Viking and Collagen type IV: Biological Overview | References
Gene name - viking and Collagen type IV
Cytological map position - 25C1-25C1 and 25C1-25C1
Symbols - vkg and Cg25C
Classification - C4: C-terminal tandem repeated domain in type 4 procollagen
Cellular location - secreted
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 has been shown that Perlecan can regulate myosin localization in the follicle cells of the Drosophila ovary (Mirouse, 2009). 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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
Aouacheria, A., et al. (2006). Insights into early extracellular matrix evolution: spongin short chain collagen-related proteins are homologous to basement membrane type IV collagens and form a novel family widely distributed in invertebrates. Mol. Biol. Evol. 23: 2288-2302. PubMed ID: 16945979
Borchiellini, C., Coulon, J., and Le Parco, Y. (1996). The function of type IV collagen during Drosophila muscle development. Mech. Dev. 58: 179-191. PubMed ID: 8887326
Bradshaw, A. D., Puolakkainen, P., Dasgupta, J., Davidson, J. M., Wight, T. N. and Sage, E. H. (2003). SPARC-null mice display abnormalities in the dermis characterized by decreased collagen fibril diameter and reduced tensile strength. J. Invest. Dermatol. 120: 949-955. PubMed ID: 12787119
Bunt, S., Hooley, C., Hu, N., Scahill, C., Weavers, H. and Skaer, H. (2010). Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila. Dev. Cell 19(2): 296-306. PubMed ID: 20708591
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
Fessler, J. H. and Fessler, L. I. (1989). Drosophila extracellular matrix. Annu. Rev. Cell Biol. 5: 309-339. PubMed ID: 2557060
Fitzgerald, M. C. and Schwarzbauer, J. E. (1998). Importance of the basement membrane protein SPARC for viability and fertility in Caenorhabditis elegans. Curr. Biol. 8: 1285-1288. PubMed ID: 9822581
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date revised: 16 January 2012
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