peroxynitrite-nitrated and 4-hydroxy-2-nonenal alkylated proteins with abnormal mitochondria [28]. Mitochondrial fusion in Malpighian epithelium is pronounced in the col4a1 G552D1 mutant at the restrictive temperature (Figure 3), as a sign of the mutation-induced stress, whereas the level of protein nitration assessed qualitatively remain unchanged in the wild-type control and mutant strains under both the permissive and restrictive conditions ( Figure 3).

Compromised Excretory System in the Mutants
Excretion is accomplished by the kidneys in vertebrates and by the Malpighian tubules in insects. As part of the clinical spectrum amino acid substitutions in the ubiquitous human BM component, COL4A1 can cause nephropathy. Affected patients develop renal dysfunction and chronic kidney failure with or without hematuria. Mouse Col4a1 mutants present the same symptoms as humans. The Malpighian tubules are functionally similar to the mammalian kidneys and offer a versatile and tractable model [48]. The freely floating tubules are in continuous movement within the hemocoel, the blood-filled body cavity with open circulation. The mechanical load of periodic movements contributes to stress-induced cytoskeletal reorganization in col4a1 mutant animals. We therefore tested the effects of mechanical load in the col4a1 G552D2 mutant and recorded the development of actin stress fibers and irregular, uneven actin staining in epithelial cells of the Malpighian tubules [27]. In a separate study, we noted mitochondrial fusion and co-localization of peroxynitrite-nitrated and 4-hydroxy-2-nonenal alkylated proteins with abnormal mitochondria [28]. Mitochondrial fusion in Malpighian epithelium is pronounced in the col4a1 G552D1 mutant at the restrictive temperature (Figure 3), as a sign of the mutation-induced stress, whereas the level of protein nitration assessed qualitatively remain unchanged in the wild-type control and mutant strains under both the permissive and restrictive conditions (Figure 3).

Intestinal Manifestation of col4a1 Mutations in Drosophila
The skeletal and cardiac muscles appeared 700 million years ago in a common ancestor prior to the evolutionary divergence of vertebrates and arthropods. The smooth muscle of vertebrates is likely to have evolved later, independent of the skeletal and cardiac muscle types [49]. Accordingly, the visceral muscles of Drosophila are striated. We studied the larval midgut through ultrathin sectioning and electron microscopy and observed that both epithelial and visceral muscle cells were severely degenerated at the restrictive temperature in col4a1 G552D2 mutant animals [26]. Both cell types detached from the distorted and thick BM, while epithelial cells accumulated lipid droplets, autophagic vacuoles and membrane ghosts. Membrane whorls practically filled the cell and the cytoplasm and muscle fibers were rarely observed. The space between the epithelial cell and the muscle cell was enlarged [26]. Functional damage of the alimentary tract in the col4a1 G552D2 mutant was reflected by intestinal dysfunction, degeneration of the gut epithelial cells marked by TUNEL-positive nuclei and the abrupt onset of the SMURF phenotype, demonstrated by blue food dye leaking out from the gut [25]. Intestinal dysfunction, a motility-associated disorder including chronic intestinal pseudoobstruction, is a direct, life-threatening and frequent complication of muscular dystrophies, requiring immediate surgical intervention [50]. Notwithstanding, complications arising from intestinal dysfunction reported in Drosophila col4a1 mutants in conjunction with COL4A1 mutations have not yet been noted in humans and mice. We suggest that intestinal dysfunction in the col4a1 G552D2 mutant is a consequence of disrupted visceral muscle BM junction in the gut, without apparent sarcomere structure and amorphous, uneven actin deposition (Figure 4).

Intestinal Manifestation of col4a1 Mutations in Drosophila
The skeletal and cardiac muscles appeared 700 million years ago in a common ancestor prior to the evolutionary divergence of vertebrates and arthropods. The smooth muscle of vertebrates is likely to have evolved later, independent of the skeletal and cardiac muscle types [49]. Accordingly, the visceral muscles of Drosophila are striated. We studied the larval midgut through ultrathin sectioning and electron microscopy and observed that both epithelial and visceral muscle cells were severely degenerated at the restrictive temperature in col4a1 G552D2 mutant animals [26]. Both cell types detached from the distorted and thick BM, while epithelial cells accumulated lipid droplets, autophagic vacuoles and membrane ghosts. Membrane whorls practically filled the cell and the cytoplasm and muscle fibers were rarely observed. The space between the epithelial cell and the muscle cell was enlarged [26]. Functional damage of the alimentary tract in the col4a1 G552D2 mutant was reflected by intestinal dysfunction, degeneration of the gut epithelial cells recorded by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in the nuclei and the abrupt onset of leakiness of the gut (SMURF phenotype), demonstrated by blue food dye leaking out from the gut [25]. Intestinal dysfunction, a motility-associated disorder including chronic intestinal pseudo-obstruction, is a direct, life-threatening and frequent complication of muscular dystrophies, requiring immediate surgical intervention [50]. Notwithstanding, complications arising from intestinal dysfunction reported in Drosophila col4a1 mutants in conjunction with COL4A1 mutations have not yet been noted in humans and mice. We suggest that intestinal dysfunction in the col4a1 G552D2 mutant is a consequence of disrupted visceral muscle BM junction in the gut, without apparent sarcomere structure and amorphous, uneven actin deposition (Figure 4). . Loss of sarcomere structure in visceral muscle fibers. Regular sarcomere structure in the wild-type control at both temperatures (A1, A4) and loss of sarcomeres in the mutant that deposits actin in an irregular fashion (B1, B4). Bars, lower left: 10 micrometers. Experiments were carried out as described [24,25,26,28,29].

Materials and Methods
Wild-type Oregon flies and col4a1 mutant lines were maintained at 20 °C and 29 °C on yeastcornmeal-sucrose-agar food, consisting of nipagin to prevent fungal infection. The mutant stocks were kept heterozygous over the recombination-preventing CyRoi balancer chromosome. Malpighian tubules, common oviducts and the gut were dissected under carbon dioxide anesthesia from adults that were grown at either the permissive or the restrictive temperature for 14 days. Dissected organs were fixed in a previously prepared 4% paraformaldehyde dissolved in phosphate buffered saline (PBS) solution for 10 minutes, washed three times in PBS, permeabilized for 5 minutes in 0.1% Triton-X dissolved in PBS solution and washed three times in PBS. Blocking was achieved in a 5% BSA dissolved in PBS solution for 1 hour, and washed three times in PBS.
Complementation tests were performed by ethyl-methanesulfonate-induced point mutants, Pelement insertional mutants and X-ray-generated deletion (null) alleles [24]. The mutation sites in eight temperature-sensitive lines were sequenced. All mutations were G to A transitions and replaced glycine with glutamate, aspartate and serine. The a-30 allele carried the p.G233E amino acid substitution; the b-9 carried the p.G467E substitution. The isoallelic lines DTS-L2 and DTS-L3 carried the p.G552D1 and p.G552D2 substitutions, respectively. The second isoallelic pair DTS-L4 and DTS-L5 carried the p.G1205E1 and p.G1205E2 substitutions, respectively, and the DTS-L10 carried the p.G1043S substitution, whereas the b-17 carried the p.G1393E substitution. Importantly, the lesions covered the collagenous domain of the COL4A1 protein [29].

Materials and Methods
Wild-type Oregon flies and col4a1 mutant lines were maintained at 20 • C and 29 • C on yeast-cornmealsucrose-agar food, consisting of nipagin to prevent fungal infection. The mutant stocks were kept heterozygous over the recombination-preventing CyRoi balancer chromosome. Malpighian tubules, common oviducts and the gut were dissected under carbon dioxide anesthesia from adults that were grown at either the permissive or the restrictive temperature for 14 days. Dissected organs were fixed in a previously prepared 4% paraformaldehyde dissolved in phosphate buffered saline (PBS) solution for 10 min, washed three times in PBS, permeabilized for 5 min in 0.1% Triton-X dissolved in PBS solution and washed three times in PBS. Blocking was achieved in a 5% bovine serum albumin dissolved in PBS solution for 1 h, and washed three times in PBS.
Complementation tests were performed by ethyl-methanesulfonate-induced point mutants, P-element insertional mutants and X-ray-generated deletion (null) alleles [24]. The mutation sites in eight temperature-sensitive lines were sequenced. All mutations were G to A transitions and replaced glycine with glutamate, aspartate and serine. The a-30 allele carried the p.G233E amino acid substitution; the b-9 carried the p.G467E substitution. The isoallelic lines DTS-L2 and DTS-L3 carried the p.G552D1 and p.G552D2 substitutions, respectively. The second isoallelic pair DTS-L4 and DTS-L5 carried the p.G1205E1 and p.G1205E2 substitutions, respectively, and the DTS-L10 carried the p.G1043S substitution, whereas the b-17 carried the p.G1393E substitution. Importantly, the lesions covered the collagenous domain of the COL4A1 protein [29].

Conclusions
The basement membrane is likely to be the primordial form of the extracellular matrix and type IV collagen the key molecule in the transition from uni-to multicellularity [51]. The visco-elasticity of the BM is provided by the type IV collagen network, with defects in this component compromising the network's elasticity and function. Type IV collagen proteins are highly conserved structurally and functionally during evolution, including the mammalian-Drosophila relationship [51]. The ubiquitous human and mammalian BM consists of (COL4A1) 2 COL4A2 heterotrimers and is distributed throughout the body. Glycine substitutions trigger systemic disease [1]. The genotype-phenotype relationship was established through 93 COL4A1 and 12 COL4A2 mutations. All of the lesions were dominant with nearly equal representation in males (48%) and females (52%). Inherited incidences were recorded at 47% and sporadic incidences at 53%. As observed in other types of collagens, triple helical Gly substitutions were the most prevalent class of mutations (68 out of 93 in COL4A1) and frequent substitutions were charged amino acids; Arg (30), Glu (11) and Asp (10) [52].
Expressivity of the COL4A1 mutation-associated phenotypic elements often depends on genetic context in humans, mice and flies. In French families recapitulating key features of HANAC, different heterozygous missense mutations in the COL4A1 gene were demonstrated [34]. In a Spanish family, tortuosity of the retinal arteries and retinal hemorrhage were identified, but the lesion was not associated with muscle cramps, renal or brain anomalies, although the p.G510R mutation was previously detected in a French family with HANAC syndrome [35]. Crossing of the Col4a1 splice site mutant C57BL6/J mice with the 129/SvEvTac and CAST/EiJ inbred strains showed that the F1 progeny were phenotypically almost indistinguishable from the wild-type [42]. Similarly, in the isoallelic p.G552D1 and p.G552D2 mutants, the survival rates upon heat selection differed slightly. The line with the p.G552D1 lesion presented survival rates of 26.9% for pupae and 5.3% for adults, whereas the survival rates for the p.G552D2 line were 5.7% and 0.50%, respectively [24,29].
Here, we provided data on the systemic phenotype of the col4a1 mutations and demonstrated the shifting of Z-discs, mitochondrial fusion and loss of sarcomeres in intestinal visceral muscle fibers. Genetic, genomic and proteomic data suggest that the fruit fly harbors all of the genetic elements of the BM, although the number of genes coding for BM components is apparently reduced in comparison with mammals, increasing the genetic tractability of the fly mutants' defective genes coding for proteins of the BM that are structurally and functionally conserved during evolution, including type IV collagens [53]. The dominant col4a1 mutations therefore develop BM-specific phenotypes which are more severe than those observed in human and mouse models. Dominant temperature-sensitivity of the col4a1 alleles is a lethal condition, which cannot be maintained in mammals. Nevertheless, the col4a1-associated phenotypic elements provide numerous biomarkers that can be measured quantitatively during the screening of large numbers of drugs in high-throughput Drosophila screens, aimed at alleviating symptoms of type IV collagenopathy.

Conflicts of Interest:
The authors declare no conflict of interest.