InteractiveFly: GeneBrief

pericardin: Biological Overview | References


Gene name - pericardin

Synonyms -

Cytological map position - 68E3-68E4

Function - extracellular matrix

Keywords - collagen-like extracellular matrix protein involved in heart development

Symbol - prc

FlyBase ID: FBgn0028573

Genetic map position - chr3L:11,958,872-11,966,131

NCBI classification - High molecular weight glutenin subunit

Cellular location - secreted



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

In Drosophila, formation of the cardiac extracellular matrix (ECM) starts during embryogenesis. Assembly and incorporation of structural proteins such as Collagen IV, Pericardin, and Laminin A, B1, and B2 into the cardiac ECM is critical to the maintenance of heart integrity and functionality. The cardiac ECM connects the heart tube with the alary muscles; thus, the ECM contributes to a flexible positioning of the heart within the animal's body. Moreover, the cardiac ECM holds the larval pericardial nephrocytes in close proximity to the heart tube and the inflow tract, which is assumed to be critical to efficient haemolymph clearance. Mutations in either structural ECM constituents or ECM receptors cause breakdown of the ECM network upon ageing, with disconnection of the heart tube from alary muscles becoming apparent at larval stages. Finally, the heart becomes non-functional. This study characterised existing and new pericardin mutants and investigated biosynthesis, secretion, and assembly of Pericardin in matrices. Two new pericardin alleles, which turned out to be a null (pericardin3-548) and a hypomorphic allele (pericardin3-21), were identifed. Both mutants could be rescued with a genomic duplication of a fosmid coding for the pericardin locus. Biochemical analysis revealed that Pericardin is highly glycosylated and forms redox-dependent multimers. Multimer formation is remarkably reduced in animals deficient for the prolyl-4 hydroxylase cluster at 75D3-4 (Wilmes, 2018).

By underlying or encasing a multitude of cells or tissues, extracellular matrices (ECMs) are essential to several physiological processes including tissue protection, tissue scaffolding, and cell signalling. Biochemical analysis, which is generally impeded by the insoluble and frequently cross-linked nature of the ECM, has shown that the complexity of matrices is much higher than previously expected. It has been reported that the 'matrisome', which collectively encompasses the proteins that constitute the ECM, comprises more than 300 proteins in mammals, including collagens, proteoglycans, growth factors, and receptors. The complexity of matrices is not only reflected by the number of proteins that constitute the matrix, but also by the different ratio with which the various components contribute and by the appearance of unique components in matrices of specific tissues. For example, while a high amount of Collagen I is characteristic of tendons, basement membranes (BMs) contain large amounts of Collagen IV, Laminins, Perlecan, and other proteins. Due to its diverse physiological function, the ECM is more than a homogeneous mass of proteins and carbohydrates. Within the meshwork of its structural components, the ECM is spatially patterned and thereby provides locally restricted reaction environments and structural micro-compartments (Wilmes, 2018).

The Drosophila heart is considered as a model for a specialised ECM composition that ensures proper tissue integrity, functionality, and organ performance. In Drosophila, at present, only four collagens or collagen-like proteins have been identified. One of these proteins is Pericardin (Prc); the others are Collagen IV alpha2 (Viking, Vkg), Cg25c (Dcg1), and Multiplexin. The Pericardin precursor protein consists of 1713 amino acids and harbours an N-terminal signal peptide as well as a long repeat region separated into a collagen-like domain and a non-collagen-like domain, with the former containing 26 atypical and several typical (Gly-X-Y)n repeats. In addition, a single potential Integrin-binding site (RGD) is present at the C-terminus (Chartier, 2002; Drechsler, 2013; Volk, 2014). In contrast to the ubiquitously distributed Collagen IV, Pericardin assembles specifically within distinct matrices: these include the matrix of the heart tube, the surface of pericardial cells and oenocytes, and the cap cells of chordotonal organs. Lack of Pericardin, or its ECM adapter protein Lonely heart (Loh), causes heart failure upon ageing (Drechsler, 2013; Rotstein, 2018). During development, Pericardin is synthesised and secreted by different tissues: first, during embryogenesis, the pericardial cells secrete Pericardin; later, in first and second instar larvae, the main source of Pericardin secretion is the adipocytes. After biosynthesis, secretion, and release into the haemolymph, Pericardin specifically assembles at the outer surface of the cardiac tube and incorporates into the meshwork formed by typical structural components of basement membranes such as Collagen IV, Perlecan, and Nidogen. Adipocyte-specific knock-down of Sar1 expression inhibits Pericardin secretion and thereby affects the formation of a proper heart ECM in Drosophila (Drechsler, 2013). When Pericardin is not expressed, not secreted, or mislocalised, heart integrity is lost, which ultimately results in heart failure and heart collapse. These findings demonstrate that the assembly of a single structural protein, such as Pericardin, in the larval heart is essential for organ integrity and that adipocytes are the major source of distinct ECM components delivered to the heart tube (Wilmes, 2018).

Aiming to extend the current knowledge on how the specific meshwork of structural ECM constituents that characterise the heart matrix is established, this study investigated aspects of the biosynthesis, secretion and deposition of Pericardin in the cardiac matrix in more detail. The Pericardin protein displays collagen-like features that led to the assumption that Pericardin forms, like Collagen IV, trimeric helices that incorporate into matrices (Drechsler, 2013; Wilmes, 2018 and references therein).

Thus, this study focused particularly on components that are known to play an important role in Collagen IV processing, asking whether these enzymes also process Pericardin. Hydroxylation of proline and lysine residues of collagen proteins, taking place within the ER of the collagen-synthesising cells, leads to dimer- and trimerisation by converting proline or lysine into hydroxyproline or hydroxylysine. This reaction is catalysed by various proteins such as Prolyl 4-hydroxlases (PH4), which map to different loci within the genome. Lysine hydroxylation is performed by Lysyl-hydroxylases of which only one, dPlod, is present in the fly genome. Prolyl 4-hydroxylases are comprised of an α2β2 tetramer; the β-subunit is encoded, in Drosophila, by the pdi gene (Pdi, Protein-disulfide isomerase) (Wilmes, 2018).

This study found that Pericardin processing, i.e. multimerisation, is not blocked in mutants for pdi and dplod, and - to some extent - is inhibited in deficiencies that delete a cluster of PH4-encoding genes, which is in contrast to Collagen IV processing phenotypes seen in mutants for pdi, dplod, or PH4 genes. Possibly, redundant or residual activity of the enzymes is sufficient for Pericardin (but not for Collagen IV) maturation and cardiac assembly. Furthermore, recent results show that Pericardin deposition at the embryonic cardiac matrix is, unlike deposition of Collagen IV (Pastor-Pareja, 2011), not necessary for the recruitment and incorporation of additional structural ECM proteins such as Laminin, Nidogen, or Perlecan. Western blot analyses provide initial evidence that Pericardin forms intermediate dimers as well as multimeres. Like many other secreted matrix proteins, Pericardin is extensively glycosylated, indicating cross-linking of Pericardin with other ECM proteins via carbohydrate chains. Finally, previous analyses of pericardin mutant phenotypes were extended by characterising two new EMS-induced pericardin alleles, which were identified in a genetic screen for mutants displaying post-embryonic heart malformations. One of the new pericardin alleles turned out to be a protein null allele, whereas the other one represents most likely a hypomorphic allele with Pericardin being expressed but misassembled (Wilmes, 2018).

Pericardin plays a fundamental role in supporting the structural integrity of the cardiac matrix in the developing Drosophila embryo and larvae. Lack of Pericardin or inhibition of Pericardin recruitment to the cardiac matrix results in destabilisation of the larval cardiac ECM meshwork and loss of the alary muscles-pericardial nephrocytes-heart tube connection upon initiation of heart beat activity. Finally, upon ageing, this leads to luminal heart collapse and renders the heart nonfunctional. This study introduced two new EMS-induced pericardin mutants that both display the characteristic cardiac phenotypes; one of the alleles turned out to be a null allele characterised by complete absence of the protein. All pericardin alleles and transheterozygous combinations of pericardin alleles not only show ECM disintegration upon ageing but also heart collapse associated with disorientation of the cardiomyocyte sarcomeres. In wild-type animals the sarcomeres are highly organised and show a helical orientation. In pericardin mutants this orientation is lost, presumably due to the fact that the costameres lose their link -- via integrins -- to the extracellular matrix upon ECM disintegration (Wilmes, 2018).

Pericardin co-localises with type IV Collagen (Viking). However, in contrast to Viking, which assembles into the basal lamina of virtually all tissues within the animal, Pericardin is highly restricted to the cardiac ECM. ECM fibres harbouring Viking and Pericardin connect the heart tube to the alary muscles. A Pericardin::GFP fusion protein expressed from an engineered fosmid carrying an approximately 40 kb genomic region including the pericardin locus with the pericardin gene tagged with GFP, is synthesised, secreted, distributed by haemolymph flow and assembles at the cardiac matrix. Co-staining for Prc::GFP and endogenous Pericardin shows a complete overlap. However, the Prc::GFP fusion protein fails to rescue the cardiac phenotype of pericardin mutants. By contrast, Pericardin, expressed from an identical fosmid but lacking the C-terminal GFP tag, harbours rescue capability. This demonstrates the importance of the C-terminus of Pericardin for full functionality. While future studies are needed to analyse why Prc::GFP fails to rescue, it appears likely that the C-terminal tag affects accessibility of the RGD-site, which is located close to the C-terminus and which might play a role in anchoring Pericardin to the cell surface via Integrin interaction (Wilmes, 2018).

Based on distinct sequence similarities, including a central Collagen-like repeat domain with typical (Gly-X-Y)n repeats, Pericardin was classified as a type IV Collagen-like protein. In addition, it has been speculated that, analogous to collagens, Pericardin has the ability to form triple helices. However, experimental evidence for dimer-, trimer- or multimerisation of Pericardin has not been provided yet. By analysing protein samples under defined redox conditions, this study found that non-reducing conditions result in formation of high molecular weight Prc multimers. Based on the apparent molecular mass (>500 kDa), the largest multimers most likely correspond to trimeric or even higher order multimeric Prc. Thus, similar to collagens, Prc appears to form redox-dependent multimers, probably disulfide-bonded. Considering the fact that Prc is embedded into the cardiac extracellular matrix, which resides in an oxidising environment, multimeric Prc likely represent the mature, functional form, while the monomeric species presumably constitute biosynthesis intermediates. In addition to confirming Prc multimerisation, this study also found that the protein is extensively glycosylated. Application of both, N- as well as O-glycosidic bond-specific enzymes resulted in distinct mass shifts. While the apparent shift of about 2-3 kDa resulting from PNGase F incubation suggests presence of 1-2 N-linked glycans, the huge mass shift that is obvious upon O-glycosidase incubation (~65 kDa) indicates substantial O-glycosylation of the protein in the Golgi. This indication is supported by sequence analysis, which predicts only two N-glycosylation (MotifScan) but 231 O-glycosylation sites (NetOGlyc 4.0). Glycosylation represents a highly prevalent post-translational modification of ECM proteins and accounts for cell-cell and cell-matrix attachment by promoting the formation of ramified networks between the glycosylated proteins present in the matrix. Considering this, as well as the severe effects of Prc knock out, glycosylated Pericardin appears to be a core component of the ECM network present at the heart (Wilmes, 2018).

Trimerisation of type IV Collagen has been shown to depend on the enzymatic activity of Prolyl (PH4)- and Lysyl (LH)-Hydroxylases. Prolyl-Hydroxylases form a tetrameric complex, with Protein disulfide-Isomerase (Pdi) being present in the complex. Hydroxylation occurs in the Collagen producing cells in the lumen of the ER prior to secretion of Collagen IV molecules. Interestingly, the primary sequence of Pericardin contains a high number of prolines (158, 9.2% of total) as well as numerous type IV Collagen-like repeats, which indicates that Pericardin may undergo a similar biosynthesis pathway as collagens. Therefore, this study analysed whether formation of high molecular mass forms of Pericardin (dimers and trimers) is affected in Pdi, PH4 or LH mutants. Only in PH4 mutants was observed the absence of these multimers observed in Western blot analysis. The PH4 mutants that were used harbour deletions that remove six annotated Prolyl-Hydroxylases at once, the so-called 75D cluster. Single mutant lines for each of these PH4s are not available; therefore whether RNAi-mediated down-regulation of the individual PH4 genes in the cluster results in an inhibition of the formation of high molecular mass forms of Pericardin, which was not the case. This is either caused by either inefficient down-regulation of the target gene or by a redundant function of more than one of the PH4s in the 75D cluster. Pdi and LH seem to play no major role in Pericardin biosynthesis (Wilmes, 2018).

In H5 cells, which are widely used to express high amounts of recombinant protein for further biochemical characterisation, Pericardin is not expressed endogenously at detectable levels. Using a full-length FLAG-tagged version of Pericardin it was found that, after the cells were transfected with the construct, only the monomeric form of Pericardin is produced. It is concluded that H5 cells derived from the cabbage looper Trichoplusia ni lack activity of certain enzymes critical to the biosynthesis of multimeric forms of Pericardin. Interestingly, it has been noticed earlier that the expression of human Collagens is highly efficient in H5 cells, but multimerisation fails due to absence of the appropriate PH4 enzymes in the cultured cells. The current result that a yet unidentified PH, or a combination of several PHs from the 75D cluster, appears to be essential to proper formation of Pericardin multimers indicates that, like in Collagens, Pericardin multimer formation fails due to absence of the required PH in the H5 cells. However, the cell culture system was successfully used to determine the epitope recognised by the widely used EC11 antibody. EC11 recognises Pericardin in the native and the denatured state and the current experiments indicate that the epitope bound by this antibody locates to the N-terminus of Pericardin (Wilmes, 2018).

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, this study observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, attempts were made to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan (Sessions, 2017).

Distinct domains in the matricellular protein Lonely heart are crucial for cardiac extracellular matrix formation and heart function in Drosophila

The biomechanical properties of extracellular matrices (ECMs) are critical to many biological processes, including cell-cell communication and cell migration and function. The correct balance between stiffness and elasticity is essential to the function of numerous tissues, and depends on ECM constituents (the matrisome). However, despite its physiological relevance, the matrisome composition and organization remain poorly understood. Previously, it was reported that the ADAMTS-like protein Lonely heart (Loh) is critical for recruiting the type IV collagen-like protein Pericardin to the cardiac ECM. Thus study utilized Drosophila as a simple and genetically amenable invertebrate model for studying Loh-mediated recruitment of tissue-specific ECM components such as Pericardin to the ECM. Focus was placed on the functional relevance of distinct Loh domains to protein localization and Pericardin recruitment. Analysis of Loh deletion constructs revealed that one thrombospondin type 1 repeat (TSR1-1), which has an embedded WXXW motif, is critical for anchoring Loh to the ECM. Two other thrombospondin repeats, TSR1-2 and TSR1-4, the latter containing a CXXTCXXG motif, appeared to be dispensable for tethering Loh to the ECM, but were crucial for proper interaction with and recruitment of Pericardin. Moreover, the results also suggested that Pericardin in the cardiac ECM primarily ensures the structural integrity of the heart, rather than increasing tissue flexibility. In conclusion, this work provides new insight into the roles of thrombospondin type 1 repeats and advances understanding of cardiac ECM assembly and function (Rotstein, 2018).

Extracellular matrices (ECMs), which support and protect cells and provide mechanical linkage between tissues like muscles and epidermis, are generally assembled in a similar manner. After incorporation of transmembrane receptors, such as integrins and dystroglycans, meshwork-forming components like laminin and collagen IV are able to anchor. By interacting with each other, they form a complex network with distinct biomechanical properties, which is furthermore stabilized by nidogen and allows other proteins (e.g., perlecan) to bind to the matrix as well (Rotstein, 2018).

Whereas these steps can be found ubiquitously, the Drosophila cardiac ECM is different from the matrices of other tissues or organs in several ways. It forms a 3D meshwork that connects the contractile heart tube to the alary muscles and, thereby, to the epidermis. Within this meshwork, embedded pericardial cells differentiate into a distinct population of cell types, such as nephrocytes or wing hearts (Rotstein, 2018).

In flies, the cardiac ECM combines two important biomechanical features: elasticity that accounts for a flexible connection between heart and alary muscle cells and a high tensile strength that withstands forces produced by lifelong heart contractions. One major difference between cardiac ECMs and matrices of other tissues is the presence of the ADAMTS-like adapter protein Lonely heart (Loh), which can be found exclusively at the surface of the heart and chordotonal organs. Lonely heart is essential to proper recruitment of the type IV collagen-like protein Pericardin. Pericardin (Prc) is secreted into the hemolymph by pericardial nephrocytes and adipocytes, and, as soon as it becomes recruited to the cardiac matrix by Lonely heart, it starts to form a stable network. By this mechanism the heart is provided with an exceptional ECM that allows it to withstand the strong mechanical forces of a heartbeat. Lack of Pericardin or its anchor Lonely heart leads to a total collapse of the dorsal vessel and dissociation of the pericardial cells and alary muscles from the heart tube. Concomitants are severely impaired heartbeat and absence of heart-mediated hemolymph transport. Accordingly, corresponding mutant animals exhibit decreased fitness and shortened lifespan (Rotstein, 2018).

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) superfamily consists of two classes of proteins: ADAMTS and ADAMTS-like proteins. Their main difference is that ADAMTS-like proteins, such as Loh, lack the proteolytically active motif within the ADAM spacer. Both classes share several domains, with most of them being poorly defined. In addition to a spacer region, a changing number of thrombospondin type 1 repeats (TSR1) can be found next to a protease and lacunin (PLAC) domain and a signal peptide. These ancillary domains apparently ensure proper substrate specificity as well as cell-surface or ECM tethering (Rotstein, 2018).

TSR1 motifs were initially discovered in thrombospondins (TSPs), which belong to the family of calcium-binding glycoproteins that are secreted into the extracellular matrix of all complex organisms. TSPs have been shown to bind to fibronectin, laminin, collagen, and other matricellular proteins to form complex networks on the cell surface. TSP superfamily members are involved in regulation of spinal cord outgrowth (e.g., F-spondin) or act as specific anti-angiogenic factors in brain development (e.g., BAI-1). In addition, they can be critical to directed ECM proteolysis. TSPs are modular proteins containing several types of repetitive sequence motifs. One of the most characteristic motifs is the evolutionarily conserved thrombospondin type 1 repeat (TSR1), which is ~60 amino acids in length and supposed to form an antiparallel three-stranded structure that interacts with glycoproteins of the extracellular matrix. The human genome harbors ~90 genes encoding TSR1-containing proteins, whereas ~14 corresponding proteins are present in D. melanogaster. Among these, some have been shown to contribute to heart development during embryogenesis. These proteins are the transmembrane receptor Uncoordinated 5 (Unc5) and the ADAMTS-like protein Lonely heart (Rotstein, 2018).

To understand the molecular mechanism by which Lonely heart ensures proper cardiac ECM formation in more detail, this study analyzed a large set of individually mutated Loh proteins for their capability to incorporate into the ECM and recruit Pericardin. To allow quantitative measurements of Pericardin recruitment efficiency, an in vivo recruitment assay was applied. In addition to the ECM of somatic muscles, this study investigated other types of matrices present in Drosophila for their capability to recruit Pericardin in a Loh-dependent manner. Furthermore, this study analyzed whether Pericardin, once recruited to a target matrix, has an intrinsic capacity to self-assemble into a meshwork independent of Loh. To perform the analysis, imaginal discs were used to express full-length Loh in distinct compartments of the disc and potential spreading was evaluated of the Pericardin meshwork over neighboring zones that lacked Loh (Rotstein, 2018).

Finally, to achieve an initial understanding of the biomechanical relevance of Pericardin, physiological consequences were sought of ectopic Pericardin deposition. In this respect, body wall muscles represent an effective readout system to investigate, for example, altered animal locomotion or lifespan. This study found that incorporation of Pericardin into the matrix of somatic muscles has no influence on lifespan but impairs contraction, thereby affecting the general locomotion performance (Rotstein, 2018).

In contrast to matrices present at the surface of most other tissues, the cardiac ECM is exposed to permanent mechanical stress generated by the regular and repetitive contraction cycles of the heart. These unique biomechanical conditions require ECM adaptation, which is achieved predominantly by incorporating specific structural components into the respective matrices. In Drosophila, one of these components is the type IV collagen-like protein Pericardin, which is recruited specifically to cardiac tissue by its adaptor protein Lonely heart. However, until now, neither the recruitment process itself nor the relevance of Pericardin to the biomechanical properties of the cardiac ECM have been studied in detail. By conducting a recruitment assay based on systematically generated domain-specific Loh mutants, this study found that presence of the first TSR1 domain is critical to localizing Loh to the ECM. Interestingly, mutating only the speculative GAG-binding site embedded within the first TSR1 domain is sufficient to abrogate Loh anchoring, indicating a high functional relevance of this distinct sequence motif. This result was confirmed by expressing the same constructs in Sf21 cells. Also in this system, deletion of the first TSR1 domain or mutation of the embedded putative GAG site resulted in considerably reduced surface localization of the respective Loh constructs. Significantly, Western blot analysis detected the proteins in the culture medium, which indicates that production and secretion still occurred, whereas incorporation into the ECM was impaired. Thus, the data suggest that TSR1-1, with its embedded putative GAG-binding site, is crucial for anchoring Loh to the ECM, which represents a prerequisite for the subsequent recruitment of Pericardin. On the other hand, the second speculative GAG binding site, embedded within the TSR1-4 domain, appears to be dispensable for localizing Loh but is required for efficient Pericardin recruitment. Of note, previous work identified the respective CXXTCXXG motif as a consensus site for O-fucosylation and showed that mutating this motif results in impaired protein secretion. Because the substitution in UAS-LohGAG2* covers this motif, slightly impaired secretion of this construct appears possible. However, its complete inability to recruit Pericardin cannot be attributed to minor deficiencies in secretion. Thus, the findings indicate that both speculative GAG-binding sites are of high functional relevance, with the first site being essential to proper anchoring of Loh, whereas the second one appears to be required for Pericardin recruitment. Interestingly, also lack of the second TSR1 domain results in failure to recruit Pericardin, whereas localization of Loh is not affected. Thus, the TSR1-2 and TSR1-4 domains as well as the putative GAG2-binding site seem to be dispensable for localizing Loh but crucial to proper Pericardin interaction and recruitment. In this context, the distinct position of the respective domains within Loh is probably decisive. According to structural modeling, TSR1-2 and TSR1-4, the latter containing the predicted GAG2 site, exhibit close spatial proximity. The fact that lack of either domain completely abolishes the capacity of Loh to recruit Pericardin indicates that these two domains constitute the interaction site between Loh and Prc, with the embedded speculative GAG binding site being of critical relevance. Subsequent to the initial binding, the nearby TSR1-3 and TSR1-5 domains may support interaction; however, their functional relevance is minor compared with TSR1-2 and TSR1-4. Taking these data into account, it appears likely that the N-terminal part of Loh, including the first TSR1 repeat and the embedded predicted GAG binding site, is facing the plasma membrane and anchors the protein to the cell surface, probably via glycosaminoglycan binding. The C-terminal part of Loh would then be available for interaction with Pericardin, and possibly also with other ECM components, via the second and fourth TSR1 repeats. Of note, a function of the PLAC domain, which is present in several enzymes and ECM proteins, such as ADAMTS-2, -3, -10, and others, was not uncovered by the approach of this study. Deleting the C-terminal PLAC domain in Loh has no distinct consequences, either for Loh secretion or for Pericardin recruitment efficiency, as far as can be stated in view of the sensitivity limitations of the test system (Rotstein, 2018).

Regarding the question of whether ECMs are generally capable of recruiting and incorporating Pericardin, this was found not to be the case. Whereas Loh-dependent recruitment was observed for fat body cells, somatic muscles, glial cells of the central nervous system, and wing discs, salivary gland cells did not incorporate Pericardin into the ECM, although Loh was present at the surface. This result indicates that other, yet unknown ECM components are required, in addition to Loh, for proper recruitment of Pericardin and that at least one of these factors is not present in salivary gland cells. Identification of the respective constituents represents an important objective of future studies because it would complement the current understanding of the interconnections that form the cardiac extracellular matrix. The alternative explanation, the presence of an inhibitory protein that prevents Pericardin incorporation into the ECM of salivary glands, appears unlikely, although this possibility cannot be ruled out for sure (Rotstein, 2018).

The impact of SPARC on age-related cardiac dysfunction and fibrosis in Drosophila

Tissue fibrosis, an accumulation of extracellular matrix proteins such as collagen, accompanies cardiac ageing in humans and this is linked to an increased risk of cardiac failure. The mechanisms driving age-related tissue fibrosis and cardiac dysfunction are unclear, yet clinically important. Drosophila is amenable to the study of cardiac ageing as well as collagen deposition; however it is unclear whether collagen accumulates in the ageing Drosophila heart. This work examined collagen deposition and cardiac function in ageing Drosophila, in the context of reduced expression of collagen-interacting protein SPARC (Secreted Protein Acidic and Rich in Cysteine) an evolutionarily conserved protein linked with fibrosis. Heart function was measured using high frame rate videomicroscopy. Collagen deposition was monitored using a fluorescently-tagged collagen IV reporter (encoded by the Viking gene) and staining of the cardiac collagen, Pericardin. The Drosophila heart accumulated collagen IV and Pericardin as flies aged. Associated with this was a decline in cardiac function. SPARC heterozygous flies lived longer than controls and showed little to no age-related cardiac dysfunction. As flies of both genotypes aged, cardiac levels of collagen IV (Viking) and Pericardin increased similarly. Over-expression of SPARC caused cardiomyopathy and increased Pericardin deposition. The findings demonstrate that, like humans, the Drosophila heart develops a fibrosis-like phenotype as it ages. Although having no gross impact on collagen accumulation, reduced SPARC expression extended Drosophila lifespan and cardiac health span. It is proposed that cardiac fibrosis in humans may develop due to the activation of conserved mechanisms and that SPARC may mediate cardiac ageing by mechanisms more subtle than gross accumulation of collagen (Vaughan, 2017).

Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response

Blood cell development in Drosophila shares significant similarities with vertebrate. The conservation ranges from biphasic mode of hematopoiesis to signaling molecules crucial for progenitor cell formation, maintenance, and differentiation. Primitive hematopoiesis in Drosophila ensues in embryonic head mesoderm, whereas definitive hematopoiesis happens in larval hematopoietic organ, the lymph gland. This organ, with the onset of pupation, ruptures to release hemocytes into circulation. It is believed that the adult lacks a hematopoietic organ and survives on the contribution of both embryonic and larval hematopoiesis. However, these studies revealed a surge of blood cell development in the dorsal abdominal hemocyte clusters of adult fly. These active hematopoietic hubs are capable of blood cell specification and can respond to bacterial challenges. The presence of progenitors and differentiated hemocytes embedded in a functional network of Laminin A and Pericardin within this hematopoietic hub projects it as a simple version of the vertebrate bone marrow (Ghosh, 2015).

Employing hemolectin-Gal4, UAS-GFP, this study has identified four hematopoietic blood cell clusters along the dorsal midline in the abdominal segments A1-A4 of adult flies. Of the four clusters, the one in the abdominal segment A1 has the maximum aggregation of cells that occupies the area that spans the lateral and dorsal sides of the heart. Located just below the dorsal cuticle of the abdominal cavity, the cells are assembled in a groove defined by transverse heart muscles and body wall muscles. The longitudinal heart muscle forming the dorsal diaphragm separates the heart and the cluster from abdominal cavity. With respect to the pericardial diaphragm formed by pericardial cells present on either side of the cardiac tube, these hemocytes are located dorsally. Thus, these clusters remain secluded from rest of the abdominal cavity by the dorsal and the pericardial diaphragm (Ghosh, 2015).

The hemocytes within the clusters are embedded in an extensive network of extracellular matrix proteins surrounding the heart and the pericardial cells. One of the important components of this network is the type IV collagen-like protein, Pericardin. In homozygous mutant for lonely heart (loh) , a gene encoding a secreted receptor of Pericardin (Drechsler, 2013), the hemocytes fail to form the cluster, as this network gets significantly affected. Similar results are observed upon knocking down the expression of Laminin A (another important component of the network) by driving UAS-laminin A RNAi in the cardiac tube by mef2-Gal4. Based on expression and functional analyses, it is concluded that both Pericardin and Laminin A function in maintaining adhesive interaction with the hemocytes, thus aiding in formation of the clusters. Interestingly, Laminin A polypeptides and collagen IV are also prevalent in vertebrate bone marrow. The finding that the blood cells are fenestrated in a functional network of Laminin A and Pericardin raises the speculation that these sites might function as bone marrow-like tissues in adult flies and thereby demands an in-depth analysis of the cell types present therein (Ghosh, 2015).

For detailed characterization of the cell types, this study focused on the largest aggregation present in the segment A1. Primarily based on the expression of peroxidasin-GFP (pxn-GFP) and NimC1/ P1, the cluster was found to house a large number of plasmatocytes, the most predominant differentiated blood cell. Interestingly, the cells in the cluster express croquemort (crq), an embryonic marker for plasmatocytes. The embryonic origin of the plasmatocytes was further validated by using G-TRACE construct that enables detection of cells that had once expressed any particular gene prior to the time of investigation (lineage traced) as well as those in which the gene is expressed at the time of observation (live expression). Activation of G-TRACE system by a Gal4 for glial cell missing (gcm), a gene known to express exclusively in embryonic plasmatocytes, results in the detection of few P1-positive gcm lineage traced (enhanced Green Fluorescent Protein [EGFP]) cells, thereby confirming that the cluster harbors plasmatocytes of embryonic origin. In addition to these markers, the cells in the cluster express several lymph gland hemocyte-specific markers like ZCL2897, and are Serpent (Srp) and dorothy-GFP positive, even some of them are lineage traced for collier. Thus, the hemocyte clusters are a medley of embryonic and larval lineages (Ghosh, 2015).

Despite one report that suggests the presence of C4 expressing crystal cells in adult circulation, it is considered that crystal cells are not present in adults. Primarily, this is due to the absence of any Prophenoloxidase (proPO) expressing crystal cell in circulation. This study, however, observed that 5-days post-eclosion (dpe), there are some Hindsight (Hnt)-positive crystal cells present within the cluster. Co-localization of lozenge-GFP (lz) with proPO further supports these findings. To have a functional correlate, activation of proPO was heat induced in crystal cells. This results in formation of melanized crystal cells on dorsal side of the abdomen, corresponding to the position of first cluster. These results clearly establish the presence of resident functional crystal cells in the clusters (Ghosh, 2015).

GATA factor Serpent (Srp) is expressed in low levels in all hemocytes, including plasmatocytes and crystal cells. However, the hemocyte precursor cell can be identified by the presence of high levels of Srp expression. Analysis of developing cluster at 2 dpe reveals the presence of cells positive for both Srp and Hemolectin (plasmatocytes), and a small subset of cells exclusively expressing Srp. No crystal cells (Hnt) are present in the cluster. In contrast, at 5 dpe, along with the two cell types mentioned above, some Srp- and Hnt-positive crystal cells are seen. Quantitative analysis of the above observations clearly demonstrate an increase in the number of differentiated cells (plasmatocytes and crystal cells) with a concomitant decline in the number of cells exclusively expressing Srp at 5 versus 2 dpe. These results also indicate that the Srp-positive cells within the cluster that do not express either hml or Hnt might be the precursor cells, yet to turn on differentiation (Ghosh, 2015).

Crystal cell development was followed in the cluster. Since activation of Notch (N) pathway precedes Lz expression in crystal cells, a recombinant fly line was generated with 12XSu(H)lacZ in the background of lz-GFP. Su(H) lacZ-positive cells are first seen in the cluster 2 dpe, whereas the expression of lz-GFP is observed only on 3 dpe. Interestingly, some of these lzGFP-positive cells still have low levels of Su(H)lacZ expression. By 5 dpe, an increase was observed in the number of cells that are either expressing lz-GFP or have low levels of Su(H)lacZ expression along with lz-GFP expression. However, at 7 dpe, while an increase in number of lz-GFP cells can be seen, there is a decrease in the number of double-positive cells. The number of cells expressing only Su(H)lacZ that remain more or less unaltered till 5 dpe demonstrates a sharp decline on day 7. Quantitative analysis of the cell types present in the cluster based on the expressions of Su(H)lacZ and lz-GFP further ascertains the above observations. These results, therefore, clearly demonstrate de novo origin of lz-GFP-positive crystal cells from Su(H)lacZ-positive cells within the cluster (Ghosh, 2015).

To determine whether Su(H)lacZ-positive cells originate from the Srp positive precursors, the the expression of both Srp and Su(H)lacZ within the cluster was monitored. Initially, while some cells that turn on Su(H)lacZ have high levels of Srp expression in subsequent days, as the Su(H)lacZ expression gets stabilized, a reduction in Srp expression is observed. This result establishes that crystal cells develop in adult cluster from high Srp-positive precursor cells, and this process requires N signaling. As a functional correlate to establish the presence of precursor cells in the cluster, N signaling was tweaked to determine its effect on differentiation of crystal cells. Since the onset of Su(H)lacZ and lz-GFP expression in the cluster is observed at 2 and 3 dpe, respectively, N signaling was impaired in the precursors by driving UAS-N RNAi using hemese-Gal4 from 2 dpe. This resulted in complete loss of crystal cells compared with that observed in WT clusters. Interestingly, the marginal increase in the number of plasmatocytes observed by knocking down N correlates with the number of crystal cells missing in this genetic background when compared to control. Likewise, overexpressing N in these cells results in almost 7-fold increase in the number of crystal cells with a significant drop in the number of plasmatocytes (Ghosh, 2015).

It is therefore quite evident from the results that the clusters of blood cells on the dorsal side of the adult fly are not a mere aggregation of hemocytes of embryonic and larval origin but also houses true progenitors. The very fact that they house blood cell precursors and exhibit dynamicity as de novo crystal cells get differentiated within them qualifies them to be considered as active hubs of hematopoiesis in adult (Ghosh, 2015).

Upon identifying the hemocyte precursors, attempts were made to define their origin. The results demonstrate that collier lineage traced progenitors in the hub originate from the hemocyte precursors present in the tertiary and quaternary lobes of larval lymph gland and that they can give rise to both plasmatocytes and crystal cells (Ghosh, 2015).

In summary this study unravels the presence of active hematopoietic hubs in Drosophila adults. Refuting the existing notion that adults rely on long-lived hemocytes originating from embryonic and larval stages, this study was successful in establishing that a surge of hematopoiesis happens in these hubs as the precursors present within differentiate into both crystal cells and plasmatocytes. The functionality of the hub gets further validated, since it was observed that besides exhibiting phagocytic activity the otherwise quiescent cells re-enter into proliferative mode in response to bacterial infection. These findings bring about a paradigm shift in understanding of the process of hematopoiesis in Drosophila. With its well-characterized embryonic and larval hematopoietic activities, Drosophila has been serving as a powerful model for hematopoietic studies. In spite of that, the system seemed to be incomplete due to lack of detailed developmental analysis of hematopoiesis in adults. This effort in establishing that the process of definitive hematopoiesis extends to adults expands the scope of exploiting this model system (Ghosh, 2015).

The conserved ADAMTS-like protein lonely heart mediates matrix formation and cardiac tissue integrity

This paper reports on the identification and functional characterization of the ADAMTS-like homolog lonely heart (loh) in Drosophila melanogaster. Loh displays all hallmarks of ADAMTSL proteins including several thrombospondin type 1 repeats (TSR1), and acts in concert with the collagen Pericardin (Prc). Loss of either loh or prc causes progressive cardiac damage peaking in the abolishment of heart function. This study shows that both proteins are integral components of the cardiac ECM mediating cellular adhesion between the cardiac tube and the pericardial cells. Loss of ECM integrity leads to an altered myo-fibrillar organization in cardiac cells massively influencing heart beat pattern. Evidence is shown that Loh acts as a secreted receptor for Prc and works as a crucial determinant to allow the formation of a cell and tissue specific ECM, while it does not influence the accumulation of other matrix proteins like Nidogen or Perlecan. These findings demonstrate that the function of ADAMTS-like proteins is conserved throughout evolution and reveal a previously unknown interaction of these proteins with collagens (Drechsler, 2013).

The establishment and maintenance of extracellular matrices (ECM) are important tasks to allow proper organ function in metazoans. Among other factors, changes in ECM composition, turnover and homeostasis are crucial mediators of human cardiovascular disease leading to life threatening conditions and premature death. The ECM allows cells to resist mechanical forces, protects complex tissues from being damaged and promotes specific physical properties like elasticity or stiffness in order to maintain organ functionality. While the composition of the ECM is very complex and extremely variable the basic structural constituents can be grouped as collagens, glycoproteins and proteoglycans, which are highly conserved throughout metazoan specie. Consequently, defects in ECM proteins or matrix composition cause major developmental defects and strongly contribute to prevalent human disease like fibroses or cancer. During the last years fibrotic disease and mutations in various ECM proteins were correlated to cardiovascular disease. For example mutations in human Col4a1 cause the weakening of the major vasculature leading to life threatening aneurysms or stroke while mutations in murine Col4a1 and Col4a2 induce vascular defects causing internal bleedings and prenatal lethality. Even more recently ADAMTS-like (ADAMTSL, A Disintegrin and Metalloprotease with Thrombospondin repeats) proteins have gained significant importance in the understanding of certain types of fibrillinopathies. Mutations in human ADAMTSL4 were identified in patients suffering from isolated ectopia lentis (EL), a recessive disorder of the occular lense, and, more severely, aberrations in ADAMTSL2 cause geleophysic dysplasia a syndrome which, amongst others, manifests in the thickening of the vascular valves and progressive cardiac failure causing premature death. Unfortunately, despite the pathological mutations no ADAMTSL alleles in genetically treatable model systems were described so far (Drechsler, 2013).

This study used Drosophila melanogaster as a model of ECM function in the cardiac system. In Drosophila the maintenance of cardiac integrity is of great importance, since no mechanisms of cardiac cell replacement or tissue repair exist. A variety of mutations in ECM genes have been analyzed with respect to their function in different tissues and processes like neurogenesis, muscle attachment, wing development and others. Cardiogenesis in the fly embryo depends on several ECM components including the evolutionarily conserved toolkit of proteins forming the basement membrane. The basement membrane constitutes a specialized type of ECM consisting of Laminins, Collagen IV, Perlecan and Nidogen found at the basal side of epithelial cells. The interaction of laminins with cellular receptors like integrins or dystroglycan and its self-assembly into a higher meshwork forms the initial step of basement membrane formation in animals. Consequently, mutations in any of the four laminin encoding genes in Drosophila lead to severe embryonic cardiac defects. For example loss of lanB1, encoding the only β-subunit of the laminin trimer, prevents the accumulation of collagen IV and perlecan towards cardiac cells, while mutations in lanA and lanB2 (encoding the α3,5-subunit and the γ-subunit, respectively) cause the detachment of pericardial cells, a specific type of nephrocytes in arthropods, from the heart tube. The highly abundant proteins forming the basement membrane have in common that they are distributed ubiquitously and cover all internal organs of the fly (Drechsler, 2013).

Compared to that the cardiac ECM is unique, since it contains the collagen Pericardin (Prc), which is rather specifically decorating the heart tube. Prc displays certain homologies to mammalian collagen IV and was shown to be crucial for heart morphogenesis and cardiac cell to pericardial cell adhesion. However, the question of how Prc accumulates in a cell specific manner in the fly embryo or how specific matrices are specified in the rather open body cavity of insects in general was not addressed in detail so far. This study introduces the gene lonely heart (loh), which is crucial to maintain cardiac integrity during postembryonic developmental stages. This study shows that Loh is a member of the ADAMTSL protein family and constitutes the essential mediator of Prc accumulation and matrix formation already in embryonic cardiac tissue. ADAMTSL proteins belong to the evolutionary conserved family of ADAM proteases with the exception that these proteins lack a proteolytically active domain in their primary sequence and therefore its function is unclear. This study found evidence that Loh is sufficient to specifically recruit Prc to the ECM of different tissues indicating that Loh regulates the assembly of tissue and organ specific matrices. This is of great interest since the composition of the ECM determines its mechanical properties crucial for correct organ function and cellular behavior. The physiological relevance of cardiac integrity is addressed, and and this study shows that lack of either loh or prc prevents proper blood circulation in the animals and cause a reduction of the fly's life span. These findings demonstrate that mutations in ADAMTSL proteins lead, like in human disease, to progressive heart failure and premature death in flies, strongly arguing for an evolutionary conserved function (Drechsler, 2013).

Based on the primary sequence the domain architecture of Loh is extremely similar to that of vertebrate ADAMTSL6 and is likely to be its ortholog. Furthermore, ADAMTSL6 is the only protein of this family known to produce two transcriptional isoforms from one gene locus. In contrast to Loh the shorter ADAMTSL6 isoform was found to be functional in organizing the ECM in mice. The current data demonstrate that LohA, the larger protein, is functional and sufficient to mediate matrix formation in Drosophila while the role of the shorter isoform C remains elusive by now. However, since the lohC transcript is not expressed during embryogenesis, the critical time window of loh function, any role of LohC in mediating cardiac ECM formation is excluded (Drechsler, 2013).

By testing different ECM proteins this study demonstrated that Prc, a collagen with a very restricted distribution in the animal, is particularly affected in all isolated loh mutant alleles, emphasizing the specific function of Loh to promote Prc matrix formation. Consequently, the first prc mutant allele, which phenocopies the cardiac defects found in loh mutant strains, was isolated. In loh mutant animals Prc mislocalizes along the heart already during embryogenesis, leading to a progressive loss of tissue integrity, which eventually causes the observed collapse of the heart tube and an abolishment of cardiac activity. The main function of both proteins is therefore the mediation of cellular adhesion between the heart, the pericardial cells and the alary muscles which further connect the whole organ system to the body cavity (Drechsler, 2013).

In addition to the cell adhesion defects it was also found that the process of heart lumen formation was impaired in prc but not loh mutants. Since the details of this phenotype have not been followed up the role of Prc in lumen formation remains elusive for now. However, the data implicates that the presence of Prc is critical to allow cardioblasts to seal the lumen correctly, while the correct localization of Prc into the matrix seems not to be essential for this process (Drechsler, 2013).

Analyzing the embryonic and larval expression patterns of loh and prc revealed that both genes are predominantly active during the growing stages of the animal and become deactivated after the heart has grown to its final size. In the embryo, both genes are transcribed in either the same or very proximate cells indicating that the proteins are not distributed over longer distances once they are secreted. Importantly, the final localization of Prc therefore mainly follows the expression of loh. This can be seen best in the oenocytes of the embryo, where Prc becomes secreted but later on mainly localizes to the overlying chordotonal organs that in turn express loh. Thus, loh expression is a prerequisite for the successful establishment of a Prc matrix. This local protein distribution changes during larval stages. As demonstrated by an inhibited secretion in adipocytes of prc>sar1-IR animals, Prc becomes strongly expressed by the fat body during early larval stages. Hence, the protein becomes distributed over longer distances in the larva but still decorates organs and tissues that initially expressed loh. Based on these data, a conceptual model is provided in which Loh predetermines the ECM to allow Prc to become coupled to the cell surface and to be organized into a reticular matrix. Previously it was shown that Collagen IV, the major collagen in the basement membrane, becomes also secreted by adipocytes and distributes through the hemolymph. It can now proven that Prc as a second collagen is also synthesized by the larval fat body, which enhances the importance of this organ for ECM biogenesis. The developmental change in prc expression might therefore be explained by the ongoing differentiation of pericardial cells into mature nephrocytes during larval stages. While embryonic pericardial cells are able to secrete large amounts of protein into the extracellular space, the major function of pericardial nephrocytes is endocytosis, thus requiring adipocytes to take over Prc production. Finally the results show that the cardiac matrix is maintained during larval growing phases presumably by the consecutive incorporation of fat body derived Prc (Drechsler, 2013).

The ectopic expression of Loh showed that the secreted protein is readily incorporated into different matrices raising the question how Loh itself interacts with the ECM in general. At the moment it is not fully understood if ADAMTSL proteins interact with miscellaneous ECM components or require specific cell surface receptors. Based on the spatial proximity of Loh to βPS integrin it is speculate that Loh may interact with integrin receptors and link these to Prc bundles, thereby promoting the connection of the Prc network to the cell surface. This idea is supported by the observed changes in fiber orientation of mutant cardiomyocytes. Since it is known that integrins are connected to the underlying Z-disks of muscle cells by a structure called the costamere it is proposed that lack of integrin-ECM binding induces the redistribution of myofibrils. However, there is no evidence of an interaction between ADAMTSL proteins and integrins or any other cellular receptor so far. Nevertheless, in such a model Loh would allow the specific binding of specialized ECM molecules to only some unique matrices. Since Drosophila possesses only two β integrin subunits the number of α/β-dimers is limited and the use of Loh as an adapter molecule increases the diversity of matrix composition and opens up the possibility to create sub-functional matrices. Furthermore, integrin mediated binding seems to influence the correct assembly of Prc since previous findings already showed that lack of αPS3- or βPS integrin can interfere with the distribution of Prc and induce pericardial cell detachment phenotypes (Drechsler, 2013).

In addition to a receptor mediated ECM incorporation of Loh, binding might also be achieved by some or all of the five TSR1 domains found in the primary sequence of the protein. Previously it was demonstrated that ADAMTS(L) proteins can bind to the ECM via the various TSR1 motifs that interact with glycosaminoglycans. This would not need special receptors and allow Loh to incorporate into any matrix. The cell specific expression of loh would then mainly decide which matrix will incorporate Prc and this would in turn strongly depend on the cis-regulation of the gene's expression (Drechsler, 2013).

On the molecular level it is proposed that Loh basically acts as a linker protein. Based on the ectopic expression of Loh and the co-immunoprecipitation experiments it was demonstrated that Loh and Prc interact in vivo. Loh behaves like a secreted receptor molecule that specifically recruits Prc to the cell surface. The findings indicate that the main molecular function might therefore be binding, but does not exclude additional functions of the protein. It was suggested previously that ADAMTSL proteins act as regulators of extracellular proteases and thereby regulate ECM content and composition. For example it was demonstrated that Drosophila Papilin, another member of ADAMTSL related proteins, is sufficient to inhibit a vertebrate procollagen proteinase in vitro. Thus, it is possible that also Loh regulates a so far unknown proteinase that renders the matrix unsuitable for the accumulation of Prc in some way. In such a model the activity of Loh would then influence the pre-existing microenvironment around a cell to allow Prc to assemble into a network. However, there is no evidence for such a function or the involvement of proteinases so far (Drechsler, 2013).

The observed roles of Loh in Drosophila partially reflect the function of ADAMTSL proteins in vertebrates, which were shown to organize Fibrillin-1 (FBN1) microfibrils in specialized matrices. Genetic and biochemical analyses showed that ADAMTSL4 and ADAMSTL6 are sufficient to mediate the formation of FBN1 fibrils in cultured fibroblasts as well as in vivo. ADAMTSL4 acts as a FBN1 binding protein that mediates microfibril assembly in the zonule fibers of the human eye leading to isolated ectopia lentis (IEL) if mutated. Thus, IEL is caused predominantly by altered mechanical properties of the zonular fibers leading to a progressive dislocation of the lens. In Drosophila, where no FBN1 homolog exists, Loh interacts with Prc and mediates its distribution within the ECM in a very similar manner. Therefore, the correct assembly of Prc between the pericardial cells and the heart tube could promote the mechanical properties needed to sustain the permanent mechanical forces during heartbeat. The clinical phenotypes of geleophysic dysplasia (GD) observed in ADAMTSL2 mutant patients exceed a function of simply promoting mechanical stability of the ECM. It was shown that ADAMTSL2 binds to FBN1 but also interacts with LTBP1, a regulator of TGFβ signaling, and therefore the phenotypes of GD also include growing defects, muscular hypertrophy and thickening of the skin [9]. None of these additional phenotypes were observed in Drosophila loh mutants. Therefore, it is obvious that ADAMTSL proteins developed novel functions during evolution making them essential mediators of ECM development and homeostasis. So far there are no reports of interactions between any ADAMTSL proteins with collagens but the obviously similar functions in flies and vertebrates strongly argue for a conserved function in organizing fibrillar matrix proteins (Drechsler, 2013).

Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure

The steps that lead to the formation of a single primitive heart tube are highly conserved in vertebrate and invertebrate embryos. Concerted migration of the two lateral cardiogenic regions of the mesoderm and endoderm (or ectoderm in invertebrates) is required for their fusion at the midline of the embryo. Morphogenetic signals are involved in this process and the extracellular matrix has been proposed to serve as a link between the two layers of cells. Pericardin (Prc), a novel Drosophila extracellular matrix protein is a good candidate to participate in heart tube formation. The protein has the hallmarks of a type IV collagen alpha-chain and is mainly expressed in the pericardial cells at the onset of dorsal closure. As dorsal closure progresses, Pericardin expression becomes concentrated at the basal surface of the cardioblasts and around the pericardial cells, in close proximity to the dorsal ectoderm. Pericardin is absent from the lumen of the dorsal vessel. Genetic evidence suggests that Prc promotes the proper migration and alignment of heart cells. Df(3)vin6 embryos, as well as embryos in which prc has been silenced via RNAi, exhibit similar and significant defects in the formation of the heart epithelium. In these embryos, the heart epithelium appears disorganized during its migration to the dorsal midline. By the end of embryonic development, cardial and pericardial cells are misaligned such that small clusters of both cell types appear in the heart; these clusters of cells are associated with holes in the walls of the heart. A prc transgene can partially rescue each of these phenotypes, suggesting that prc regulates these events. These results support, for the first time, the function of a collagen-like protein in the coordinated migration of dorsal ectoderm and heart cells (Chartier, 2002).


REFERENCES

Search PubMed for articles about Drosophila Pericardin

Chartier, A., Zaffran, S., Astier, M., Semeriva, M. and Gratecos, D. (2002). Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure. Development 129: 3241-3253. PubMed ID: 12070098

Drechsler, M., Schmidt, A. C., Meyer, H. and Paululat, A. (2013). The conserved ADAMTS-like protein lonely heart mediates matrix formation and cardiac tissue integrity. PLoS Genet 9: e1003616. PubMed ID: 23874219

Ghosh, S., Singh, A., Mandal, S. and Mandal, L. (2015). Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response. Dev Cell 33(4):478-88. PubMed ID: 25959225

Pastor-Pareja, J. C. and Xu, T. (2011). Shaping cells and organs in Drosophila by opposing roles of fat body-secreted Collagen IV and perlecan. Dev Cell 21(2): 245-256. PubMed ID: 21839919

Rotstein, B., Post, Y., Reinhardt, M., Lammers, K., Buhr, A., Heinisch, J. J., Meyer, H. and Paululat, A. (2018). Distinct domains in the matricellular protein Lonely heart are crucial for cardiac extracellular matrix formation and heart function in Drosophila. J Biol Chem 293(20): 7864-7879. PubMed ID: 29599288

Sessions, A. O., Kaushik, G., Parker, S., Raedschelders, K., Bodmer, R., Van Eyk, J. E. and Engler, A. J. (2017). Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan. Matrix Biol 62: 15-27. PubMed ID: 27793636

Vaughan, L., Marley, R., Miellet, S. and Hartley, P. S. (2018). The impact of SPARC on age-related cardiac dysfunction and fibrosis in Drosophila. Exp Gerontol 109: 59-66. PubMed ID: 29032244

Volk, T., Wang, S., Rotstein, B. and Paululat, A. (2014). Matricellular proteins in development: perspectives from the Drosophila heart. Matrix Biol 37: 162-166. PubMed ID: 24726952

Wilmes, A. C., Klinke, N., Rotstein, B., Meyer, H. and Paululat, A. (2018). Biosynthesis and assembly of the Collagen IV-like protein Pericardin in Drosophila melanogaster. Biol Open 7(4). PubMed ID: 29685999


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date revised: 22 July 2018

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