InteractiveFly: GeneBrief

Viking and Collagen type IV alpha 1: Biological Overview | References

Gene name - viking and Collagen type IV alpha 1

Synonyms -

Cytological map position - 25C1-25C1 and 25C1-25C1

Function - ligand-binding extracellular matrix structural components

Keywords - fat body, mesoderm, hemocyte, BMP pathway hemolymph, major determinant of organ shape and animal form, basement membrane

Symbols - vkg and Col4a1

FlyBase IDs: FBgn0016075 and FBgn0000299

Genetic map positions - chr2L:5012144-5027412 and chr2L:5029394-5037306

Classification - C4: C-terminal tandem repeated domain in type 4 procollagen

Cellular location - secreted

NCBI links for Viking: EntrezGene
NCBI links Col4a1 : EntrezGene

vkg orthologs: Biolitmine
Col4a1 orthologs: Biolitmine

Recent literature
Zang, Y., et al. (2015). Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes. Elife [Epub ahead of print]. PubMed ID: 26090908
Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. This study shows that plasma membrane overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. Loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of plasma membrane is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess plasma membrane being caused in this case by increased secretion. Finally, trimeric Collagen accumulation, downstream of Toll or endocytic defects, was shown to activate a tissue damage response. This work indicates that traffic imbalances and plasma membrane topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases.

Van De Bor, V., Zimniak, G., Papone, L., Cerezo, D., Malbouyres, M., Juan, T., Ruggiero, F. and Noselli, S. (2015). Companion blood cells control ovarian stem cell niche microenvironment and homeostasis. Cell Rep [Epub ahead of print]. PubMed ID: 26456819
The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. This study uncovers an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. A distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward was identified. Expressing tagged collagen IV tissue specifically, it was shown that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

Cummings, C. F., et al. (2016). Extracellular chloride signals collagen IV network assembly during basement membrane formation. J Cell Biol 213: 479-494. PubMed ID: 27216258
Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. This study reports that extracellular Cl(-) ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl(-) in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins. Phylogenetic evidence indicates this conformational switch is a fundamental mechanism of collagen IV network assembly throughout Metazoa. Using recombinant triple helical protomers, this study proves that NC1 domains direct both protomer and network assembly and shows in Drosophila that NC1 architecture is critical for incorporation into basement membranes. These discoveries provide an atomic-level understanding of the dynamic interactions between extracellular Cl(-) and collagen IV assembly outside cells, a critical step in the assembly and organization of basement membranes that enable tissue architecture and function. Moreover, this provides a mechanistic framework for understanding the molecular pathobiology of NC1 domains.
Crest, J., Diz-Munoz, A., Chen, D. Y., Fletcher, D. A. and Bilder, D. (2017). Organ sculpting by patterned extracellular matrix stiffness. Elife 6. PubMed ID: 28653906
How organ-shaping mechanical imbalances are generated is a central question of morphogenesis, with existing paradigms focusing on asymmetric force generation within cells. This study shows that organs can be sculpted instead by patterning anisotropic resistance within their extracellular matrix (ECM). Using direct biophysical measurements of elongating Drosophila egg chambers, this study documents robust mechanical anisotropy in the ECM-based basement membrane (BM) but not the underlying epithelium. Atomic force microscopy (AFM) on wild-type BM in vivo reveals an A-P symmetric stiffness gradient, which fails to develop in elongation-defective mutants. Genetic manipulation of ECM components Collagen IV, Laminin, and Perlecan showed that the BM is instructive for tissue elongation and the determinant is relative rather than absolute stiffness, creating differential resistance to isotropic tissue expansion. The stiffness gradient requires morphogen-like signaling to regulate BM incorporation, as well as planar-polarized organization to homogenize it circumferentially. These results demonstrate how fine mechanical patterning in the ECM can guide cells to shape an organ.
Dai, J., Ma, M., Feng, Z. and Pastor-Pareja, J. C. (2017). Inter-adipocyte adhesion and signaling by Collagen IV intercellular concentrations in Drosophila. Curr Biol 27(18): 2729-2740.e2724. PubMed ID: 28867208
Sheet-forming Collagen IV is the main component of basement membranes, which are planar polymers of extracellular matrix underlying epithelia and surrounding organs in all animals. Adipocytes in both insects and mammals are mesodermal in origin and often classified as mesenchymal. However, they form true tissues where cells remain compactly associated. Neither the mechanisms providing this tissue-level organization nor its functional significance are known. This study shows that discrete Collagen IV intercellular concentrations (CIVICs), distinct from basement membranes and thicker in section, mediate inter-adipocyte adhesion in Drosophila. Loss of these Collagen-IV-containing structures in the larval fat body caused intercellular gaps and disrupted continuity of the adipose tissue layer. Integrin and Syndecan matrix receptors attach adipocytes to CIVICs and direct their formation. Finally, Integrin-mediated adhesion to CIVICs was shown to promote normal adipocyte growth and prevents autophagy through Src-Pi3K-Akt signaling. These results evidence a surprising non-basement membrane role of Collagen IV in non-epithelial tissue morphogenesis while demonstrating adhesion and signaling functions for these structures.
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
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.
Ke, H., Feng, Z., Liu, M., Sun, T., Dai, J., Ma, M., Liu, L. P., Ni, J. Q. and Pastor-Pareja, J. C. (2018). Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements. J Genet Genomics. PubMed ID: 29935791
This study reports an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, distribution of GFP-tagged Collagen IV was examined in live animals, and 88 gene hits were found for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4alphaEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in the hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. Additionally a characterization of a screening hit, trabuco (tbc), is presented, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, the success is discussed of this screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells.
Weaver, L. N. and Drummond-Barbosa, D. (2018). Maintenance of proper germline stem cell number requires adipocyte collagen in adult Drosophila females. Genetics. PubMed ID: 29884747
Stem cells reside in specialized niches and are regulated by a variety of physiological inputs. Adipocytes influence whole-body physiology and stem cell lineages; however, the molecular mechanisms linking adipocytes to stem cells are poorly understood. This study reports that collagen IV produced in adipocytes is transported to the ovary to maintain proper germline stem cell (GSC) number in adult Drosophila females. Adipocyte-derived collagen IV acts through beta-integrin signaling to maintain normal levels of E-cadherin at the niche, thereby ensuring proper adhesion to GSCs. These findings demonstrate that extracellular matrix components produced in adipocytes can be transported to and incorporated into an established adult tissue to influence stem cell number.
Ramos-Lewis, W., LaFever, K. S. and Page-McCaw, A. (2018). A scar-like lesion is apparent in basement membrane after wound repair in vivo. Matrix Biol. PubMed ID: 29981372
Basement membrane is a highly conserved sheet-like extracellular matrix in animals, underlying simple and complex epithelia, and wrapping around tissues like muscles and nerves. Like the tissues they support, basement membranes become damaged by environmental insults. Although it is clear that basement membranes are repaired after damage, virtually nothing is known about this process. For example, it is not known how repaired basement membranes compare to undamaged ones, whether basement membrane components are necessary for epithelial wound closure, or whether there is a hierarchy of assembly that repairing basement membranes follow, similar to the hierarchy of assembly of embryonic basement membranes. This report addresses these questions using the basement membrane of the Drosophila larval epidermis as a model system. By analyzing the four main basement membrane proteins - laminin, collagen IV, perlecan, and nidogen - it was found that although basement membranes are repaired within a day after mechanical damage in vivo, thickened and disorganized matrix scars are evident with all four protein components. The new matrix proteins that repair damaged basement membranes are provided by distant adipose and muscle tissues rather than by the local epithelium, the same distant tissues that provide matrix proteins for growth of unwounded epithelial basement membranes. To identify a hierarchy of repair, the dependency of each of the basement membrane proteins on the others was tested for incorporation after damage. For proper incorporation after damage, nidogen requires laminin, and perlecan requires collagen IV, but surprisingly collagen IV does not to depend on laminin. Thus, the rules of basement membrane repair are subtly different than those of de novo assembly.
Sauerwald, J., Backer, W., Matzat, T., Schnorrer, F. and Luschnig, S. (2019). Matrix metalloproteinase 1 modulates invasive behavior of tracheal branches during entry into Drosophila flight muscles. Elife 8. PubMed ID: 31577228
Tubular networks like the vasculature extend branches throughout animal bodies, but how developing vessels interact with and invade tissues is not well understood. This study investigated the underlying mechanisms using the developing tracheal tube network of Drosophila indirect flight muscles (IFMs) as a model. Live imaging revealed that tracheal sprouts invade IFMs directionally with growth-cone-like structures at branch tips. Ramification inside IFMs proceeds until tracheal branches fill the myotube. However, individual tracheal cells occupy largely separate territories, possibly mediated by cell-cell repulsion. Matrix metalloproteinase 1 (MMP1) is required in tracheal cells for normal invasion speed and for the dynamic organization of growth-cone-like branch tips. MMP1 remodels the CollagenIV-containing matrix around branch tips, which show differential matrix composition with low CollagenIV levels, while Laminin is present along tracheal branches. Thus, tracheal-derived MMP1 sustains branch invasion by modulating the dynamic behavior of sprouting branches as well as properties of the surrounding matrix.
Van De Bor, V., Loreau, V., Malbouyres, M., Cerezo, D., Placenti, A., Ruggiero, F. and Noselli, S. (2021). A dynamic and mosaic basement membrane controls cell intercalation in Drosophila ovaries. Development 148(4). PubMed ID: 33526583
Basement membranes (BM) are extracellular matrices assembled into complex and highly organized networks essential for organ morphogenesis and function. However, little is known about the tissue origin of BM components and their dynamics in vivo, This study unravel the assembly and role of the BM main component, Collagen type IV (ColIV), in Drosophila ovarian stalk morphogenesis. Stalks are short strings of cells assembled through cell intercalation that link adjacent follicles and maintain ovarian integrity. Stalk ColIV has multiple origins and is assembled following a regulated pattern leading to a unique BM organisation. Absence of ColIV leads to follicle fusion, as observed upon ablation of stalk cells. ColIV and integrins are both required to trigger cell intercalation and maintain mechanically strong cell-cell attachment within the stalk. These results show how the dynamic assembly of a mosaic BM controls complex tissue morphogenesis and integrity.
Luo, W., Liu, S., Zhang, W., Yang, L., Huang, J., Zhou, S., Feng, Q., Palli, S. R., Wang, J., Roth, S. and Li, S. (2021). Juvenile hormone signaling promotes ovulation and maintains egg shape by inducing expression of extracellular matrix genes. Proc Natl Acad Sci U S A 118(39). PubMed ID: 34544864
It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. This study describes a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction (Luo, 2021).
Ready, D. F. and Chang, H. C. (2021). Calcium waves facilitate and coordinate the contraction of endfeet actin stress fibers in Drosophila interommatidial cells. Development. PubMed ID: 34698814
Actomyosin contraction shapes the Drosophila eye's panoramic view. The convex curvature of the retinal epithelium, organized in ∼800 close-packed ommatidia, depends upon a fourfold condensation of the retinal floor mediated by contraction of actin stress fibers in the endfeet of interommatidial cells (IOCs). How these tensile forces are coordinated is not known. This study discovered a novel phenomenon: Ca2+ waves regularly propagate across the IOC network in pupal and adult eyes. Genetic evidence demonstrates that IOC waves are independent of phototransduction, but require inositol 1,4,5-triphosphate receptor (IP3R), suggesting these waves are mediated by Ca2+ releases from ER stores. Removal of IP3R disrupts stress fibers in IOC endfeet and increases the basal retinal surface by ∼40%, linking IOC waves to facilitating stress fiber contraction and floor morphogenesis. Further, IP3R loss disrupts the organization of a collagen IV network underneath the IOC endfeet, implicating ECM and its interaction with stress fibers in eye morphogenesis. It is proposed that coordinated cytosolic Ca2+ increases in IOC waves promote stress fiber contractions, ensuring an organized application of the planar tensile forces that condense the retinal floor.
Peng, Q., Chen, J., Wang, R., Zhu, H., Han, C., Ji, X. and Pan, Y. (2022). The sex determination gene doublesex regulates expression and secretion of the basement membrane protein Collagen IV. J Genet Genomics. PubMed ID: 35017121
The highly conserved doublesex (dsx) and doublesex/mab-3 related (Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, Dsx(M) in males and Dsx(F) in females, which function differentially and often oppositely to establish sexual dimorphism. This study report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane (BM) protein Collagen IV in the fat body. Dsx isoforms regulate the expression of Collagen IV in the fat body and its secretion into the BM of other tissues. The procollagen lysyl hydroxylase (dPlod) gene, which is involved in the biosynthesis of Collagen IV, was identified as a direct target of Dsx. It was further shown that Dsx regulates Collagen IV through dPlod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen IV and remotely establish sexual dimorphism.
Topfer, U., Guerra Santillan, K. Y., Fischer-Friedrich, E. and Dahmann, C. (2022). Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila. Development 149(10). PubMed ID: 35575071
The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. This study systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. The Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, this study showed that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy it was shown that basement membrane stiffness mainly depends on Collagen IV. This analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.


Basement membranes (BMs) are resilient polymer structures that surround organs in all animals. Tissues, however, undergo extensive morphological changes during development. It is not known whether the assembly of BM components plays an active morphogenetic role. To study in vivo the biogenesis and assembly of Collagen IV, the main constituent of BMs, a GFP-based RNAi method (iGFPi) was used that was designed to knock down any GFP trapped protein in Drosophila. With this method it was found that Collagen IV is synthesized by the fat body, secreted to the hemolymph (insect blood), and continuously incorporated into the BMs of the larva. Incorporation of Collagen IV determines organ shape, first by mechanically constricting cells and second through recruitment of trol, the Drosophila Perlecan, which counters constriction by Collagen IV. These results uncover incorporation of Collagen IV and Perlecan into BMs as a major determinant of organ shape and animal form (Pastor-Pareja, 2011).

Basement membranes (BMs) are layered polymers of extracellular matrix proteins that underlie epithelia in all animals and surround organs, including muscles, fat, endothelium, and nervous tissue. Among BM components, Collagen IV is the most abundant, comprising 50% of the proteins of the BM (Kalluri, 2003). Collagen IV molecules consist of a chains bound in long helical trimers that assemble into a network through lateral and enddomain interactions (Yurchenco, 1987). Stacked layers of this polymer provide the main structural feature of BMs (Pastor-Pareja, 2011).

The biogenesis of functional collagen is very complex. Deficient or aberrant production is crucially involved in many diseases, including syndromes caused by mutations in collagens and collagen-modifying enzymes (Myllyharju, 2004). Among the first steps in collagen biogenesis, a chains undergo extensive posttranslational modification in the endoplasmic reticulum (ER). A number of chaperones and enzymes assist folding and trimerization, including lysyl- and prolylhydroxylases, which require vitamin C as a cofactor (Lamande, 1999). CopII coated vesicles, which mediate ER-to-Golgi transport of secreted proteins, are typically 60- 80 nm in diameter, whereas collagen trimers are 300 nm long. Consequently, studies on collagen secretion have fuelled a controversy as to whether alternative mechanisms exist for the secretion of large proteins. It is not clear either how collagen trimers avoid self-aggregation and aggregation with other BM components inside the producing cell or at the plasma membrane. Therefore, while collagens are the most abundant proteins in the human body (30% of its protein mass), many of the steps of their biogenesis are poorly understood (Pastor-Pareja, 2011).

Collagen IV is found in all animals, from sponges to humans, indicating a central role of BMs in the development of complex body plans (Hynes, 2000). It is the ancestral type of collagen, from which the 27 remaining vertebrate types have evolved. Type IV Collagens are divided into two subfamilies, α1-like and α2-like, split already in Cnidaria (Aouacheria, 2006). Drosophila has two genes encoding a chains of Collagen IV, named viking (vkg) and Collagen at 25C (Cg25C) (Le Parco, 1986; Natzle, 1982; Rodriguez, 1996; Yasothornsrikul, 1997), belonging to the α2-like and α1-like subfamilies respectively. vkg and Cg25C are adjacently located head-to-head in the genome, an arrangement conserved in the three α1-like/α2-like pairs of Collagen IV genes in mammals. Drosophila Collagen IV genes are essential, as loss of function of either of them causes embryonic lethality (Borchiellini, 1996; Rodriguez, 1996). In addition, Cg25C and Vkg modulate TGF-β gradient formation in the blastoderm embryo before a BM exists (Wang, 2008). Early lethality, although informative of the importance of Collagen IV for normal development, has precluded further analysis of its function in later stages, when organs and systems increase in size and complexity (Pastor-Pareja, 2011).

Apart from Collagen IV, the three other major components of BMs are Laminin, Nidogen, and the heparan-sulfate proteoglycan Perlecan, all conserved in Drosophila as well. Multiple interactions between BM components have been mapped in vitro. However, little evidence exists in vivo to show which interactions are crucial for BM assembly, maintenance and regeneration. Furthermore, a study of mice homozygous for a targeted deletion of the Collagen IV α1/α2 pair reported embryonic lethality but no defect in deposition of other BM components (Pöschl, 2004). This led to the proposal that Collagen IV is a terminal BM component, dispensable for BM assembly (Pastor-Pareja, 2011).

This study used a GFP protein trap inserted into the Drosophila vkg gene, producing a functional GFP-tagged version of Vkg, to study Collagen IV biogenesis and function. An approach (in vivo GFP interference, iGFPi) was developed to specifically knock down Vkg-GFP. Using iGFPi, it was found that Collagen IV is synthesized in the larva by the fat body and continuously incorporated into BMs, where it exerts a constricting force on tissues. It was additionally found that Collagen IV is not a terminal BM component, but, on the contrary, essential for deposition of Perlecan into BMs. Surprisingly, Perlecan incorporation counters, rather than reinforces, constriction by Collagen IV (Pastor-Pareja, 2011).

iGFPi significantly adds to the tools available in Drosophila to study gene function. Advantages of iGFPi are the easy assessment of knockdown effectiveness by examining GFP expression, and versatility, since a single RNAi construct can be used to target all GFP traps. Ongoing trapping projects, with improved techniques such as less biased transposons and recombineering- mediated tagging, will increase the number of trap lines and make the iGFPi approach of broader application. Insertional protein trapping has proved successful in mammalian cells too. Therefore, iGFPi, or RNAi targeting any other tag, can be a powerful method providing a convenient avenue from protein expression/localization to functional studies in Drosophila and other organisms (Pastor-Pareja, 2011).

The fat body, formed by polyploid adipocytes, has essential roles in metabolic regulation. It is also an important effector of immune responses though secretion of antibacterial peptides to the hemolymph. To these functions, the production of Collagen IV is now added. The experiments using iGFPi show that the fat body is the source of this protein in the larva. In contrast, there is no contribution from overlying cells to the Collagen IV content of a BM. Similar nonautonomy in the production of Collagen IV was observed in C. elegans (Graham, 1997). It has been shown that hemocytes, are the main source of Collagen IV earlier in the Drosophila embryo (Bunt, 2010; Martinek, 2008; Olofsson, 2005). Since fat body and hemocytes are twin mesodermal lineages, it seems likely that the fat body takes over the task of Collagen IV production when the need of protein increases, as poliploidy makes fat body cells especially suited for synthesis of large amounts of protein. Secretion of Collagen IV by the fat body, in addition, raises the possibility that immune or metabolic inputs might regulate its synthesis (Pastor-Pareja, 2011).

Further evidence was obtained that the larval fat body is the source of BM Collagen IV from experiments in which several genes were silenced and the effect on Vkg localization was studied. Loss of SPARC caused retention of Vkg in the membranes of fat body cells in the form of thick fibers, reminiscent of fibrosis. Loss of CopII coatomer components, mediating vesicular traffic from the ER to the Golgi, or the CopII cargo adaptor Tango1 caused large intracellular cumules of Vkg. Finally, loss of the prolyl-hydroxylase PH4alphaEFB prevented trimerization of Collagen IV, causing accumulation in the hemolymph of monomeric Vkg. These data, showing accumulation of Vkg at different locations when different steps of its biogenesis are prevented, indicate that Collagen IV is secreted by the fat body to the hemolymph as a soluble trimer and from there incorporated into BMs. Importantly, the results provide a genetic model to advance understanding of collagen biogenesis and secretion. Due to their large size, fat body cells are ideal for subcellular imaging and localization studies, allowing detailed analysis of Collagen IV biogenesis in vivo (Pastor-Pareja, 2011).

The role of the cytoskeleton in shaping cells has received extensive attention. The effect of the BM on cell shape, in contrast, is less well dissected. The current results evidence a major role of Collagen IV in shaping Drosophila larval organs. Loss of Collagen IV caused deformations consistent with Collagen IV and the BM exerting a constricting physical force on the ensheathed tissues. It has been recently shown that Collagen IV constricts Drosophila eggs into an elliptical shape, becoming spheric instead in its absence (Haigo, 2011). The current study has shown that in the wing imaginal disc, an epithelium consisting of highly columnar cells, knockdown of Collagen IV caused planar expansion and shortening in the apico-basal axis, resulting in flattening of the tissue. Similar changes in cell and organ shape were quickly elicited by collagenase treatment (Pastor-Pareja, 2011).

These data also show that Collagen IV is required for Perlecan incorporation into the BM. Interactions among BM components have been mapped in vitro, but the relevance of these interactions in vivo remains to be established. It has been shown that Laminin B1, another BM component, is required for presence of Collagen IV in BMs of the embryo (Urbano, 2009). This study has shown that the reverse is not true, with Laminin unaffected by absence of Collagen IV. Additionally it was found that Collagen IV was still present in BMs in the absence of Perlecan and that integrin expression in disc cells is required for Collagen IV to be correctly deposited. These results suggest that a strict hierarchy of assembly of BM components exists, as opposed to self-assembly guided by the simultaneous effect of multiple component interactions. Genetic data in model organisms like Drosophila will be essential to complement biochemical studies and help finally unravel the hierarchy of assembly of BM components during development and regeneration (Pastor-Pareja, 2011).

Lastly, it was found that the presence of Perlecan counters BM constriction. Both loss- and gain of-function experiments show that the effect of Perlecan is opposite to that of Collagen IV. Therefore, Collagen IV has a dual role in shaping organs: first, it constricts tissues, and, second, it mediates incorporation into the BM of Perlecan, which alleviates this constriction. Additional effects of BM components on morphogenesis are possible. Both Collagen IV and Perlecan affect signaling pathways (Kalluri, 2003; Wang, 2008) and, indeed, intercellular signaling has been implicated in cell shape changes in imaginal discs. The current data, nonetheless, indicate a major role of the BM in shaping cells and organs through mechanical tension (Pastor-Pareja, 2011).

Further studies are needed to explain how Perlecan opposes Collagen IV and whether the core protein, the heparan sulfate chains or both mediate this effect. From the current results, however, it can be hypothesized that the ratio of the contents in Collagen IV and Perlecan will determine the tension exerted by a BM on a tissue. In this way, opposition between Collagen IV and Perlecan could be a major mechanism regulating cell and organ shape. It would be interesting to know to what extent cells passively change shape under tension from the BM or, on the contrary, reform their cytoskeleton in response. Also, by manipulating the amount of Collagen IV or Perlecan, it will be possible now to test recent models of wing disc growth that postulate a role of physical tension in controlling proliferation (Pastor-Pareja, 2011).

A moving source of matrix components is essential for de novo basement membrane formation

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly, this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components, including Col IV, Perl and LanA appears to be hierarchical, yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types, and it is unclear how large, possibly insoluble protein complexes are delivered into the matrix. This study exploited the ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, it was shown that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components (Matsubayashi, 2017).

To analyze de novo basement membrane (BM) formation, developing Drosophila embryos were used. The developmental profile of BM components was analyzed from the Drosophila modENCODE project. This revealed that, while Laminin mRNAs are observed early in development, extracellular matrix (ECM) components associated with a mature BM, such as Collagen IV (Vkg in Drosophila) and Perlecan (Trol in Drosophila), are expressed later, suggesting that there is a temporal hierarchy of BM production during embryogenesis (Matsubayashi, 2017).

Embryonic BM protein production was examined using endogenously tagged BM fly lines. Homozygous viable GFP-protein traps were used in Collagen IV (Col IV) and Perlecan (Perl) as well as a recently generated line containing GFP-tagged Lamininα (LanA). This LanA-GFP is capable of biochemically interacting with other Laminin subunits to form a mature Laminin trimer, and it rescued LanA mutant embryos. Furthermore, when expressed in a Lamininβ (LanB1) mutant background, LanA levels were severely reduced, suggesting that subunit trimerization is indeed essential for Laminin production and secretion. Using these GFP-tagged lines, the dynamics of BM production were analyzed by quantifying GFP intensity over time during development. This revealed that expression of BM components peaked immediately prior to embryonic hatching. Furthermore, components showed precise temporal regulation with LanA expressed first, followed by Col IV, and finally Perl. A second GFP-tagged construct was examined of the sole Drosophila Lamininβ isoform (LanB1), which was previously confirmed to be fully functional, and this also revealed Laminin expression to occur prior to Col IV or Perl (Matsubayashi, 2017).

In Drosophila embryos, hemocytes are known to produce BM. However, it has been unclear what proportion of the embryonic BM is hemocyte dependent. When GFP-tagged BM proteins were expressed in a mutant background in which hemocytes failed to develop, it was revealed that BM components are differentially hemocyte dependent. This showed that 70% of Col IV and 50% of Perl are dependent on hemocytes. In contrast, hemocytes contribute only 30% of embryonic LanA, with most of the hemocyte-derived Laminin induced at later stages of development. As the mesoderm expresses LanA, it was hypothesize that its early expression is likely dependent on this tissue. For Col IV and Perl, the remaining protein was expressed in the fat body at late stages of development, which is known to be the major source of larval BM (Pastor-Pareja, 2011; Matsubayashi, 2017 and references therein).

To investigate the functional importance of the temporal hierarchy of BM component expression, embryos were generated expressing the GFP-tagged LanA, Col IV, and Perl in all possible mutant backgrounds of opposite components. This revealed that, while LanA incorporation or levels were unaffected by the absence of subsequent components, Col IV and Perl formed disorganized extracellular deposits in the absence of Laminin. It was hypothesized that these aggregates are the specific result of Col IV aggregation, as the Perl deposits were absent in a Col IV/Laminin double mutant. Finally, Perl, which is expressed last in the temporal hierarchy, required prior production of Laminin and Col IV for proper expression and incorporation into the BM, which is similar to what was previously reported (Hollfelder, 2014). These results suggest that proper de novo BM formation requires temporal regulation of component production. A similar temporal hierarchy of BM production may be critical for BM formation in other species, as disorganized ECM deposits have also been observed in laminin mutant mice (Smyth, 1999) and C. elegans (Huang, 2003; Matsubayashi, 2017).

Differences were observed in the appearance of Col IV and Laminin in the wild-type background, with Laminin showing a much more diffuse distribution. These differences were investigated by time-lapse microscopy during hemocyte migration along the ventral nerve cord (VNC), which is a known migratory route that is readily amenable to live imaging. Both LanA and LanB1 subunits were observed to form 'halos' of graded expression surrounding migrating hemocytes, with trails of Laminin forming as cells moved within the acellular fluid-filled cavity of the embryo (hemocoel). These halos of Laminin were identical to expression of secreted-GFP, suggesting that Laminin is simply filling the hemocoel. In contrast, while Col IV and Perl decorated the surface of the VNC, there was no observable fluorescence filling the hemocoel. Whether the differences in BM component localization were the result of their differing diffusive characteristics was examined by performing fluorescence recovery after photobleaching (FRAP) analysis. This showed that LanA had a significant mobile fraction unlike Col IV, which failed to show any recovery. To understand why Laminin formed halos surrounding hemocytes along the VNC, the ventral hemocoel was examined by transmission electron microscopy (TEM). This revealed that the ventral hemocoel is highly confined, with the VNC in physical contact with the overlying epithelium. Therefore the halos of Laminin and its trails following hemocyte movement represent hemocytes separating the VNC from the overlying epithelium, allowing Laminin diffusion. These data highlight that different BM components have distinct diffusive properties within the developing embryo (Matsubayashi, 2017).

The apparent absence of soluble Col IV in the hemocoel suggested that Col IV might require a local mechanism of deposition by hemocytes. However, while it was possible to observe some BM material deposited beneath migrating hemocytes by TEM, it was difficult to examine the dynamics of Col IV deposition beneath hemocytes by standard confocal microscopy due to the low level of fluorescence and small size of the deposits. Therefore, lattice light-sheet microscopy, which allows for enhanced spatiotemporal resolution with reduced phototoxicity, was used. Indeed, hemocyte motility within the ventral hemocoel was highly amenable to lattice light-sheet imaging at early stages of hemocyte dispersal with minimal photobleaching (Matsubayashi, 2017).

Imaging by lattice light-sheet microscopy revealed that, at the stage when hemocytes are aligned on the ventral midline, Col IV is primarily localized beneath hemocytes on the surface of the nerve cord and in the segmentally spaced dorsoventral channels of the VNC. Subsequently, when hemocytes left the midline and migrated laterally, they appeared to deposit Col IV in a local fashion leaving puncta of matrix that eventually developed into longer fibrils. Additionally, simultaneous imaging of Col IV and the hemocyte actin cytoskeleton showed that Col IV colocalized with actin fibers within lamellae, suggesting hemocyte secretion of Col IV may require release along actin fibers or that recently released Col IV is rapidly remodelled by hemocytes using their actin network. Indeed, tracking movements in the Col IV matrix at high temporal resolution by particle image velocimetry revealed strong regions of ECM deformation beneath hemocyte lamellae, suggesting hemocyte traction forces are being exerted on the developing BM (Matsubayashi, 2017).

As time-lapse imaging suggested that hemocytes are 'plastering' embryonic surfaces with Col IV, it was hypothesized that hemocyte developmental dispersal may be a critical part of the BM deposition process. Hemocytes develop in the anterior of the embryo, and after stage 10 of embryogenesis they disperse within the hemocoel using a combination of external guidance cues and contact inhibition of locomotion, resulting in an evenly tiled cellular distribution. Therefore, how the timing of BM component production correlated with the dispersal of hemocytes was examined. While LanA was expressed during initial stages as hemocytes migrated from their source in the head of the embryo, Col IV production lagged behind by approximately 5 hr. As the induction of Col IV expression occurred largely after hemocyte dispersal, this suggested that hemocyte spreading within the embryo might be a prerequisite for Col IV delivery (Matsubayashi, 2017).

It was previously proposed that hemocytes were required for BM deposition specifically around the renal tubules during embryogenesis (Bunt, 2010); however, this was only interrogated in mutant embryos that were defective in both hemocyte migration and their survival. To directly examine the role of hemocyte migration in BM component deposition, aberrant hemocyte dispersal was caused by misexpression of Pvf2, a platelet-derived growth factor (PDGF)-like chemotactic cue for hemocytes. Overexpressing Pvf2 during hemocyte dispersal caused hemocytes to aggregate in the embryonic head, which was likely due to a distraction of hemocytes from their normal Pvf source. LanA in wild-type embryos initially spread down the midline of the VNC, and this was unaffected by the inhibition of hemocyte migration. Subsequently, in control embryos, a sheet-like structure containing Laminin extended from the middle of the VNC to lateral positions. These nascent Laminin sheets were stable in time compared to the halos/trails of Laminin following migrating hemocytes, which fluctuated on the order of seconds. Therefore, it was hypothesize that the extension of the Laminin sheets reflects the incorporation and growth of the polymerized matrix from a soluble source of Laminin residing predominantly on the midline. The initiation of Laminin incorporation was unaffected by Pvf2 overexpression. However, in Pvf2-expressing embryos, the Laminin sheets failed to continue extending, leaving large gaps that increased in size by later stages of development. This apparent breakdown of the Laminin matrix was similar to embryos lacking hemocytes. Therefore, Laminin produced by hemocytes may be critical for proper Laminin incorporation or that hemocyte movement, which opens up spaces between tissues, could be aiding the growth of the Laminin matrix by enhancing its diffusion in the hemocoel. In contrast, despite an increase in Col IV upon Pvf2 overexpression, confocal microscopy and lattice light-sheet imaging revealed that there was an uneven coverage of Col IV within the embryo, with most Col IV surrounding hemocytes in the head. A similar local deposition of Col IV around hemocytes was also observed when hemocyte migration was disturbed by the expression of dominant-negative Rac (RacN17) or constitutively active Rac (RacV12). These results further suggest that Laminin deposition requires its diffusion within the embryonic hemocoel while Col IV is locally deposited by hemocytes (Matsubayashi, 2017).

While these data suggested a highly local mechanism of Col IV delivery by migrating hemocytes, a more complex picture emerged over longer time periods of imaging. At later stages of development, Col IV appeared to spread at a distance from hemocytes and fill the hemocoel. Therefore Col IV was imaged within embryos over a longer period of approximately 12 hr, which represents the time frame just prior to embryonic hatching. Inducing hemocyte aggregation in the anterior of the embryo through overexpression of Pvf2 or RacN17/RacV12 revealed an accumulation of Col IV around hemocytes approximately 6 hr after Col IV induction. However, by 12 hr the fluorescence of Col IV was distributed throughout the embryo despite a continued aggregation of hemocytes. These data suggest that Col IV is eventually capable of spreading within the hemocoel but suffers from very slow effective diffusion (Matsubayashi, 2017).

Whether hemocyte migration and even BM deposition are functionally important for embryogenesis was subsequently tested. Therefore VNC condensation, a known morphogenetic event that requires hemocytes and BM, was examined. As the BM surrounds the outer surface of the VNC, it is readily accessible to ultrastructural analysis. Fillet preparations of the embryonic VNC were generated, and the developing BM was examined by scanning electron microscopy (SEM). At stage 14 of development, the matrix surrounding the VNC was surprisingly fibrillar in appearance. However, by stage 15 these matrix fibrils were rapidly remodelled into a contiguous sheet containing holes that progressively closed during VNC condensation. Next the distribution of the BM surrounding the VNC was examined after inhibition of hemocyte migration, which severely affected the condensation process and led to a reduced embryonic viability. This revealed that, while the wild-type VNC showed a relatively even distribution of BM, Pvf2 overexpression led to a dense matrix in the head region with a sparse matrix surrounding the VNC in the tail. This highlights that uniform hemocyte dispersal is indeed essential for even incorporation of BM and that the catching up in fluorescence levels upon the inhibition of hemocyte migration is likely the result of diffusing Col IV within the hemocoel rather than proper incorporation (Matsubayashi, 2017).

Whether the severity of hemocyte migration defects correlated with embryonic lethality was examined. Hemocytes are completely essential for embryogenesis, as killing off hemocytes led to 100% lethality as measured by the frequency of embryonic hatching. Varying degrees of hemocyte migration defects were examined. Expression of a dominant-negative Myosin II specifically in hemocytes led to minor clumping defects but no obvious effects on embryonic lethality. In contrast, Pvf2 overexpression or hemocyte-specific expression of RacN17 led to intermediate migration defects and resulted in approximately 50% embryonic lethality. Finally, hemocyte-specific expression of RacV12, which induced severe migration defects with hemocytes failing to disperse from their origin in the head, led to the most severe embryonic phenotype with 96% lethality. Importantly, these differences in lethality were not correlated with levels of Col IV expression, indicating that the lethality was not related to a change in Col IV levels. These data show that hemocyte migration is indeed essential for embryonic viability (Matsubayashi, 2017).

Finally, whether a genetic interaction could be observed between hemocyte migration defects and BM mutant alleles was examined. Causing aberrant hemocyte migration in the presence of a heterozygous colIV mutant allele, which led to a 50% reduction in Col IV expression, abolished VNC condensation and induced a synergistic effect on embryonic lethality with 100% of embryos failing to hatch. This lethality was higher than homozygous colIV mutants, showing that the synergy between hemocyte migration and Col IV reduction is not simply the result of a loss of Col IV expression; it also suggests that uneven Col IV deposition may be worse for the embryo than a complete loss of Col IV. In contrast, combining hemocyte migration defects with heterozygous laminin mutants led to a slight increase in lethality, which was similar to homozygous laminin mutant embryos. These data further show that Col IV deposition is more dependent on hemocyte migration than other BM components, such as Laminin (Matsubayashi, 2017).

This study has shown that during Drosophila embryogenesis, a subset of BM components requires local deposition by migrating hemocytes. This highlights that the ability of hemocytes to evenly spread throughout the embryo, part of a wider mechanism to uniformly deliver ECM. Therefore, as is increasingly realized for vertebrate macrophages, which are also involved in morphogenetic processes that involve matrix remodelling, hemocytes have important non-immune roles critical for development. Interestingly, mammalian macrophages have recently been revealed to produce various ECM components; along with the current data, this suggests that a critical role for macrophage-derived ECM may be more ubiquitous than previously recognized (Matsubayashi, 2017).

It is unclear why embryonic BM components like Col IV require local delivery by hemocytes, while in larvae they are thought to diffuse from the fat body. This may be related to physiological differences between embryo and larva. In larvae, the heart pumps hemolymph around the animal, which may aid in the spreading of BM proteins. In contrast, the embryonic heart does not begin beating until stage 17, which is after the start of Col IV deposition; in lieu of flowing hemolymph, BM factors with low effective diffusion may therefore require a moving source. Interestingly, recent work has revealed that at least one larval tissue, the developing ovary, requires hemocyte-specific production of Col IV, and it is possible that tissues not in direct contact with hemolymph require other mechanisms of BM deposition. However, it is unclear whether hemocytes associated with the ovary plaster Col IV in a manner similar to embryonic hemocytes or shed soluble Col IV similarly to the larval fat body (Matsubayashi, 2017).

It is also likely that there are differences between the mechanisms of de novo BM formation in the embryo versus homeostatic mechanisms involved in BM growth in the larva; when Col IV is first deposited in the embryo, its binding sites in the nascent Laminin matrix will be completely unsaturated leading to its rapid capture, thus preventing it from spreading far from its source. As Col IV saturates the BM at later stages of development, this would allow for its subsequent long-distance diffusion in older embryos and larvae. The larva may also have specific mechanisms that aid in Col IV solubility. Indeed, Sparc mutant larvae have abnormal extracellular BM deposits, and recent data from both Drosophila and C. elegans suggest that Sparc is a carrier for components like Col IV. It is interesting to note that there is no embryonic phenotype in Drosophila in the absence of Sparc, suggesting that embryonic Col IV does not need to be solubilized, which is hypothesized to be due to its specific hemocyte-dependent mechanism of delivery during de novo BM formation (Matsubayashi, 2017).

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, this study identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. Amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. Evidence is provided that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans (Zhang, 2020).

Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos

SPARC is an evolutionarily conserved collagen-binding extracellular matrix (ECM) glycoprotein whose morphogenetic contribution(s) to embryonic development remain elusive despite decades of research. This study used Drosophila genetics to gain insight into the role of SPARC during embryogenesis. In Drosophila embryos, high levels of SPARC and other basal lamina components (such as network-forming collagen IV, laminin (see Laminin A and Laminin B1) and perlecan) are synthesized and secreted by haemocytes, and assembled into basal laminae. A SPARC mutant was generated by P-element mutagenesis that is embryonic lethal because of multiple developmental defects. Whereas no differences in collagen IV immunostaining were observed in haemocytes between wild-type and SPARC-mutant embryos, collagen IV was not visible in basal laminae of SPARC-mutant embryos. In addition, the laminin network of SPARC-mutant embryos appeared fragmented and discontinuous by late embryogenesis. Transgenic expression of SPARC protein by haemocytes in SPARC-mutant embryos restored collagen IV and laminin continuity in basal laminae. However, transgenic expression of SPARC by neural cells failed to rescue collagen IV in basal laminae, indicating that the presence of collagen IV deposition requires SPARC expression by haemocytes. A previous finding that haemocyte-derived SPARC protein levels are reduced in collagen-IV-mutant embryos and the observation that collagen-IV-mutant embryos showed a striking phenotypic similarity to SPARC-mutant embryos suggests a mutual dependence between these major basal laminae components during embryogenesis. Patterning defects and impaired condensation of the ventral nerve cord also resulted from the loss SPARC expression prior to haemocyte migration. Hence, SPARC is required for basal lamina maturation and condensation of the ventral nerve cord during Drosophila embryogenesis (Martinek, 2008).

Metazoan radiation gave rise to a complex variety of organisms with distinctive body plans, adaptations and survival strategies. This necessitated the co-evolution of specialized extracellular matrix (ECM) macromolecules capable of forming elaborate matrices that provide tissues with their unique biomechanical, biochemical and functional properties. Among the most ancient ECM molecules are those that comprise the basal lamina, a specialized, cell-surface-associated ECM sheet underlying epithelial and endothelial cells and surrounding muscle, neural and adipose tissues. In addition to serving as adhesive substrata for cell adhesion and migration, basal laminae regulate signal transduction pathways through interactions with cell-surface receptors, such as members of the integrin superfamily. Whereas the molecular complexity of basal laminae varies among tissues, the most broadly distributed components include laminin, collagen IV, perlecan, nidogen and SPARC. Mammalian genomes encode six genetically distinct collagen IV α chains. The major embryonic and most broadly distributed isoform of collagen IV is a heterotrimer composed of two α1(IV) and one α2(IV) chain, designated as α1(IV)2α2(IV). The folding and maturation of collagen IV is dependent on molecular chaperones such as the endoplasmic reticulum (ER)-resident 47-kDa heat shock protein (HSP47). Even though embryonic expression of collagen IV begins in mouse embryos at day 5 post-coitus, mutations in collagen IV do not lead to developmental arrest until embryonic day (E) 10.5-11.5 (Poschl, 2004). Since embryonic lethality is coincident with the onset of muscle contractions, it has been hypothesized that collagen IV is required at this stage of development to provide tensile strength to basal laminae, enabling them to withstand contractile forces associated with embryonic movements (Yurchenco, 2004). However, the underlying cause of lethality is likely to be more complex because dynamic interactions exist between collagen IV and other basal laminae components that affect multiple signaling pathways during embryogenesis (Martinek, 2008).

SPARC is a 32-35 kD Ca2+-binding matricellular glycoprotein whose modular organization is phylogenetically conserved (Martinek, 2002). Biochemical studies indicate that SPARC binds to several collagenous and non-collagenous ECM molecules, including a Ca2+-dependent interaction with network-forming collagen IV. The binding of SPARC to collagen IV might serve to concentrate SPARC in a subset of embryonic basal laminae and basal lamina EHS tumors. However, studies indicate that SPARC is either associated with the plasma membrane or concentrated at the interface between epithelial and basal lamina. Whereas the precise role of SPARC in vertebrate basal lamina assembly and maturation is poorly understood, in vivo studies indicate that the stability of the lens capsule is compromised in SPARC-null mice. The lens capsule (hereafter referred to as a basement membrane) is a continuous thick avascular collagen-IV-rich specialized basal-lamina-like matrix that surrounds the lens. In SPARC-null mice, cataract formation is preceded by disruptions in the ultrastructural organization of capsular collagen IV and laminin networks. Coincident with the altered matrix organization is the presence of filopodia-like cellular extensions in the lens capsule derived from cells that form the lens capsule (Martinek, 2008 and references therein).

SPARC is an integral component of most embryonic laminae in invertebrates. In the nematode Caenorhabditis elegans, SPARC protein is distributed in basal laminae body wall and sex muscles and overlaps with the distribution of collagen IV (Fitzgerald, 1998). The reduction of SPARC protein production by RNA interference results in embryonic and larval lethality. Previously studies have shown that SPARC is a component of embryonic basal laminae in Drosophila (Martinek, 2002). In collagen-IV-α1-mutant embryos, the level of SPARC immunostaining within haemocytes was dramatically decreased and present at very low levels in the basal laminae. This study now reports that inhibition of SPARC expression in Drosophila leads to several developmental anomalies, impaired ventral nerve cord (VNC) condensation and the absence of collagen IV from haemocyte-derived embryonic basal laminae (Martinek, 2008).

SPARC is required for normal embryonic development in Drosophila. In the absence of SPARC, haemocyte-derived collagen IV is not observed in basal laminae during mid- to late embryonic development. The absence of collagen IV leads to discontinuous laminin distribution during late embryonic development, indicative of decreased basal lamina stability. That SPARC selectively affects the presence of collagen IV in basal laminae is further supported by data demonstrating that collagen-IV-mutants have phenotypic similarities to SPARC-mutant embryos (Martinek, 2008).

Studies using vertebrates and invertebrates have shown that laminin is the first basal lamina component to be expressed and secreted during embryonic development. The expression and deposition of laminin along cell surfaces are promoted by its binding to cell-surface receptors such as α1-integrin and β-dystroglycan. In SPARC-mutant embryos, the association of laminin with cell surfaces is unaffected until late embryogenesis, a stage in development when collagen IV and SPARC have been integrated into basal laminae of wild-type embryos. In support of the proposal that the discontinuous laminin network observed in SPARC mutants is because collagen IV is absent from the basal lamina, discontinuous laminin networks are also observed in late-stage collagen-IV-mutant embryos. Laminin networks are likewise disrupted in mouse and C. elegans mutants that lack the expression of collagen IV (see Poschl, 2004). The data indicate that the compromised structural integrity of the laminin network is probably owing to the absence of collagen IV in basal lamina rather than a molecular interaction between SPARC and laminin. However, the presence of a thicker laminin network in lens capsules of SPARC-null mice might reflect a more complex relationship between laminin and SPARC (Martinek, 2008).

Molecular interactions have not been demonstrated between SPARC and perlecan or nidogen, two other universal components of basal laminae. The current data indicate that absence of SPARC does not affect the distribution of perlecan and nidogen in basal laminae during embryogenesis. A potential explanation is that nidogen and perlecan do not form extended crosslinked polymers such as laminin and collagen IV. Hence, they are expected to be less susceptible to distortion by mechanical forces associated with late embryonic development. Another possibility is that, whereas perlecan and nidogen bind to, and bridge with, laminin and collagen IV, their interactions with transmembrane receptors promotes pericellular associations that are independent of laminin and collagen IV networks (Martinek, 2008).

Whereas the current data indicate that SPARC and collagen IV are integral components of the majority of embryonic basal laminae in Drosophila, no SPARC was detected in basal laminae overlying the dorsal vessel and somatic muscles of wild-type embryos, which suggests that molecules other than SPARC promote the deposition of collagen IV molecules in these basal laminae. Interestingly, pericardial cells only express the α2 chain of collagen IV, raising the possibility that the basal lamina overlying the dorsal vessel is composed of collagen IV α2 homotrimers. Adding to the complexity of this basal lamina, Pericardin, a collagen-IV-like ECM molecule is also required for proper dorsal vessel formation (Chartier, 2002). Hence, diverse regulatory factors and mechanisms are likely to control collagen IV deposition and/or stability during development, consistent with cumulative data indicating that the precise molecular composition and function of basal laminae varies between tissues and at different stages of development (Martinek, 2008).

A direct Ca2+-dependent interaction has been demonstrated between collagen IV and the EC domain of SPARC. Phylogenetic analysis reveals a striking evolutionary conservation of amino acids in the EC domain essential for collagen binding in organisms ranging from nematodes to mammals. Site-directed mutagenesis of these conserved amino acids results in a loss of binding between SPARC and collagen triple helices (Maurer, 1995; Mayer, 1991; Martinek, 2002; Martinek, 2007; Pottgiesser, 1994). Since this study has demonstrated that the presence of collagen IV in basal laminae requires SPARC, whether mutations in collagen IV generate a similar phenotype as SPARC mutants was examined to further substantiate their proposed interrelationship (Martinek, 2008).

This study partially characterized alleles of the gene encoding the α1 subunit of collagen IV (DCg1412 and DCgl234) and a deficiency line that lacks both collagen IV genes (Df(2L)sc19-8). Mutant embryos homozygous for collagen IV show reduced protein expression of collagen IV and, similar to SPARC-mutant embryos, are embryonic lethal. As in SPARC-mutant embryos, ventral cuticle holes are observed in these collagen-IV-mutant embryos; however, the holes are smaller in the latter. In both SPARC- and collagen-IV-mutants, tracheal integrity is also compromised. A major function of collagen IV is to provide tensile strength to basal laminae, a biomechanical contribution that increases in importance during late embryogenesis due to an increase in the frequency and strength of muscle contractions. The discontinuous laminin network surrounding the ventral nerve cord and other organs by late embryogenesis in collagen IV and SPARC mutants is probably due to the absence of collagen IV from basal laminae (Martinek, 2008).

A similarity between SPARC-mutant and collagen-IV-mutant embryos during late embryogenesis is the absence of VNC condensation. VNC condensation has been shown by a variety of genetic approaches to be dependent on the deposition of collagen IV in basal laminae and on electrical conductivity (Olofsson, 2005). Hence, failure to undergo VNC condensation in SPARC-mutant embryos is probably because of the absence of collagen IV from basal lamina surrounding the VNC. Whereas the molecular and cellular events regulating VNC condensation are poorly understood, intracellular signaling events are affected by integrins binding to collagen IV during late embryogenesis (Fessler, 1989). These data suggest both a biomechanical and regulatory role for collagen IV that is crucial in VNC condensation. Transgenic expression of SPARC in haemocytes and glia (under the control of gcm-GAL4) as well transgenic expression only in haemocytes (under the control of SrpHemo-GAL4) in a SPARC mutant background, restored the presence of collagen IV in the basal lamina surrounding the VNC, but did not promote its condensation. The combined data indicate that SPARC plays a role in neural patterning that is independent of its contribution to the deposition of collagen IV in basal laminae (Martinek, 2008).

The coexpression of SPARC and collagen IV in haemocytes, combined with the direct demonstrated biochemical interactions (Maurer, 1995: Mayer, 1991: Pottgiesser, 1994), raises the possibility that SPARC and collagen IV form a complex in the ER that promotes the proper folding and secretion of collagen IV. In support of this hypothesis, the presence of collagen IV in basal laminae is restored when haemocyte expression of SPARC is rescued transgenically. Ectopic expression of SPARC by neuroblasts or glia in SPARC-mutant embryos does not induce collagen IV expression by neural and glial cells, nor does it induce the presence of haemocyte-derived collagen IV in basal laminae. Whereas collagen IV and SPARC colocalize in basal laminae of tissues that do not express either protein, their coexpression by haemocytes appears to be required for their proper integration into basal laminae (Martinek, 2008).

The data indicate that inhibition of SPARC expression leads to the absence of collagen IV in the basal laminae during Drosophila embryogenesis, without affecting the secretion and deposition of the other major basal lamina components. The combined data raise the possibility that SPARC functions intracellularly to promote correct folding and secretion of collagen IV and/or its stability in basal laminae during Drosophila embryogenesis. Consistent with a collagen-chaperone-like activity is the recent report that SPARC affects the processing of fibrillar collagen I at the plasma membrane, which could in part account for the distinct collagen phenotype between wild-type and SPARC-null mice (Rentz, 2007). Moreover, it is also possible that collagen IV is not properly assembled extracellularly into a stable network and is therefore rapidly degraded by matrix remodeling proteases. Whereas this possibility cannot be discounted on the basis of the current data, proteases capable of selectively degrading collagen IV during Drosophila embryogenesis have yet to be identified. Moreover, as stated above, the secretion of SPARC by non-haemocyte cells does not rescue the association of collagen IV with basal laminae, which indicates that the formation of a stable collagen IV network is not generated by an extracellular interaction with SPARC. Whereas a potential role for SPARC in regulating the maturation of collagen IV in extracellular membrane compartments cannot be eliminated, the vesicular colocalization of SPARC and collagen IV in haemocytes is indicative of an intracellular functional relationship (Martinek, 2008).

The folding, assembly and processing of collagens from cells via the secretory pathway is dependent on molecular chaperones. Misfolded or incompletely assembled proteins are retained in the ER and are eventually targeted for degradation. In vertebrates, heat shock protein 47 (Hsp47) is a 47 kD collagen-specific protein that binds to and promotes the maturation of collagen molecules (Ishida, 2006: Marutani, 2004: Nagata, 2003). In the absence of Hsp47, both fibril-forming collagen I, and network-forming collagen IV secretion and assembly into matrices are severely compromised, leading to embryonic lethality at ES10.5-ES11.5 in mice (Marutani, 2004). Immunoelectron microscopy shows that collagen IV accumulates within the dilated ER of mutant cells. The accumulation of misfolded or unfolded protein within the ER activates an ER-stress response, in which the expression of molecular chaperones is induced. In Hsp47-null mouse embryos, massive apoptotic cell death occurs just before the death of the embryo at ES10.5. Collagen molecules that bypass the ER-quality control in mouse Hsp47-null fibroblasts and embryonic stem (ES) cells show increased sensitivity to protease degradation, indicative of incorrectly folded procollagen molecules (Marutani, 2004: Matsuoka, 2004). Since an Hsp47 ortholog is not encoded by invertebrate genomes, it is possible that one or more alternative chaperones ensure correct collagen assembly, maturation and secretion (Martinek, 2008).

Studies have indicated that the basal lamina components are highly conserved in metazoans. These data and findings from other laboratories indicate that a functional relationship between SPARC and collagens is also evolutionarily conserved. Analyses of SPARC-null mice demonstrate that SPARC affects the supramolecular assembly of both network and fibrillar collagens (Bradshaw, 2003: Norose, 2000: Sangaletti, 2003). Two months after birth, SPARC-null mice develop early onset cataracts, which suggest of a role for SPARC in lens transparency. Ultrastructural analysis of the lens capsule revealed that cellular extensions from the lens epithelium penetrate and invade the overlying basal lamina, and that the lens capsule contains an altered distribution of collagen IV and laminin (Yan, 2002). Therefore, the early onset cataracts observed in SPARC-null mice probably result from compromised assembly and stability of the lens basal lamina. The data indicate that, in Xenopus, decreased SPARC expression during embryogenesis also leads to the formation of cataracts (Martinek, 2008).

In this study it was observed that early loss of SPARC expression in SPARC-mutant embryos and SPARC knockdown using da-GAL4 prior to haemocyte migration produces a variety of patterning defects within the developing nervous system that cannot be rescued by SPARC expression in haemocytes. Moreover, loss of tracheal, fat-body and ventral-epidermal integrity were observed by the end of embryogenesis together with disorganized neurons and glia. These observations suggest that SPARC has a non-cell-autonomous role in the development of the CNS that impacts on guidance of muscles, neurons, glia and the tracheal system (Martinek, 2008).

The novel neural phenotype observed in SPARC-mutant embryos points to a role for SPARC in CNS patterning that is independent of collagen IV. This is not surprising in light of vertebrate studies that lend strength to the idea that SPARC is a multifunctional glycoprotein with both extracellular and intracellular functions (Martinek, 2008).

Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila

Details of the mechanisms that determine the shape and positioning of organs in the body cavity remain largely obscure. This study shows that stereotypic positioning of outgrowing Drosophila renal tubules depends on signaling in a subset of tubule cells and results from enhanced sensitivity to guidance signals by targeted matrix deposition. VEGF/PDGF ligands from the tubules attract hemocytes, which secrete components of the basement membrane to ensheath them. Collagen IV sensitizes tubule cells to localized BMP guidance cues. Signaling results in pathway activation in a subset of tubule cells that lead outgrowth through the body cavity. Failure of hemocyte migration, loss of collagen IV, or abrogation of BMP signaling results in tubule misrouting and defective organ shape and positioning. Such regulated interplay between cell-cell and cell-matrix interactions is likely to have wide relevance in organogenesis and congenital disease (Bunt, 2010).

As the renal tubules extend through the body cavity, two processes occur; they elongate through cell rearrangements and they make precise, guided movements with respect to other tissues. A major source of the motive force required for tubule extension is the convergent-extension movements of the tubule cells themselves. As the tubules are continuous with the hindgut and thus have a fixed point proximally, these movements result in a distal-directed extensive force. This study shows that in addition the normal morphogenesis of the anterior tubules depends on tissue guidance involving the coordinated activity of the PDGF/VEGF and BMP signaling pathways. Abrogation of either pathway has no effect on convergent-extension movements in the tubules but leads to failure of their normal pathfinding through the body cavity (Bunt, 2010).

PVF ligands (Pvf1-3) expressed by the tubules attract migrating hemocytes to form short-term associations with them, during which hemocytes secrete components of the BM. The presence of collagen IV in the matrix ensheathing anterior tubule cells primes their response to local sources of the BMP pathway ligand, Dpp. Thus, interference with hemocyte secretion of collagen IV, whether by preventing hemocyte migration, by preventing their attraction to the tubules, or by abrogating hemocyte expression and/or processing of collagen IV, results in failure of BMP pathway activation in tubule cells and consequent misrouting of the anterior tubules. The tissue interactions that govern the guided outgrowth of the anterior tubules are summarized in Tissue Interactions Underlie Anterior Tubule Morphogenesis (Bunt, 2010).

As the tubules elongate, a distinct but dynamic subset of cells in the kink region responds sequentially to Dpp guidance cues from dorsal epidermal cells, the midgut, and, more anteriorly, gastric cecal visceral mesoderm and leads forward extension. Activation of the pathway targets, pMad and Dad, in these leading cells ensures that as the tubules project through the body cavity they take a stereotypical route. Loss of Dpp expression in the midgut or repression of BMP signaling in the tubules leads to stalling of their forward movement. Misexpression of Dpp is sufficient to cause tubule misrouting, in which the kink regions project toward the ectopic source. In accordance with these findings defective tubule morphogenesis has been described in embryos lacking the BMP receptors Thick veins (type 1) or Punt (type II), as well as in embryos mutant for schnurri, which encodes a pathway transcriptional regulator shown to be active during embryogenesis (Bunt, 2010 and references therein).

Strikingly only cells in the kink show pathway activation. The current evidence suggests that leading kink cells respond directionally to local gradients of Dpp and that they receive the highest level of ligand, which would account for the restricted domain of activation. However, as the kink region extends beyond the Dpp source, more posterior cells experience high levels of signal but show no pathway activation, indicating that other factors must differentiate between the leading and trailing cells. Segregation into leading and following populations is a common feature of collective cell migration and tubule branching and extension during organogenesis. Leading cells in outgrowing Drosophila trachea, migrating border cells and mammalian ureteric bud formation show distinct patterns of gene expression, respond differentially to external signals, and may repress pathway activation in their neighbors. Thus, tubule kink cells could themselves restrict the domain of pathway response (Bunt, 2010)

As well as their roles in determining cell fate, survival, and growth in Drosophila, TGF-β superfamily signals regulate tissue morphogenesis and have been shown to influence the invasive behavior of metastatic tumors. This study shows, through loss- and gain-of-function analysis, that Dpp also acts as a chemoattractant during organogenesis to determine the path of renal tubule extension though the body cavity. TGF-β superfamily signaling can induce epithelial-to-mesenchymal (EMT) transition through the expression of Snail- and ZEB-family members, which act to repress cell adhesion and polarity, leading to increased motility and, in the case of cancers, to single-cell metastatic activity. Such changes in kink cells could explain their role in pathfinding. However, recent evidence suggests that collective cell migration of epithelial tissues can occur without full EMT and kink cells remain polarized, ensheathed in ECM during tubule elongation (Bunt, 2010).

Ninov (2010) has shown that pathway activation through pMAD leads to increased actin dynamics and E-cadherin turnover in outgrowing histoblasts, resulting in reduced cell adhesion and enhanced cell motility through filopodial/lamellipodial extensions. The current results reveal similar lamellipodial extensions in kink cells, in line with Vasilyev (2009), who demonstrated directional basal lamellipodia in cells of the extending pronephric tubules of zebrafish. It is possible that the production of lamellipodia and tubule navigation also depends on Mad-independent effects on cytoskeletal regulators such as cdc42 (Bunt, 2010).

The current analysis reveals that deposition of ECM is a prerequisite for BMP signaling in tubule guidance. TGF-β/BMP signaling can be modified both by soluble ECM components such as HSPGs and also by architectural, fibrillar elements. The current evidence indicates that for normal tubule outgrowth collagen IV is the crucial component of the BM; it is deposited before tubule elongation (cf. perlecan deposited after elongation), is uniquely contributed by the hemocytes (the tubules express laminins as well as the hemocytes), and the effects of collagen IV loss of function mimic the failure of hemocyte migration to the tubules (whether in collagen IV mutants or in embryos lacking the function of lysyl hydroxylase or dSparc, factors that are required for normal collagen IV processing and deposition) (Bunt, 2010).

Collagen IV sharpens the dorsoventral gradient of BMP signaling in early Drosophila embryos through enhanced ligand-mediated activation (Wang, 2008), which depends on a conserved BMP-binding domain in the C-terminal region of collagen IV. Wang (2008) propose a two-step process in which the binding of Dpp/Screw ligand hetereodimers to collagen IV facilitates the formation of a complex between Dpp/Scw dimers, Sog, and Tsg. Tolloid cleavage of the complex releases ligand dimers, which become active on rebinding to collagen IV dorsally where Sog is absent. This study now shows that basement membrane collagen IV also acts during organogenesis to facilitate BMP signaling in a specialized region of tubule cells. Whereas the mechanism of activation could be as outlined by Wang (2008), early requirements for Dpp signaling in tubule development (Hatton-Ellis, 2007) complicate further analysis (Bunt, 2010).

Although the forward extension of the anterior tubules is important for their morphogenesis, it is likely that other factors regulate their navigation through the body cavity. The kink region dips ventrally and the distal tips extend dorsally late in embryogenesis so that specialized cells at the distal tip contact dorsal structures. Further, morphogenesis of the posterior tubules is unaffected by the repression of BMP signaling; they migrate posteriorly, crossing the hindgut and adopt their normal position in the body cavity, with their tip cells contacting hindgut visceral nerves. It is probable that the coordination of multiple inputs controls the morphogenetic movements of all four tubules (Bunt, 2010).

This study has highlighted the importance of multiple tissue interactions in the outgrowth of Drosophila renal tubules, between the tubules and hemocytes, and, as a consequence of this interaction, with guidepost tissues such as the midgut visceral mesoderm. Similar interactions occur during the specification and recruitment of renal tubule cells, in the branching of the ureteric bud and in the formation of the glomerulus. In vertebrate nephrogenesis kidney medullary and cortical tubules extend, taking up stereotypical positions with respect to blood vessels, with which they later interact to maintain tissue homeostasis. TGF-β superfamily signaling plays multiple roles early in vertebrate kidney development so that analysis of signaling during renal tubule morphogenesis requires conditional alleles or specialized reagents. Such studies reveal requirements for TGF-β superfamily signaling in the morphogenesis of the pronephric tubules and duct in Xenopus, and for the maintenance and morphogenesis of mammalian nephrogenic mesenchyme. VEGF is expressed in early renal mesenchyme and ureteric bud and later in glomeruli, where it is essential for glomerular capillary growth. It will be exciting to discover whether a combination of VEGF/PDGF ligands in renal tissues and spatially restricted TGF-beta superfamily guidance cues underpins the coordinated morphogenesis of these spatially linked renal/blood systems, as has now been shown ro occur in Drosophila (Bunt, 2010).

Global tissue revolutions in a morphogenetic movement controlling elongation.

Polarized cell behaviors drive axis elongation in animal embryos, but the mechanisms underlying elongation of many tissues remain unknown. Eggs of Drosophila undergo elongation from a sphere to an ellipsoid during oogenesis. Live imaging of follicles (developing eggs) was used to elucidate the cellular basis of egg elongation. Elongating follicles were found to undergo repeated rounds of circumferential rotation around their long axes. Follicle epithelia mutant for integrin or collagen IV fail to rotate and elongate, which results in round eggs. Evidence is presented that polarized rotation is required to build a polarized, fibrillar extracellular matrix (ECM) that constrains tissue shape. Thus, global tissue rotation is a morphogenetic behavior that uses planar polarity information in the ECM to control tissue elongation (Haigo, 2011).

Elongation of a tissue along a major body axis is a central and conserved feature of animal development, and defects in this process cause human developmental abnormalities. Studies of elongating tissues have uncovered morphogenetic behaviors such as convergent extension, part of a small repertoire of cell movements known to shape animal body plans. However, for many tissues, the mechanism underlying their elongation is unknown (Haigo, 2011).

The development of the ellipsoid Drosophila egg is an elegant case of tissue elongation. Drosophila eggs develop from individual follicles, each consisting of a somatic follicle cell epithelium that surrounds the germline. Follicles are initially spherical and grow isotropically but acquire anisotropic growth along the antero-posterior (A-P) axis from stage 4 of oogenesis to form a mature (stage 14) egg. 74 percent of this 2.5-fold elongation is achieved in 20 hours between stages 5 and 9. How the developing follicle breaks symmetry to channel a 20-fold increase in volume from a sphere to an ellipsoid is poorly understood. Evidence indicates that egg shape requires activities within the follicle epithelium, specifically from proteins linking intracellular actin to the ECM, but how follicle cells confer egg shape has remained elusive (Haigo, 2011).

Analysis of fixed samples suggested that polarized cell divisions and cell shape changes, which are associated with elongation of other tissues, are not readily apparent in elongating follicles. To determine whether dynamic cell behaviors are involved, live imaging of elongating follicles was used. This analysis revealed a previously undocumented morphogenetic behavior. The entire follicle epithelium undergoes a dramatic migration, in a circumferential direction around the elongating A-P axis, leading to global rotation of this geometrically continuous tissue (Haigo, 2011).

Polarized rotation is observed in > 95% of wild type (WT) follicles with a velocity of 0.26–0.78 μm/min and both left- and right-handed chirality. Polarized rotation is developmentally regulated, occurring prominently between stages 5 and 9, which parallels the major phase of follicle elongation. The data suggest that a developing follicle undergoes approximately 3 revolutions during elongation (Haigo, 2011).

Visualization of germline nuclei revealed rotation in concert with follicle cells, both in direction and angular velocity. By contrast, follicle cells move across static Collagen IV, demonstrating active rotation over an ECM substrate. 'Follicle rotation' therefore involves global polarized revolutions of each developing egg within the basement membrane that encases each follicle (Haigo, 2011).

The strong correlation between the phases of follicle rotation and egg elongation suggests that this behavior might play a role in morphogenesis. Follicles mosaic for null mutants in the integrin βPS subunit (myospheroid; mys), which is required for egg elongation were examined. mys mosaic follicles are significantly rounder than WT controls from stage 5, the time when follicle rotation normally occurs. Live imaging of round mys mutant follicles revealed failure to rotate or off-axis rotation in most samples (Haigo, 2011).

mys or vkg mosaic follicles do not undergo polarized rotation and do not elongate. The requirement for βPS integrin in follicle shape and rotation suggest that cell-ECM interactions link both processes. Unique among ECM components, Collagen IV forms circumferentially planar polarized fibrils around the follicle during the entire elongation phase. This led to a hypothesis that Collagen IV, like Laminin and Perlecan, may control egg shape. Indeed, follicles with epithelia entirely mutant for Collagen IV α2 (viking; vkg) deviate in shape at stage 8, ultimately forming round eggs. Live imaging revealed polarized rotation until stage 7, when there is a notable breakdown in rotation. These data demonstrate that mutations in genes that block follicle rotation also block elongation in a similar time frame, suggesting tight coupling of these two processes (Haigo, 2011).

What mechanism might link follicle rotation to egg shape? Intriguingly, Collagen IV fibrils increase in length and density during follicle rotation and change their organization. Collagen IV forms an initial basal matrix around young follicles with distinct puncta that mature into circumferentially oriented fibrils from stage 5 onward. Fibril orientation is tightly regulated, in the same orientation that the follicle rotates. This suggests that coordinated migration of follicle cells during global rotation may direct the polarization of the fibrillar ECM (Haigo, 2011).

To test this in vivo, clonal analysis was carried out with a chromosome in which Collagen IV-GFP is genetically linked to a red fluorescent nuclear marker, allowing the position of RFP-marked follicle cells to be compared with the distribution of Collagen IV-GFP that they have secreted. It was observed that Collagen IV-GFP fibrils are present in unmarked domains of the follicle epithelium, indicating that the cells that produced them moved relative to these fibrils. This was not observed when follicle rotation is blocked. Moreover, when Collagen IV-GFP producing cells are distributed across the A-P axis of the follicle, they distribute marked fibrils across this axis. In contrast, if Collagen IV-GFP producing follicle cells occupy primarily the anterior or posterior half of the follicle, marked fibrils are associated only with that region of the follicle. These data are consistent with a model in which global rotation builds the polarized basement membrane that surrounds developing follicles (Haigo, 2011).

What is the role of the polarized basement membrane produced by follicle rotation? One model is that of a 'molecular corset,' which could control egg shape by constraining growth along the dorso-ventral axis. Planar-polarized basal actin filaments of the follicle epithelium have been proposed to form a corset, However, acutely disrupting actin filaments in elongated follicles with Latrunculin A did not perturb follicle shape (Haigo, 2011).

Collagen IV organization made it an attractive alternative for a molecular corset. Basement membrane integrity was found to be compromised in vkg mutant follicles, consistent with vertebrate studies showing that type IV Collagens maintain, but do not establish, ECM organization. It was reasoned that if the fibrillar Collagen IV matrix acts as a molecular corset, then its acute loss should affect the shape of elongated follicles. WT stage 12 follicles, which have completed global revolutions and display a polarized Collagen IV matrix, were treated with Collagenase. Acute loss of Collagen IV rounds these follicles, supporting Collagen IV matrix activity as a molecular corset (Haigo, 2011).

Finally, when follicle rotation is blocked (in round mys mosaic follicles) the Collagen IV matrix is present but its organization is perturbed. Although fibril density and length of the longest fibrils was unchanged, the shape of individual fibrils was significantly altered. Importantly, the uniform orientation of fibrils was completely lost. Together, these results suggest that the polarization of the Collagen IV matrix, via global tissue revolutions, governs elongation of the Drosophila egg (Haigo, 2011).

This work expands the repertoire of known morphogenetic behaviors by identifying a novel morphogenetic movement that elongates a developing tissue. Like many other collective cell migrations, basal cell-ECM focal contacts provide the motile force for follicle rotation, but the follicle's unique closed topology with no leading edge results in a treadmill-like migration with no net translocation. Like convergent extension, cells move orthogonal to the axis of elongation to generate a more than two-fold elongation of the tissue. However, in the radially symmetric follicle epithelium no axis of convergence is evident. Engagement of all cells of the tissue in multiple revolutions distinguishes follicle rotation from known phenomena involving partial and local rotation of cell clusters within a tissue. The signal(s) that dictate the chirality, onset and cessation of rotation remain interesting unanswered questions (Haigo, 2011).

Polarized cell movements involve planar cell polarity (PCP). PCP in the follicle was first noted two decades ago, and whereas follicle PCP and egg shape are independent of the core PCP signaling pathway, they do require proteins that link the actin cytoskeleton and ECM. Early work proposed that polarized basal actin mechanically constrains egg shape, but the discovery of follicle rotation suggests an alternative, in which actin filaments are required for polarized cell motility during egg elongation. The data indicate that polarized, global follicle rotation directs polarization of the Collagen IV matrix, echoing other systems where migrating cells influence surrounding ECM structure. Importantly, the polarized ECM can communicate PCP information and also feed back to promote directed tissue migration. Since individual follicle cells move over ECM fibrils previously oriented by neighbors, global rotation can both amplify and reinforce coordination of PCP across the entire tissue. It is suggested that PCP coordination in the follicle may thus result from dynamic movement of an epithelium across a static cue –the ECM – rather than propagation of a cue through a static epithelium (Haigo, 2011).

Finally, in providing a specific mechanism for the control of Drosophila egg shape, this work sheds light on the general role of the ECM in sculpting tissues. The circumferential Collagen IV fibrils formed by follicle rotation recall the circumferential cellulose fibrils formed by rotating the cellulose synthase complex within the plasma membrane during elongation of stationary plant cells. Whereas the proposed mechanism can account for the majority of follicle elongation, data from Drosophila mutants suggest that it is only one of several mechanisms that establish final egg shape (Haigo, 2011).

A morphogenetic movement with the attributes of follicle rotation has not been described in other animal tissues. It is noted that the existence of follicle rotation was not anticipated despite a rich history of studies on Drosophila oogenesis from fixed specimens. Live imaging of other morphogenetic events may uncover additional instances where polarized tissue rotation influences tissue and organ shape (Haigo, 2011).

Basement membrane and cell integrity of self-tissues in maintaining Drosophila immunological tolerance

The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. This study established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. Genes for the two collagen IV subunits and the four laminin subunits (see Laminin A) were nocked down individually via UAS-RNAi using ubiquitous and tissue-specific GAL4 drivers. The basement membrane was found to endow otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and it was further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, cell integrity, as determined by cell-cell interaction and apicobasal polarity, was found to function as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity (Kim, 2014)

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster

SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. It was previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. This study sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency was found to lead to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell autonomous accumulation of BL components around mutant cells that include Collagen IV (Col IV), Laminin and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. These data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV (Shahab, 2014).

The emergence of multicellular organisms was co-incident with the appearance of genes coding for extracellular matrix (ECM) molecules that gave rise to two major classes of ECMs: interstitial matrices and basal laminae (BL)/basement membranes. In contrast to vertebrate tissues where interstitial matrices predominate, BL are the principal ECMs in animals of lower phyla. Universal components of BLs include network-forming Collagen IV (Col IV), Laminin, Perlecan, and Nidogen, which are assembled into 2D sheet-like networks. In addition to serving as tissue boundaries and an adhesive substratum for cell anchoring and migration, BLs make diverse regulatory contributions to the development of tissues and organs (Hohenester, 2013; Shahab, 2014).

Col IV imparts tensile strength to BL and provides an anchoring substratum for cell adhesion, migration, and secreted signaling molecules. Much of what is known about Col IV is derived from vertebrate studies. Vertebrates express six Col IV α-chains [α1(ΙV)-α6(ΙV)] that are assembled in the endoplasmic reticulum into different combinations of heterotrimeric protomers. Upon secretion, the C-terminal globular domain of these trimeric protomers form head-to-head dimers Flexible non-helical interruptions separating collagenous domains of the protomers promote lateral associations during supramolecular assembly of 2D Col IV networks. Further contributing to the stability of these networks, the N-terminal globular domain of the heterotrimers form anti-parallel tetramers. As with fibril-forming collagens, purified Col IV protomers can self-assemble into polymeric networks. In contrast to vertebrates, the Drosophila genome codes for only two Col IV α-chains: Dcg 1/Cg25C and Viking (Vkg). The primary sources of BL components produced within Drosophila embryos and larvae are hemocytes and the fat body (Olofsson, 2005); however, how Col IV and the other BL components are assembled into a stereotypic 2D sheet of precise thickness is unknown (Shahab, 2014).

Previously studies have shown that SPARC (Secreted Protein, Acidic and Rich in Cysteine), a highly conserved matricellular glycoprotein, is a major component of embryonic Drosophila BL (Martinek, 2002; Martinek, 2008). SPARC, also known as osteonectin/ BM40, binds to fibril-forming collagens and Col IV via epitopes located within the C-terminal domain. The absence of interstitial matrices in Drosophila makes it an ideal developmental and genetic model to decipher the role of SPARC in BL assembly and maturation (Shahab, 2014).

Using imprecise P-element excision to generate a mutation/deletion of SPARC in Drosophila, a previous study reported decreased Col IV and BL stability and neural defects resulting in embryonic lethality in the absence of SPARC. However, attempts to rescue embryonic lethality by expressing exogenous SPARC were unsuccessful (Martinek, 2008), raising the possibility that aspects of this phenotype were due to a second site mutation on the 3rd chromosome. The present study, determined that both the neural phenotype and embryonic lethality reported previously, result from a disruption of the neurogenic gene, neutralized. The disruption of SPARC alone leads to larval lethality characterized by compromised fat body homeostasis. The fat body is crucial for development. It acts as the primary source of energy, and fat body together with hemocytes are the principle sources of BL components during larval development. Formed during embryonic development, the larval fat body is a bilateral, multi-lobed organ consisting of a monolayer of about 2,200 polygonal cells called adipocytes. The larval fat body is entirely surrounded by hemolymph, but does not directly interface with it owing to the presence of a BL that covers the entire surface of the fat body. The adipocytes within the fat body have no classical apical-basal polarity. Instead, cell-cell adhesion and shape is mediated by BL surrounding the adipocytes (Pastor-Pareja, 2011). This study reports that a reduction of SPARC leads to defective fat body BL assembly, inducing the resident polygonal adipocytes to round up and accumulate BL components within their microenvironment in a cell-autonomous manner. These findings define a pivotal role for SPARC in the proper assembly of BL surrounding the adipocytes of the Drosophila fat body (Shahab, 2014).

The results of this study demonstrate that loss or knockdown of SPARC expression in Drosophila result in arrest during larval development and disruption of fat body architecture and function. Based upon the SPARC mutation Df(3R)nm136, it was previously reported that loss of SPARC resulted in embryonic lethality associated with severe defects in nervous system development. This study now provide evidence that a second-site mutation present in the neuralized locus, a key regulator of Notch/Delta signalling, is the cause of the Df(3R)nm136 neural phenotype and embryonic lethality. Hence, SPARC is not required for nervous system development (Shahab, 2014).

The new Df(3R)nm136 H2AvD::GFP line, from which the neuralized mutation has been removed, demonstrates that loss of SPARC in Drosophila results in larval lethality and morphological changes of the fat body. The larval fat body is a multifunctional organ essential to fly development. Principle functions of the organ are nutrient storage and regulation of energy availability, functions that may become compromised in SPARC-deficient larvae. SPARC-deficient larvae appear transparent, which is consistent with reduced lipid or energy stores. While it is possible that knockdown of SPARC in hemocytes was responsible for the lethality and fat body morphological defects, knockdown of SPARC selectively within hemocytes using a hemolectin promoter did not result in larval lethality or a fat body phenotype, indicating that the phenotype reported in this study is due to loss of fat body SPARC expression. Moreover, larval lethality and the fat body phenotype of SPARC mutant larvae were rescued by a SPARC transgene that was expressed under the control of either endogenous SPARC or Col IV promoters (Shahab, 2014).

SPARC reduction led to a marked accumulation of BL components in the extracellular microenvironment of affected adipocytes. Temporal expression data from modENCODE indicate that maximum levels of SPARC and Col IV expression occur during the 1st and 2nd instar stages, with expression decreasing during the 3rd larval instar prior to pupariation. Consistent with the idea that SPARC effects are largely mediated prior to the late 3rd instar stage, knockdown of SPARC in 3rd instar had no impact on survival or fat body remodeling (Shahab, 2014).

Pastor-Pareja (2011) showed that knockdown of SPARC results in extracellular assembly of Col IV into thick fibers in the fat body, leading them to speculate that SPARC is required for Col IV secretion and solubility. However, the impact of SPARC knockdown on Col IV secretion, BL integrity, or adipocyte morphology was not addressed in that study. The current study suggests that SPARC deficiency does not prevent Col IV secretion. Consistent with the results of Pastor-Pareja (2011), this study shows extracellular accumulation of Col IV, suggestive of decreased solubility. Moreover, this study shows that Laminin, Perlecan, and Nidogen also accumulate at the surface of SPARC-deficient adipocytes, indicating that all BL components are affected by the loss or knockdown of SPARC (Shahab, 2014).

Biochemical studies have shown that SPARC binds to the triple-helical domains of purified invertebrate and vertebrate Col IV, an interaction that is mediated by two collagen-binding epitopes located in the C-terminal region of SPARC. Col IV is a primary regulator of cell shape and adhesion; thus, alterations in the availability or structure of Col IV fibrils impact cell morphology. Several studies have shown that SPARC has counter-adhesive activity in vitro that causes cells to detach from their substrate and round up. The current data appear paradoxical as loss of SPARC results in cell rounding but does not lead to adipocyte dissociation. However, the impact on cell shape in this instance is likely due to the dysregulation of Col IV polymerization and BL homeostasis, rather than directly to the effect of SPARC on cell-cell or cell-matrix interactions (Shahab, 2014).

A previous studies suggested that SPARC co-localizes with Col IV within secretory vesicles of adipocytes, but it remains to be determined whether SPARC and Col IV directly bind to one another intracellularly. Upon exocytosis, close proximity of SPARC with Col IV enables immediate physical association such that SPARC can regulate Col IV polymerization and sequester Col IV from its cellular receptors. Bradshaw (2009) demonstrated such a relationship between SPARC and Collagen I in mammalian cells. SPARC deficiency does not lead to an increase in intracellular Col IV, demonstrating that the impact of a lack of SPARC on Col IV assembly into BL likely occurs extracellularly. Upon secretion, SPARC may act to maintain solubility of Col IV, preventing it from immediately undergoing polymerization. In the absence of SPARC, Col IV release to the fat body extracellular space occurs; however, without SPARC to delay its polymerization, Col IV may rapidly assemble into a dense meshwork. Other ECM proteins, such as Laminin, Perlecan, and Nidogen, are synthesized and secreted; they encounter polymerized Col IV and are incorporated into the assembled structure as they would in a normal BL. This causes accumulation of multiple BL proteins on the surface of adipocytes. As ECM material accumulates, it promotes the rounding of the cells. The formation of a dense ECM meshwork likely impedes normal adipocyte function and could interfere with a variety of physiological processes such as feeding behavior and energy metabolism (Shahab, 2014).

In light of the diffusible nature of SPARC, the finding of a cell-autonomous phenotype with fat body SPARC knockdown clones was unexpected. The failure of SPARC secreted from adjacent wild-type adipocytes to compensate for the lack of production by SPARC-deficient cell clones indicates that SPARC was not able to diffuse across the BL in sufficient quantities. To date, no study that has addressed the ability of SPARC to diffuse across the BL, but the current data raise the possibility of a charge-dependent barrier that retains SPARC within the microenvironment of a cell. Alternatively, the more immediate interaction of SPARC with Col IV afforded by their intracellular co-localization may be required to effectively prevent premature polymerization of Col IV. Hence, an intracellular interaction between SPARC and Col IV may be required to regulate the kinetics of Col IV polymerization immediately upon its secretion (Shahab, 2014).

SPARC may also regulate BL deposition and remodelling through cell surface receptors. Expression of the cell-matrix adhesion molecules Dg and the βPS integrin subunit was observed on the plasma membrane of wild-type adipocytes. RNAi knockdown of SPARC did not alter the expression or localization of either of these transmembrane receptors in fat body cells indicating that it is unlikely that ECM accumulation around SPARC mutant adipocytes is associated with dysregulation of ECM receptors. However, the possibility that the interaction of BL components with these ECM receptors may have been affected cannot be excluded (Shahab, 2014).

Randomly distributed pits were observed on the surface of adipocytes, which increased in number with the knockdown of SPARC. However, the majority of the pits associated with a SPARC knockdown exhibited thickened circumferential borders underlaid by intracellular lipid-like vesicles. It is conceivable that the pits represent sites of lipid exocytosis. However, preliminary data indicates that the knockdown of SPARC does not affect protein or vesicular endocytosis and exocytosis. Moreover, differences in lipid content between wild-type and SPARC-deficient adipocytes were not observed. Hence the molecular basis of the dramatic difference in the surface topography between wild-type and SPARC-deficient adipocytes remains unknown (Shahab, 2014).

Analysis of the evolutionary history of SPARC revealed a conservation of the collagen-binding epitopes from cnidarians to mammals, which enable SPARC to bind to fibril-forming and network-forming Col IV. While SPARC-null mice develop normally, ultrastructural analysis revealed that interstitial Col IV fibrils are less abundant, smaller and more uniform in size, resulting in fibrils with decreased tensile strength. Biochemical studies indicate that SPARC increases the length of the first stage/lag phase of collagen fibrillogenesis by decreasing the rate of nucleation (Bradshaw, 2009). SPARC is also concentrated in the basal laminae of the nematode C. elegans. RNAi knockdown of SPARC leads to larval lethality for a large percentage of the progeny with a deficiency in gut granules and reduction in body size (Fitzgerald, 1998). It remains to be determined if aberrations in BL lamina assembly is the underlying cause of the phenotype (Shahab, 2014).

Hence, these findings support an emerging concept of SPARC as a critical extracellular collagen chaperone. A detrimental loss of BL homeostasis is evident in the absence of SPARC. The evolutionary conservation of SPARC parallels the advent of BL in multi-cellular organisms, indicating that this chaperone activity of SPARC is important for the maintenance of ECM homeostasis in all metazoans (Shahab, 2014).

Distinct functions of the laminin beta LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

The Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles. Using an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos, multiple mutants were obtained for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin beta and collagen IV alpha1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV alpha2 chain is detected during late embryogenesis. It is concluded that assembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. The data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the beta6/beta3/beta8 interface of the laminin beta LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases (Hollfelder, 2014).

Regulation of Drosophila matrix metalloprotease Mmp2 is essential for wing imaginal disc:trachea association and air sac tubulogenesis

The Drosophila Dorsal Air Sac Primordium (ASP) is a tracheal tube that grows toward Branchless FGF-expressing cells in the wing imaginal disc. This study shows that the ASP arises from a tracheal branch that invades the basal lamina of the disc to juxtapose directly with disc cells. The role of matrix metalloproteases (Mmps) was examined; reducing Mmp2 activity perturbed disc-trachea association, altered peritracheal distributions of collagen IV and Perlecan, misregulated ASP growth, and abrogated development of the dorsal air sacs. Whereas the function of the membrane-tethered Mmp2 in the ASP is non-cell autonomous, it was found that Mmp2 may have distinct tissue-specific roles in the ASP and disc. These findings demonstrate a critical role for Mmp2 in tubulogenesis post-induction, and implicate Mmp2 in regulating dynamic and essential changes to the extracellular matrix (Guha, 2009).

The invasive coupling of the wing disc with the Tr2 transverse connective and the ECM remodeling that accompanies ASP growth led to an investigation of how the presence of a Basal lamina (BL) impacts FGF signaling and the roles of MMPs. Based on the capacity of Bnl/FGF to signal through the disc and tracheal BL, it is concluded that these BL are functionally transparent to FGF. Based on the effects of btl-TIMP, ap-TIMP, btl-MMP2 RNAi and ap-MMP2RNAi, and on the phenotypes of Mmp2 mutants, it is concluded that Dm-MMP2 has essential roles sculpting the disc-trachea association and remodeling the ECM during ASP induction and growth. This discussion considers mechanisms underlying disc-trachea association, disc to trachea FGF signaling and the role of Dm-Mmp2 in tissue contact, ECM remodeling and organ morphogenesis (Guha, 2009).

Invasive coupling of the wing disc and trachea must involve several distinct processes. First, progenitor disc and trachea cells, which originate independently in the embryo, must establish contact. Disc cells migrate towards the trachea during embryogenesis, leading to direct juxtaposition. Collagen IV is not detected at this stage, so it seems unlikely that the BL had fully formed. The current experiments did not address whether Mmps are required for the early association of disc and trachea. However, it was found that L3 discs remain associated with trachea in Mmp2w307 and Mmp1Q273 mutants. Since the Mmp2w307 allele harbors a nonsense mutation and is a genetic null, these findings suggest that Mmp2 is not required to join these tissues (Guha, 2009).

Second, the arrangement of BL over the invasively coupled tracheal segment requires precise position-specific synthesis as well as continuous remodeling as the disc and trachea grow. Since core proteins of the ECM (e.g. collagen IV) are expressed by only a few disc cells, most BL components are presumably recruited from circulating stores in the hemolymph; little is known of the processes that bring these components to appropriate locations or regulate their assembly. It is not known, for instance, whether the absence of a distinct tracheal BL where the transverse connective contacts the disc is a consequence of insufficient levels of components. Alternatively, if availability of BL components is not limiting, it is not known whether the enzymes that synthesize BL are absent from these locations, or whether proteases that degrade the core components might be activated there. The changes in levels of Mmp2 that were engineered had significant effects on the ECM and its components. The presence of ectopic BL around disc-associated trachea in ap-TIMP animals suggests that neither components nor synthetic enzymes are limiting. Moreover, the accumulation of collagen IV and Perlecan in Mmp2 mutants revealed that proteolysis of ECM components is dependent upon Mmp2 and regulates BL assembly around disc-associated tracheae. collagen IV and Perlecan are either substrates of Mmp2 or their levels are dependent on another component that is. The distinct effects of reducing Mmp2 activity in the trachea (increased collagen IV and stunted ASP growth) or in the disc (hypertrophic ASP growth) show that location and level of Mmp2 activity is critically important for normal association and growth of the ASP. They also imply that the location and level of Mmp2 expression must be precisely regulated and that Mmp2, a membrane-tethered enzyme, might have different substrates in the disc and trachea extracellular milieu. A possible explanation is that despite the absence of a lamina densa separating the ASP and disc, distinct collagen-containing layers overly each tissue (Guha, 2009).

Third, the invasively coupled transverse connective and ASP nestle within the plane of the disc such that the overlying ECM forms a relatively flat sheet. However, reducing Mmp2 activity led to the partial extrusion of the disc-associated trachea. This phenotype revealed that the character of the disc:trachea association is sensitive to the composition of the ECM, and is impaired if the system's capacity to remodel the ECM is reduced (Guha, 2009).

The BL is rich with proteins that bind and sequester growth factors, and it therefore has the potential to block movement of proteins such as FGF. However, ectopic expression of Bnl/FGF in the disc induces invasive outgrowths from regions of the trachea that do not contact the disc and are separated from disc cells by two layers of BL. Invasive coupling and direct contact are not therefore prerequisites for signaling and growth. The ectopic expression assay is a qualitative measure of FGF signaling and does not ascertain whether direct apposition facilitates signaling; however, the results suggest that the BL is functionally transparent to FGF signaling. Tunneling may be needed for other purposes, for example, for the ASP to interact with the disc and to develop together with other thoracic structures during pupal development. It is speculated that the functional transparency of the BL is likely to be general property, that the BL may be generally transparent to signaling proteins and growth factors. Such transparency would be relevant to the mechanisms that distribute signaling proteins, since constraining signaling proteins to restrict their influence to only their intended targets would seem to be an essential feature (Guha, 2009).

Although tube formation is essential to generate many vertebrate organs, Drosophila offers few relevant models. Strategies for making tubes have been classified according to the apical-basal polarity of the founding cells. Some, such as the vertebrate mammary gland, hair follicle and early pancreas, form from clusters of cells that initially lack polarity but acquire apical-basal polarity as they coalesce around a central lumen. Others, such as the vertebrate liver, lung and neural tube and the Drosophila salivary glands, form directly from morphogenetic movements of polarized epithelial sheets. The progenitors of these tubes retain their apical-basal polarity as they generate tubular extrusions (Guha, 2009).

The ASP is an example of the latter type of tubulogenesis. The cells of the ASP retain the apical-basal polarity of the tracheal epithelium from which they emerge. Many of the cells in the ASP are mitotically active, distinguishing the ASP from the Drosophila salivary gland, whose cells invaginate from an epithelial sheet but do not divide. The process of ASP tubulogenesis is therefore more like that of the vertebrate liver, lung and neural tube, which also grow by coupling cell division to invagination and morphogenesis (Guha, 2009).

Mmps have been implicated in organ morphogenesis in a variety of contexts. A relevant example is HGF-induced tubulogenesis by MDCK cells cultured in 3D-matricies. Initial stages of tube morphogenesis required ERK activation, after which tube growth was dependent on Mmps but independent of ERK. Since the Drosophila ASP was induced but its growth was stunted in genetic backgrounds that reduced Mmp function, Mmps also appear to have a stage-specific role in ASP morphogenesis (Guha, 2009).

Type IV collagens regulate BMP signalling in Drosophila

Dorsal-ventral patterning in vertebrate and invertebrate embryos is mediated by a conserved system of secreted proteins that establishes a bone morphogenetic protein (BMP) gradient. Although the Drosophila embryonic Decapentaplegic (Dpp) gradient has served as a model to understand how morphogen gradients are established, no role for the extracellular matrix has been previously described. This study shows that type IV collagen extracellular matrix proteins bind Dpp and regulate its signalling in both the Drosophila embryo and ovary. Evidence is provided that the interaction between Dpp and type IV collagen augments Dpp signalling in the embryo by promoting gradient formation, yet it restricts the signalling range in the ovary through sequestration of the Dpp ligand. Together, these results identify a critical function of type IV collagens in modulating Dpp in the extracellular space during Drosophila development. On the basis of findings that human type IV collagen binds BMP4, it is predicted that this role of type IV collagens will be conserved (Wang, 2008).

There are two type IV collagen proteins in Drosophila, Viking (Vkg) and Dcg1 (also known as Cg25C). The ability was tested of secreted biologically active, epitope-tagged Dpp purified from the media of transfected Drosophila S2 tissue culture cells to bind to the amino- and carboxy-terminal non-collagenous domains of Vkg and Dcg1. Dpp-haemagglutinin (HA) binds to the C-terminal but not the N-terminal domains of both Vkg and Dcg1, whereas denatured Dpp-HA protein does not. Dpp/Scw heterodimers also bind to the Vkg and Dcg1 C-terminal domains. Surface plasmon resonance analyses show that the binding between Dpp and glutathione S-transferase (GST)-VkgC or GST-DcgC is saturable and has dissociation constants (Kd) of 0.75 and 0.65 microM, respectively (Wang, 2008).

Deletion analysis of VkgC identified a region required for Dpp interaction which, when aligned with the equivalent region of Dcg1, shows a short conserved sequence. Deletion of five of these amino acids from the Vkg C-terminal domain severely attenuates the interaction between Vkg and both Dpp and Dpp/Scw ligands. As these binding studies used GST fusion proteins purified from bacteria, the results were confirmed using GST-VkgC and GST-VkgCDelta proteins secreted into the media of transfected S2 cells. In addition, further mutational analysis was performed of the Vkg sequences required for Dpp interaction. The amino acids in Vkg that are necessary for Dpp interaction are present in a sequence that is conserved in mosquito, worm, mouse and human type IV collagens. Alignment of all the known type IV collagen sequences from these species identifies a consensus Y/FI/VSRCXVCE, which may function as a conserved BMP-binding module. In support of this, saturable binding has been shown between human full-length triple-helical type IV collagen and BMP4 with a Kd of 92 nM (Wang, 2008).

The data demonstrate that the interaction between Dpp and collagen IV is an essential aspect of correct signalling in the Drosophila germarium and early embryo. In wild-type germaria, it is suggested that Dpp secreted from the niche binds to Vkg, which restricts Dpp signalling range from the source. In mutant germaria with reduced Vkg protein, less Dpp will be bound by Vkg, resulting in an increased Dpp signalling range which downregulates bam transcription in more cells, thereby increasing GSC number (Wang, 2008).

In the embryo, a model is favored whereby binding of Dpp/Scw to type IV collagens facilitates assembly of the Dpp/Scw-Sog-Tsg complex. Tolloid (Tld) cleavage of this complex releases Dpp/Scw, which can rebind type IV collagens. In the presence of Sog, the inhibitory complex will be reassembled, whereas in the absence of Sog, type IV collagens will promote Dpp/Scw-receptor interactions. This latter function may require the unusual apical distribution of Vkg protein in the embryo, since Dpp seems to predominantly interact with its receptor apically in the embryo. In the dorsal ectoderm, initial Dpp signalling enhances subsequent Dpp signalling by the activation of an as yet unidentified target gene in a positive feedback loop. By promoting Dpp-receptor interactions at the dorsal midline leading to target gene activation, type IV collagens will facilitate the amplification of signalling by positive feedback (Wang, 2008).

The model explains the phenotype of embryos from vkg/+ females; the reduced amount of type IV collagens would impair assembly of the Dpp/Scw-Sog-Tsg complex and initial gradient formation. Disruption of the early gradient, in combination with reduced receptor interactions in type IV collagen mutant embryos, will reduce target gene expression and positive feedback, further decreasing subsequent signalling. As a result, the peak Dpp target genes are lost and intermediate thresholds are thinner (Wang, 2008).

In addition to the role of type IV collagens in regulating Dpp signalling in the early embryo that is described here, integrins (other principal constituents of basal lamina) are required for apposition of the amnioserosa and yolk sac to mediate proper germ band retraction and dorsal closure during later embryonic development. Therefore, basal lamina components have repeated roles in dorsal-ventral patterning of the fly embryo. Different types of extracellular matrix proteins also modulate BMP signalling at other development stages, for example, heparan sulphate proteoglycans regulate Dpp movement in the Drosophila wing. In vertebrates, type IV collagens are not only transcriptional targets of BMP signalling, but they also bind BMP4 and have been suggested to potentiate signalling in tissue culture cells. It is proposed that the conserved sequence that were identified will function as a BMP-binding module, and that type IV collagens will affect BMP signalling during vertebrate development (Wang, 2008).

Multistep molecular mechanism for bone morphogenetic protein extracellular transport in the Drosophila embryo

In the Drosophila embryo, formation of a bone morphogenetic protein (BMP) morphogen gradient requires transport of a heterodimer of the BMPs Decapentaplegic (Dpp) and Screw (Scw) in a protein shuttling complex. Although the core components of the shuttling complex--Short Gastrulation (Sog) and Twisted Gastrulation (Tsg)--have been identified, key aspects of this shuttling system remain mechanistically unresolved. Recently, it was discovered that the extracellular matrix protein collagen IV is important for BMP gradient formation. This study formulates a molecular mechanism of BMP shuttling that is catalyzed by collagen IV. Dpp is shown to be the only BMP ligand in Drosophila that binds collagen IV. A collagen IV binding-deficient Dpp mutant signals at longer range in vivo, indicating that collagen IV functions to immobilize free Dpp in the embryo. In vivo evidence is provided that collagen IV functions as a scaffold to promote shuttling complex assembly in a multistep process. After binding of Dpp/Scw and Sog to collagen IV, protein interactions are remodeled, generating an intermediate complex in which Dpp/Scw-Sog is poised for release by Tsg through specific disruption of a collagen IV-Sog interaction. Because all components are evolutionarily conserved, it is proposed that regulation of BMP shuttling and immobilization through extracellular matrix interactions is widely used, both during development and in tissue homeostasis, to achieve a precise extracellular BMP distribution (Sawala, 2012).

There is ample experimental and theoretical support for the notion that BMP gradient formation in the early embryo involves the concentration of the most potent signaling species, the Dpp/Scw heterodimer, at the dorsal midline in a process involving Sog and Tsg. This study presents in vivo evidence for a role of collagen IV in two key aspects of this shuttling model, which have remained mechanistically unresolved. First, collagen IV functions to immobilize free Dpp, explaining why Sog and Tsg are needed for Dpp movement. Second, collagen IV acts as a scaffold for assembly of the Dpp/Scw-Sog-Tsg shuttling complex. The advantage to BMP gradient formation of assembling the shuttling complex on collagen IV has been suggested by analysis of organism-scale mathematical models. These models reveal that the in vitro binding affinity between BMPs and Sog is too low to account for the rate of shuttling complex formation required in vivo. However, by acting as a scaffold, collagen IV would increase complex formation by locally concentrating Dpp/Scw and Sog. Models with a 10–20% reduction in diffusion rates for Dpp/Scw and Sog and an increased apparent affinity of Dpp/Scw for Sog, show the best fit to in vivo data (Sawala, 2012).

The molecular model of shuttling complex assembly occurs in three steps. The first step involves independent binding of Dpp/Scw and Sog to collagen IV. The ability of Dpp-Δa to signal long range in sog embryos, where wild-type Dpp is trapped in its expression stripe, provides in vivo evidence that the Dpp-collagen IV interaction restricts movement of free Dpp ligands. The result also demonstrates that Sog and Tsg promote long-range movement of Dpp because they release Dpp from collagen IV, and not simply because they prevent Dpp–receptor interactions. Restriction of Dpp diffusion by collagen IV may stabilize the gradient by preventing ventral movement of Dpp/Scw after release from Sog/Tsg and promoting Dpp/Scw–receptor interactions at the dorsal midline. It will be interesting, ultimately, to directly visualize Dpp and Dpp-Δa directly in sog and tsg mutant embryos. Although current methods allow detection of high levels of receptor-bound Dpp, there are technical limitations associated with specifically detecting the pools of Dpp that would be informative here, i.e., Dpp/Scw heterodimer within the shuttling complex or Dpp-Δa/Scw diffusing between cells. The data show that Scw is unable to bind the NC1 domain of collagen IV. This lack of collagen IV-dependent immobilization can explain why Scw, unlike Dpp, is capable of long-range signaling in the absence of Sog (Sawala, 2012).

Step 2 of shuttling complex assembly involves remodeling of the protein interactions to generate a poised intermediate. Specifically, step 2 is driven by Scw-mediated disruption of the Sog CR4–collagen IV interaction, so that Dpp/Scw is transferred from collagen IV to the Sog CR3-CR4 domains. Scw displacement of the Sog CR4 domain from collagen IV provides molecular insight as to why Scw is needed for Dpp transport. In addition to the binding preference of Sog and Tsg for the Dpp/Scw heterodimer, only Scw has a high affinity for the Sog CR4 domain. Therefore, Dpp/Scw can be released from collagen IV into the shuttling complex, whereas the Dpp homodimer remains trapped on collagen IV (Sawala, 2012).

In the final step of the model, Tsg mobilizes the shuttling complex by disrupting the Sog CR1–collagen IV interaction. It has been noted that tsg mutants display a more severe reduction in BMP signaling than sog and sog tsg double mutants. This observation has been attributed to a potential Sog-independent pro-BMP activity of Tsg at the level of receptor binding. A second contributing factor is suggested by the model, where Sog and Tsg act at distinct steps to allow formation of the shuttling complex. In tsg mutants, Dpp/Scw is loaded onto Sog by collagen IV, but remains locked in this inhibitory poised complex, so that the only BMPs capable of signaling are Dpp and Scw homodimers, which are less potent than the Dpp/Scw heterodimer. By contrast, in sog or sog tsg mutants, Dpp/Scw is not shuttled dorsally but is still capable of signaling locally, adding to signaling by Dpp and Scw homodimers. The weaker level of Dpp/Scw signaling in tsg mutants also provides support for the proposed order of steps 2 and 3 in the assembly process, because this order gives rise to the inhibitory intermediate of Dpp/Scw-Sog. Previously it was shown that an N-terminal fragment of Sog, called Supersog, which contains the CR1 domain and a portion of the stem, can partially rescue the loss of peak Dpp/Scw signaling in tsg embryos. The model suggests that this property of Supersog comes from the ability of its CR1 domain to compete with full-length Sog for binding to collagen IV, thereby releasing Sog-Dpp/Scw, similar to the role of Tsg in shuttling complex assembly. It is noted that the CR1–collagen IV interaction appears weaker than that of CR4–collagen IV, which may facilitate release of Dpp/Scw by Tsg or Supersog-like fragments. After Tsg-mediated release from collagen IV, the mobile shuttling complex can diffuse randomly. Upon Tolloid cleavage of Sog, the liberated Dpp/Scw heterodimer rebinds collagen IV, which either promotes receptor binding or a further round of shuttling complex assembly, depending on the local concentration of Sog (Sawala, 2012).

In addition to collagen IV, the basic region in Dpp/BMP2/4 also binds to heparan sulfate proteoglycans (HSPGs), which can either restrict or enhance BMP long-range movement. Indeed, this study found that an HSPG-binding mutant, Dpp-ΔN, also binds only weakly to collagen IV, suggesting that the collagen IV- and HSPG-binding sites on Dpp overlap. It will be interesting to test how HSPGs and collagen IV interact to regulate BMP activity in tissues where they are coexpressed, such as the early vertebrate embryo. In the early Drosophila embryo, the absence of glycosaminoglycan chains, which largely mediate binding of HSPG to Dpp, make it possible to specifically focus on the Dpp–collagen IV interaction (Sawala, 2012).

A shuttling-based mechanism of BMP transport is also used in a number of other developmental contexts, including the early vertebrate embryo, specification of the vertebral field in mice, and establishment of the posterior cross-vein territory in the Drosophila wing disk. Restriction of BMP movement may also be important in other contexts, including several where collagen IV was already shown to regulate a short-range Dpp signal, such as the ovarian stem cell niche and the tip of malpighian tubules. The basic collagen IV binding motif is highly conserved among the Dpp/BMP2/4 subfamily and is also found in some other BMPs, including BMP3, consistent with reports that BMP3 and BMP4 can bind collagen IV. Overall, these findings support the idea that the collagen IV–BMP interaction is a conserved aspect of extracellular BMP regulation and suggest that the function of collagen IV in both long-range BMP shuttling and local restriction of BMP movement will impact on a number of other contexts in both flies and vertebrates (Sawala, 2012).

Drosophila SPARC is a self-protective signal expressed by loser cells during cell competition

During development and aging, animals suffer insults that modify the fitness of individual cells. In Drosophila, the elimination of viable but suboptimal cells is mediated by cell competition, ensuring that these cells do not accumulate during development. In addition, certain genes such as the Drosophila homolog of human c-myc (dmyc) are able to transform cells into supercompetitors, which eliminate neighboring wild-type cells by apoptosis and overproliferate, leaving total cell numbers unchanged. This study identified Drosophila Sparc as an early marker transcriptionally upregulated in loser cells that provides a transient protection by inhibiting Caspase activation in outcompeted cells. Overall, the unexpected existence of a physiological mechanism is described that counteracts cell competition during development (Portela, 2010).

This study describes the existence of a physiological mechanism that counteracts cell competition. Evidence is provided that dSPARC is a specific marker of cell competition, and not a general marker of apoptosis. Transcriptional activation of dsparc sets a higher threshold for Caspase activation in loser cells, possibly by inactivating an unknown secreted Killing Signal (KS), which is produced upon survival factor withdrawal. dSPARC is not a general inhibitor of apoptosis, despite its potent inhibition of cell competition-induced cell death. dSPARC may allow useful cells to recover from transient and limited damage before they are unnecessarily eliminated by their neighbors. These results show that dSPARC and Flower (Fwe) function in parallel and opposing pathways during cell competition, with dSPARC providing transient protection, whereas the 'Fwe Code' promotes cell elimination by labeling cells as 'losers' (Rhiner, 2010). Therefore, it seems likely that during early stages of cell competition, the decision of whether the potential loser cell will finally undergo apoptosis or not is still reversible. This intermediate state, where dSPARC protects outcompeted cells, may prevent the removal of valid cells that suffer only a temporary fitness deficit. However, if the differences in cellular fitness persist and/or are too ample, cell competition-induced apoptosis is, nevertheless, triggered (Portela, 2010).

One possibility is that secreted dSPARC blocks the unknown KS(s) directly in the extracellular space. dSPARC could bind directly to the KS(s) or just form a matrix that serves as a barrier for the KS(s) to reach the loser cells. The other possibility is that dSPARC could activate a protective pathway in an autocrine way that counteracts the effects of the KS. For example, mammalian SPARC has been shown to protect cells from apoptosis in vitro via activation of integrin-linked kinase and AKT (Weaver, 2008; Shi, 2004). The identity of the killing cell(s) is not yet known (Portela, 2010).

If cell competition is conserved in mammals, this role of dSPARC specifically repressing cell competition may have important consequences for understanding of mammalian development, homeostasis, stem cell replacement, or cancer. In particular, deregulation of this mechanism is likely to be important in cancer, for example by allowing metastatic cells to survive in a new environment or during the expansion of cancerization fields (Portela, 2010).

The Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn06946). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca2+-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S+O+) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S+O+ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S+O+H+Pv+) pre-prohemocytes initiate the expression of dome-gal4 (dg4), thereby maturing into prohemocytes. The prohemocyte population (S+O+H+Pv+dg4+) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S+O+H+Pv+dg4lowM+). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S+O+H+Pv+dg4-M+), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster

Two types of screens in Drosophila were conducted using enhancer detector strains to find genes involved in immunity and tumor formation; genes expressed in the immune system (type A; hemocytes, lymph glands and fat body) and genes increased in expression by bacterial infection (type B). For type A, tissue-specific reporter gene activity was determined. For type B, a variation of enhancer detection was devised in which beta-galactosidase is assayed spectrophotometrically with and without bacterial infection. Because of immune system involvement in melanotic tumor formation, a third type was hypothesized to be found among types A and B; genes that, when mutated, have a melanotic tumor phenotype. Enhancer detector strains (2800) were screened for type A, 900 for B, and 11 retained for further analysis. Complementation tests, cytological mapping, P-element mobilization, and determination of lethal phase and mutant phenotype have identified six novel genes, Dorothy, wizard, toto, viking, Thor and dappled, and one previously identified gene, Collagen IV. All are associated with reporter gene expression in at least one immune system tissue. Thor has increased expression upon infection. Mutations of wizard and dappled have a melanotic tumor phenotype (Rodriguez, 1996).

The novel approach of identifylng mutations with a melanotic tumor phenotype based only on selection of tissue specificity and/or infection inducibility of the reporter gene was successful for tissue specific selection and has identified the genes wizard and dappled. The lethality of both wizard and dappledEf1 is early in larval development, and thus would have gone undetected in previous screens, which have selected directly for a tumor formation phenotype in late larvae and pupae. The viability of dappledMLB is not unusual for a melanotic tumor mutation, but the combination with 100% tumor formation rather than the typical variable and lower levels makes this mutation a unique practical tool for isolation of new genes that suppress or enhance melanotic tumors. In general, melanotic tumors are postulated to be a reaction to abnormal development, and this has been extended to the proposal that all melanotic tumor mutants can be categorized as belonging to one of two classes: class 1 - melanotic tumors associated with apparently normal immune systems that are responding to abnormal tissues, and class 2 - melanotic tumors associated with obvious defects of the immune system's lymph glands and hemocytes. Since the lymph glands are normal in dappledMLB and dappledEf1, these are class 1 mutations. wizard has an expression pattern in lymph glands, oenocytes and head. wizard may thus be a class 1 or 2 mutation and requires further analysis to distinguish between the two possibilities. Some class 2 tumor forming genes have been molecularly characterized. Molecular characterization of class 1 tumor formation genes is lacking, and dappled is being cloned also to provide molecular information about this category (Rodriguez, 1996).

viking: identification and characterization of a second type IV collagen in Drosophila

An enhancer trap approach has been taken to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. A molecular analysis of two P-element insertion strains was conducted that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. The results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila (Yasothornsrikul, 1997).

The function of type IV collagen during Drosophila muscle development

Type IV collagen forms a network that provides the major structural support for basement membranes. Basement membranes are specialized forms of extracellular matrix with important functions in development. One collagen gene (Dcg1 or Cg25C) was characterized in Drosophila and shown to encode a collagen chain related to vertebrate basement membrane type IV collagen chains. To access the functional importance of type IV collagen during Drosophila myogenesis, two different approaches were adopted to decrease the Dcg1 gene expression in Drosophila embryos. Decrease in Dcg1 gene expression causes, in particular, defective muscle attachments. These mutant phenotypes suggest that type IV collagen acts to stabilize cell-matrix interactions (Borchiellini, 1996).


Search PubMed for articles about Drosophila Viking or Collagen IV

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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

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Biological Overview

date revised: 15 November 2022

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