InteractiveFly: GeneBrief

Secreted protein, acidic, cysteine-rich: Biological Overview | References

BIOLOGICAL OVERVIEW

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 of 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 (Maurer, 1997: Rosenblatt, 1997). 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 (Hunzelmann, 1998: Kim, 1997: Sage, 1989). 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 (Yan, 2002). 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 (Gilmour, 1998). 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).

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

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

This study describes the existence of a physiological mechanism that counteracts cell competition. Evidence is provided that dSPARC is a specific marker of cell competition, and not a general marker of apoptosis. Transcriptional activation of dsparc sets a higher threshold for Caspase activation in loser cells, possibly by inactivating an unknown secreted Killing Signal (KS), which is produced upon survival factor withdrawal. dSPARC is not a general inhibitor of apoptosis, despite its potent inhibition of cell competition-induced cell death. dSPARC may allow useful cells to recover from transient and limited damage before they are unnecessarily eliminated by their neighbors. These results show that dSPARC and Flower (Fwe) function in parallel and opposing pathways during cell competition, with dSPARC providing transient protection, whereas the 'Fwe Code' promotes cell elimination by labeling cells as 'losers'. 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. 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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Elimination of unfit cells maintains tissue health and prolongs lifespan

Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, a molecular mechanism, based on 'fitness fingerprints' displayed on cell membranes, was identifed that allows direct fitness comparison among cells in Drosophila. This study reports the physiological consequences of efficient cell selection for the whole organism. The study found that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. A gene, ahuizotl (azot), was identified that ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan (Merino, 2015).

Individual cells can suffer insults that affect their normal functioning, a situation often aggravated by exposure to external damaging agents. A fraction of damaged cells will critically lose their ability to live, but a different subset of cells may be more difficult to identify and eliminate: viable but suboptimal cells that, if unnoticed, may adversely affect the whole organism (Merino, 2015).

What is the evidence that viable but damaged cells accumulate within tissues? The somatic mutation theory of aging proposes that over time cells suffer insults that affect their fitness, for example, diminishing their proliferation and growth rates, or forming deficient structures and connections. This creates increasingly heterogeneous and dysfunctional cell populations disturbing tissue and organ function. Once organ function falls below a critical threshold, the individual dies. The theory is supported by the experimental finding that clonal mosaicism occurs at unexpectedly high frequency in human tissues as a function of time, not only in adults an embryos (Merino, 2015).

Does the high prevalence of mosaicism in our tissues mean that it is impossible to recognize and eliminate cells with subtle mutations and that suboptimal cells are bound to accumulate within organs? Or, on the contrary, can animal bodies identify and get rid of unfit viable cells (Merino, 2015)?

One indirect mode through which suboptimal cells could be eliminated is proposed by the 'trophic theory,' which suggested that Darwinian-like competition among cells for limiting amounts of surv ead to removal of less fit cells. However, it is apparent from recent work that trophic theories are not sufficient to explain fitness-based cell selection, because there are direct mechanisms that allow cells to exchange 'cell-fitness' information at the local multicellular level (Merino, 2015).

In Drosophila, cells can compare their fitness using different isoforms of the transmembrane protein Flower. The 'fitness fingerprints' are therefore defined as combinations of Flower isoforms present at the cell membrane that reveal optimal or reduced fitness. The isoforms that indicate reduced fitness have been called FlowerLose isoforms, because they are expressed in cells marked to be eliminated by apoptosis called 'Loser cells.' However, the presence of FlowerLose isoforms at the cell membrane of a particular cell does not imply that the cell will be culled, because at least two other parameters are taken into account: (1) the levels of FlowerLose isoforms in neighboring cells: if neighboring cells have similar levels of Lose isoforms, no cell will be killed; (2) the levels of a secreted protein called Sparc, the homolog of the Sparc/Osteonectin protein family, which counteracts the action of the Lose isoforms (Merino, 2015 and references therein).

Remarkably, the levels of Flower isoforms and Sparc can be altered by various insults in several cell types, including: (1) the appearance of slowly proliferating cells due to partial loss of ribosomal proteins, a phenomenon known as cell competition; (2) the interaction between cells with slightly higher levels of d-Myc and normal cells, a process termed supercompetition; (3) mutations in signal transduction pathways like Dpp signaling; or (4) viable neurons forming part of incomplete ommatidia. Intriguingly, the role of Flower isoforms is cell type specific, because certain isoforms acting as Lose marks in epithelial cells are part of the fitness fingerprint of healthy neurons. Therefore, an exciting picture starts to appear, in which varying levels of Sparc and different isoforms of Flower are produced by many cell types, acting as direct molecular determinants of cell fitness. This study aimed to clarify how cells integrate fitness information in order to identify and eliminate suboptimal cells. Subsequently, the physiological consequences were analyzed of efficient cell selection for the whole organism (Merino, 2015).

In order to discover the molecular mechanisms underlying cell selection in Drosophila, this study analyzed genes transcriptionally induced using an assay where WT cells (tub>Gal4) are outcompeted by dMyc-overexpressing supercompetitors (tub>dmyc) due to the increased fitness of these dMyc-overexpressing cells. The expression of CG11165 was strongly induced 24 hr after the peak of flower and sparc expression. In situ hybridization revealed that CG11165 mRNA was specifically detected in Loser cells that were going to be eliminated from wing imaginal discs due to cell competition. The gene, which was named ahuizotl (azot) after a multihanded Aztec creature selectively targeting fishing boats to protect lakes, consists of one exon. azot's single exon encodes for a four EF-hand-containing cytoplasmic protein of the canonical family that is conserved, but uncharacterized, in multicellular animals (Merino, 2015).

To monitor Azot expression, a translational reporter was designed resulting in the expression of Azot::dsRed under the control of the endogenous azot promoter in transgenic flies. Azot expression was not detectable in most wing imaginal discs under physiological conditions in the absence of competition. Mosaic tissue was generated of two clonal populations, which are known to trigger competitive interactions resulting in elimination of otherwise viable cells. Cells with lower fitness were created by confronting WT cells with dMyc-overexpressing cells, by downregulating Dpp signaling, by overexpressing Flower^Lose isoforms, in cells with reduced Wg signaling, by suppressing Jak-Stat signaling or by generating Minute clones. Azot expression was not detectable in nonmosaic tissue of identical genotype, nor in control clones overexpressing UASlacZ. On the contrary, Azot was specifically activated in all tested scenarios of cell competition, specifically in the cells undergoing negative selection. Azot expression was not repressed by the caspase inhibitor protein P35 (Merino, 2015).

Because Flower proteins are conserved in mammals, tests were made to see if they are also able to regulate azot. Mouse Flower isoform 3 (mFlower³) has been shown to act as a 'classical' Lose isoform, driving cell elimination when expressed in scattered groups of cells, a situation where azot was induced in Loser cells but is not inducing cell selection when expressed ubiquitously a scenario where azot was not expressed. This shows that the mouse Flower^Lose isoforms function in Drosophila similarly to their fly homologs (Merino, 2015).

Interestingly, azot is not a general apoptosis-activated gene because its expression is not induced upon eiger, hid, or bax activation, which trigger cell death. Azot was also not expressed during elimination of cells with defects in apicobasal polarity or undergoing epithelial exclusion-mediated apoptosis (dCsk) (Merino, 2015).

azot expression was analyzed during the elimination of peripheral photoreceptors in the pupal retina, a process mediated by Flower-encoded fitness fingerprints. Thirty-six to 38hr after pupal formation (APF), when Flower^Lose-B expression begins in peripheral neurons, no Azot expression was detected in the peripheral edge. At later time points (40 and 44hr APF), Azot expression is visible and restricted to the peripheral edge where photoreceptor neurons are eliminated. This expression was confirmed with another reporter line, azot{KO; gfp}, where gfp was directly inserted at the azot locus using genomic engineering techniques (Merino, 2015).

From these results, it is concluded that Azot expression is activated in several contexts where suboptimal and viable cells are normally recognized and eliminated (Merino, 2015).

To understand Azot function in cell elimination, azot knockout (KO) flies were generated by deleting the entire azot gene. Next, Azot function was analyzed using dmyc-induced competition. In the absence of Azot function, loser cells were no longer eliminated, showing a dramatic 100-fold increase in the number of surviving clones. Loser cells occupied more than 20% of the tissue 72hr after clone induction (ACI). Moreover, using azot{KO; gfp} homozygous flies (that express GFP under the azot promoter but lack Azot protein), it was found that loser cells survived and showed accumulation of GFP. From these results, it is concluded that azot is expressed by loser cells and is essential for their elimination.

In addition, clone removal was delayed in an azot heterozygous background (50-fold increase, 15%), compared to control flies with normal levels of Azot. Cell elimination capacity was fully restored by crossing two copies of Azot::dsRed into the azot^-/- background demonstrating the functionality of the fusion protein. Silencing azot with two different RNAis was similarly able to halt selection during dmyc-induced competition. Next, in order to determine the role of Azot's EF hands, a mutated isoform of Azot (Pm4Q12) was generated and overexpressed, that carryed, in each EF hand, a point mutation known to abolish Ca²⁺ binding. Although overexpression of wild-type azot in negatively selected cells did not rescue the elimination, overexpression of the mutant AzotPm4Q12 reduced cell selection, functioning as a dominant-negative mutant. This shows that Ca²⁺ binding is important for Azot function. Finally, staining for apoptotic cells corroborated that the lack of Azot prevents cell elimination, because cell death was reduced 8-fold in mosaic epithelia containing loser cells (Merino, 2015).

The role of azot in elimination of peripheral photoreceptor neurons in the pupal retina was examined using homozygous azot KO flies. Pupal retinas undergoing photoreceptor culling (44hr APF) of azot^+/+ and azot^-/- flies were stained for the cell death marker and the proapoptotic factor. Consistent with the expression pattern of Azot, the number of Hid and TUNEL-positive cells was dramatically decreased in azot^-/- retinas compared to azot^+/+ retinas (Merino, 2015).

Those results show that Azot is required to induce cell death and Hid expression during neuronal culling. Therefore, tests were performed to see that was also the case in the wing epithelia during dmyc-induced competition. Hid was found to be expressed in loser cells and the expression was found to be strongly reduced in the absence of Azot function (Merino, 2015).

Finally, forced overexpression of Flower^Lose isoforms from Drosophila were unable to mediate WT cell elimination when Azot function was impaired by mutation or silenced by RNAi (Merino, 2015).

These results suggested that azot function is dose sensitive, because heterozygous azot mutant flies display delayed elimination of loser cells when compared with azot WT flies. Therefore advantage was taken of the functional reporter Azot::dsRed to test whether cell elimination could be enhanced by increasing the number of genomic copies of azot. Tissues with three functional copies of azot were more efficient eliminating loser cells during dmyc-induced competition and most of the clones were culled 48hr ACI. From these results, it is concluded that azot expression is required for the elimination of Loser cells and unwanted neurons (Merino, 2015).

Next, it was asked what could be the consequences of decreased cell selection at the tissue and organismal level. To this end, advantage was taken of the viability of homozygous azot KO flies. An increase of several developmental aberrations was observed. Focus was placed on the wings, where cell competition is best studied and, because aberrations, including melanotic areas, blisters, and wing margin nicks, were quantified. Wing defects of azot mutant flies could be rescued by introducing two copies of azot::dsRed, showing that the phenotypes are specifically caused by loss of Azot function (Merino, 2015).

Next, it was reasoned that mild tissue stress should increase the need for fitness-based cell selection after damage. First, in order to generate multicellular tissues scattered with suboptimal cells, larvae were exposed to UV light and Azot expression was monitored in wing discs of UV-irradiated WT larvae that were stained for cleaved caspase-3, 24hr after treatment. Under such conditions, Azot was found to be expressed in cleaved caspase-3-positive cells. All Azot-positive cells showed caspase activation and 17% of cleaved caspase-positive cells expressed Azot. This suggested that Azot-expressing cells are culled from the tissue. To confirm this, later time points (3 days after irradiation) were examined; the increase in Azot-positive cells was no longer detectable. The elimination of azot-expressing cells after UV irradiation required azot function, because cells revealed by reporter azot{KO; gfp}, that express GFP instead of Azot, persisted in wing imaginal discs from azot-null larvae. Tests were performeed to see if lack of azot leads to a faster accumulation of tissue defects during organ development upon external damage. azot^-/- pupae 0 stage were irradiated, and the number of morphological defects in adult wings was compared to those in nonirradiated azot KO flies. It was found that aberrations increased more than 2-fold when compared to nonirradiated azot^-/- flies (Merino, 2015).

In order to functionally discriminate whether azot belongs to genes regulating apoptosis in general or is dedicated to fitness-based cell selection, whether azot silencing prevents Eiger/TNF-induced cell death was exanubed. Inhibiting apoptosis (UASp35) or eiger (UASRNAieiger) rescued eye ablation, whereas azot silencing and overexpression of AzotPm4Q12 did not. Furthermore, azot silencing did not impair apoptosis during genitalia rotation or cell death of epithelial precursors in the retina. These results highlight the consequences of nonfunctional cell-quality control within developing tissues (Merino, 2015).

The next part of the analysis demonstrated that the azot promoter computes relative Flower^Lose and Sparc Levels. Epistasis analyses were performed to understand at which level azot is transcriptionally regulated. For this purpose, the assay where WT cells are outcompeted by dMyc-overexpressing supercompetitors was used. It was previously observed that azot induction is triggered upstream of caspase-3 activation and accumulates in outcompeted cells unable to die. Then, upstream events of cell selection were genetically modified. Silencing fwe^Lose transcripts by RNAi or overexpressing Sparc both blocked the induction of Azot::dsRed in WT loser cells. In contrast, when outcompeted WT cells were additionally 'weakened' by Sparc downregulation using RNAi, Azot is detected in almost all loser cells compared to its more limited induction in the presence of endogenous Sparc. Inhibiting JNK signaling with UASpuc did not suppress Azot expression (Merino, 2015).

The activation of Azot upon irradiation was examined. Strikingly, it was found that all Azot expression after irradiation was eliminated when Flower Lose was silenced and also when relative differences of Flower Lose where diminished by overexpressing high levels of Lose isoforms ubiquitously. On the contrary, Azot was not suppressed after irradiation by expressing the prosurvival factor Bcl-2 or a p53 dominant negative. These results show that Azot expression during competition and upon irradiation requires differences in Flower Lose relative levels (Merino, 2015).

Finally, the regulation of Azot expression in neurons was analyzed. Silencing fwe transcripts by RNAi blocked the induction of Azot::dsRed in peripheral photoreceptors. Because Wingless signaling induces Flower^Lose-B expression in peripheral photoreceptors, tests were performed to see if overexpression of Daxin, a negative regulator of the pathway, affected Azot levels. Axin overespression completely inhibited Azot expression. Similarly, overexpression of the cell competition inhibitor Sparc also fully blocked Azot endogenous expression in the retina. Finally, ectopic overexpression of Flower^Lose-B in scattered cells of the retina was sufficient to trigger ectopic Azot activation. These results show that photoreceptor cells also can monitor the levels of Sparc and the relative levels of Flower^Lose-B before triggering Azot expression (Merino, 2015).

These results suggest that the azot promoter integrates fitness information from neighboring cells, acting as a relative 'cell-fitness checkpoint.'

To test if fitness-based cell selection is a mechanism active not only during development, but also during adult stages, WT adult flies were exposed to UV light and monitor Azot and Flower expression were monitored in adult tissues. UV irradiation of adult flies triggered cytoplasmic Azot expression in several adult tissues including the gut and the adult brain. Likewise, UV irradiation of adult flies triggered Flower Lose expression in the gut and in the brain. Irradiation-induced Azot expression was unaffected by Bcl-2 but was eliminated when Flower Lose was silenced or when relative differences of Flower Lose where diminished in the gut. This suggests that the process of cell selection is active throughout the life history of the animal. Further confirming this conclusion, Azot function was essential for survival after irradiation, because more than 99% of azot mutant adults died 6 days after irradiation, whereas only 62.4% of WT flies died after the same treatment. The percentage of survival correlated with the dose of azot because adults with three functional copies of azot had higher median survival and maximum lifespan than WT flies, or null mutant flies rescued with two functional azot transgenes (Merino, 2015).

The next part of the study addressed the role of cell selection during aging. Lack of cell selection could affect the whole organism by two nonexclusive mechanisms. First, the failure to detect precancerous cells, which could lead to cancer formation and death of the individual. Second, the time-dependent accumulation of unfit but viable cells could lead to accelerated tissue and organ decay. We therefore tested both hypotheses (Merino, 2015).

It has been previously shown that cells with reduced levels for cell polarity genes like scrib or dlg are eliminated but can give rise to tumors when surviving. Therefore this study checked if azot functions as a tumor suppressing mechanism in those cells. Elimination of dlg and scrib mutant cells was not affected by RNAi against azot or when Azot function was impaired by mutation, in agreement with the absence of azot induction in these mutant cells. However, azot RNAi or the same azot mutant background efficiently rescued the elimination of clones with reduced Wg signaling (Merino, 2015).

Moreover, the high number of suboptimal cells produced by UV treatment did not lead to tumoral growth in azot-null background. Thus, tumor suppression mechanisms are not impaired in azot mutant backgrounds, and tumors are not more likely to arise in azot-null mutants (Merino, 2015).

Also tests were performed to see whether the absence of azot accelerates tissue fitness decay in adult tissues. Focused was placed on the adult brain, where neurodegenerative vacuoles develop over time and can be used as a marker of aging. The number was compared of vacuoles appearing in the brain of flies lacking azot (azot^-/-), WT flies (azot^+/+), flies with one extra genomic copy of the gene (azot^+/+; azot⁺), and mutant flies rescued with two genomic copies of azot (azot^-/-;azot^+/+). For all the genotypes analyzed, a progressive increase was observed in the number and size of vacuoles in the brain over time. Interestingly, azot^-/- brains showed higher number of vacuoles compared to control flies (azot^+/+ and azot^-/-;azot^+/+) and a higher rate of vacuole accumulation developing over time. In the case of flies with three genomic copies of the gene (azot^+/+; azot⁺), vacuole number tended to be the lowest (Merino, 2015).

The cumulative expression of azot was analyzed during aging of the adult brain. Positive cells were detected as revealed by reporter azot{KO; gfp}, in homozygosis, that express GFP instead of Azot. A time-dependent accumulation of azot-positive cells was observed (Merino, 2015).

From this, it is concluded that azot is required to prevent tissue degeneration in the adult brain and lack of azot showed signs of accelerated aging. This suggested that azot could affect the longevity of adult flies. Flies lacking azot (azot^-/-) had a shortened lifespan with a median survival of 7.8 days, which represented a 52% decrease when compared to WT flies (azot^+/+), and a maximum lifespan of 18 days, 25% less than WT flies (azot^+/+). This effect on lifespan was azot dependent because it was completely rescued by introducing two functional copies of azot. On the contrary, flies with three functional copies of the gene (azot^+/+; azot⁺) showed an increase in median survival and maximum lifespan of 54% and 17%, respectively (Merino, 2015).

In conclusion, azot is necessary and sufficient to slow down aging, and active selection of viable cells is critical for a long lifespan in multicellular animals (Merino, 2015).

The next part of the study demonstrates that death of unfit cells is sufficient and required for multicellular fitness maintenance. The results cited above show the genetic mechanism through which cell selection mediates elimination of suboptimal but viable cells. However, using flip-out clones and MARCM, this study found that Azot overexpression was not sufficient to induce cell death in wing imaginal discs. Because Hid is downstream of Azot, it was wondered whether expressing Hid under the control of the azot regulatory regions could substitute for Azot function (Merino, 2015).

In order to test this hypothesis, the whole endogenous azot protein-coding sequence was replaced by the cDNA of the proapoptotic gene hid (azot{KO; hid}) flies. In a second strategy, the whole endogenous azot protein-coding sequence was replaced by the cDNA of transcription factor Gal4, so that the azot promoter can activate any UAS driven transgene (azot{KO; Gal4} flies. The number of morphological aberrations was compared in the adult wings of six genotypes: first, homozygous azot{KO; Gal4} flies that lacked Azot; second, azot{KO; hid} homozygous flies that express Hid with the azot pattern in complete absence of Azot; third, azot^+/+ WT flies as a control; and finally three genotypes where the azot{KO; Gal4} flies were crossed with UAShid, UASsickle, another proapoptotic gene, or UASp35, an apoptosis inhibitor. In the case of UASsickle flies, a second azot mutation was introduced to eliminate azot function. Interestingly, the number of morphological aberrations was brought back to WT levels in all the situations where the azot promoter was driving proapoptotic genes (azot{KO; hid}, azot{KO; Gal4} × UAShid, azot{KO; Gal4} × UASsickle with or without irradiation. On the contrary, expressing p35 with the azot promoter was sufficient to produce morphological aberrations despite the presence of one functional copy of azot. Likewise, p35-expressing flies (azot{KO; Gal4}/azot⁺; UASp35) did not survive UV treatments, whereas a percentage of the flies expressing hid (26%) or sickle (28%) in azot-positive cells were able to survive (Merino, 2015).

From this, it is concluded that specifically killing those cells selected by the azot promoter is sufficient and required to prevent morphological malformations and provide resistance to UV irradiation (Merino, 2015).

The next part of the study demonstrated that death of unfit cells extends lifespan It was asked whether the shortened longevity observed in azot^-/- flies could be also rescued by killing azot-expressing cells with hid in the absence of Azot protein. It was found that azot{KO; hid} homozygous flies had dramatically improved lifespan with a median survival of 27 days at 29°C, which represented a 125% increase when compared to azot^-/- flies, and a maximum lifespan of 34 days, 41% more than mutant flies (Merino, 2015).

Similar results were obtained at 25°C. It was found that flies lacking azot (azot^-/-) had a shortened lifespan with a median survival of 25days, which represented a 24% decrease when compared to WT flies (azot^+/+), and a maximum lifespan of 40 days, 31% less than WT flies (azot^+/+). On the contrary, flies with three functional copies of the gene (azot^+/+; azot⁺) or flies where azot is replaced by hid (azot{KO; hid} homozygous flies) showed an increase in median survival of 54% and 63% and maximum lifespan of 12% and 24%, respectively (Merino, 2015).

Finally, the effects of dietary restriction on longevity of those flies was tested. It was found that dietary restriction could extend both the median survival and the maximum lifespan of all genotypes. Interestingly, dietary restricted flies with three copies of the gene azot showed a further increase in maximum lifespan of 35%. This shows that dietary restriction and elimination of unfit cells can be combined to maximize lifespan (Merino, 2015).

In conclusion, eliminating unfit cells is sufficient to increase longevity, showing that cell selection is critical for a long lifespan in Drosophila (Merino, 2015).

This study has shown that active elimination of unfit cells is required to maintain tissue health during development and adulthood. The gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).

Although cancer and aging can both be considered consequences of cellular damage, no evidence was found for fitness-based cell selection having a role as a tumor suppressor in Drosophila. The results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).

Azot expression in a wide range of 'less fit' cells, such as WT cells challenged by the presence of 'supercompetitors,' slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in 'less fit' cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell's own levels of Flower^Lose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated. It is proposed that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and - given the evolutionary conservation of Azot - pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).

Evolutionary conservation and association of SPARC with the basal lamina in Drosophila

SPARC (secreted protein, acidic, rich in cysteine, also called BM40 and osteonectin) is a multifunctional calcium-binding glycoprotein whose modular organization has been highly conserved between invertebrates and vertebrates, indicating a conservation of function during metazoan evolution. Genome analysis has revealed a single copy of the Drosophila SPARC (dSPARC) gene. As a first step towards investigating the function of SPARC in Drosophila, its spatiotemporal distribution was analyzed during development. During embryogenesis, dSPARC mRNA transcripts are restricted to mesoderm derivatives, hemocytes, and the fat body. Immunostaining with anti- Drosophila SPARC antibodies indicates that dSPARC secreted by the hemocytes and fat body cells is concentrated in basal laminae surrounding internal organs. During oogenesis, dSPARC transcripts are restricted to the somatic cells of the germarium and follicles. Consistent with embryonic development, the resultant protein is concentrated in basal laminae. Mutations in type IV collagen are associated with a dramatic decrease in dSPARC protein immunostaining in hemocytes. The data suggest that the production and assembly of dSPARC in the basal lamina is dependent on type IV collagen, and raise the possibility that dSPARC and type IV collagen interactions are a prerequisite to the assembly and structural integrity of basal laminae in Drosophila (Martinek, 2002).

SPARC preserves follicular epithelium integrity in insect ovaries

The importance of juvenile hormone regulating insect oogenesis suggests looking for genes whose expression is regulated by this hormone. SPARC is a calcium-binding glycoprotein that forms part of the extracellular membranes, which in vertebrates participates in bones mineralization or regulating cell proliferation in some cancer types. This large number of functions described for SPARC in different species might be related to the significant differences in its structure observed when comparing different species-groups. Indeed, these structural differences allow characterizing the different clades. In the cockroach Blattella germanica, a SPARC homolog emerged from ovarian transcriptomes that were constructed to find genes responding to juvenile hormone. In insects, SPARC functions have been studied in oogenesis and in embryo development of Drosophila melanogaster. The present work, using RNAi approaches, novel functions for SPARC in the B. germanica panoistic ovaries are described. Depletion of SPARC does not allow to the follicular cells to complete mitosis, resulting in giant follicular cells nuclei and in a great alteration of the ovarian follicle cytoskeleton. The SPARC contribution to B. germanica oogenesis occurs stabilizing the follicular cell program and helping to maintain the nuclear divisions. Moreover, SPARC is necessary to maintain the cytoskeleton of the follicular cells. Any modification of these key processes disables females for oviposition (Irles, 2017).

Search PubMed for articles about Drosophila Sparc

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

Bradshaw, A. D., Baicu, C. F., Rentz, T. J., Van Laer, A. O., Boggs, J., Lacy, J. M. and Zile, M. R. (2009). Pressure overload-induced alterations in fibrillar collagen content and myocardial diastolic function: role of secreted protein acidic and rich in cysteine (SPARC) in post-synthetic procollagen processing. Circulation 119: 269-280. PubMed ID: 19118257

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

Gilmour, D. T., et al. (1998). Mice deficient for the secreted glycoprotein SPARC/osteonectin/BM40 develop normally but show severe age-onset cataract formation and disruption of the lens. EMBO J. 17(7): 1860-70. PubMed ID: 9524110

Hohenester, E. and Yurchenco, P. D. (2013). Laminins in basement membrane assembly. Cell Adh Migr 7: 56-63. PubMed ID: 23076216

Hunzelmann, N., Hafner, M., Anders, S., Krieg, T. and Nischt, R. (1998). BM-40 (osteonectin, SPARC) is expressed both in the epidermal and in the dermal compartment of adult human skin. J. Invest. Dermatol. 110: 122-126. PubMed ID: 9457905

Irles, P., Ramos, S. and Piulachs, M. D. (2017). SPARC preserves follicular epithelium integrity in insect ovaries. Dev Biol 422(2): 105-114. PubMed ID: 28089848

Ishida, Y., Kubota, H., Yamamoto, A., Kitamura, A., Bächinger, H. P. and Nagata, K. (2006). Type I collagen in Hsp47-null cells is aggregated in endoplasmic reticulum and deficient in N-propeptide processing and fibrillogenesis. Mol. Biol. Cell 17: 2346-2355. PubMed ID: 9457905

Kim, S. Y., Ondhia, N., Vidgen, D., Malaval, L., Ringuette, M. and Kalnins, V. I. (1997). Spatiotemporal distribution of SPARC/osteonectin in developing and mature chicken retina. Exp. Eye Res. 65: 681-689. PubMed ID: 9367648

Merino, M.M., Rhiner, C., Lopez-Gay, J.M., Buechel, D., Hauert, B. and Moreno, E. (2015). Elimination of unfit cells maintains tissue health and prolongs lifespan. Cell 160: 461-476. PubMed ID: 25601460

Martinek, N., Zou, R., Berg, M., Sodek, J. and Ringuette, M. (2002). Evolutionary conservation and association of SPARC with the basal lamina in Drosophila. Dev. Genes Evol. 212: 124-133. PubMed ID: 11976950

Martinek, N., Shahab, J., Sodek, J. and Ringuette, M. J. (2007). Is SPARC an evolutionarily conserved collagen chaperone? J. Dent Res. 86: 296-305. PubMed ID: 17384023

Martinek, N., Shahab, J., Saathoff, M. and Ringuette, M. (2008). Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos. J. Cell Sci. 121: 1671-80. PubMed ID: 18445681

Marutani, T., Yamamoto, A., Nagai, N., Kubota, H. and Nagata, K. (2004). Accumulation of type IV collagen in dilated ER leads to apoptosis in Hsp47-knockout mouse embryos via induction of CHOP. J. Cell Sci. 117: 5913-22. PubMed ID: 15522896

Matsuoka, Y., Kubota, H., Adachi, E., Nagai, N., Marutani, T., Hosokawa, N. and Nagata, K. (2004). Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells. Mol. Biol. Cell 15: 4467-4475. PubMed ID: 15282337

Maurer, P., Hohenadl, C., Hohenester, E., Gohring, W., Timpl, R. and Engel, J. (1995). The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV. J. Mol. Biol. 253: 347-357. PubMed ID: 7563094

Maurer, P., et al. (1997). Recombinant and tissue-derived mouse BM-40 bind to several collagen types and have increased affinities after proteolytic activation. Cell Mol. Life Sci. 53(5): 478-84. PubMed ID: 9176569

Mayer, U., et al. (1991). Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV. Eur. J. Biochem. 198(1): 141-50. PubMed ID: 2040276

Nagata, K. (2003). HSP47 as a collagen-specific molecular chaperone: function and expression in normal mouse development. Semin. Cell Dev. Biol. 14: 275-282. PubMed ID: 14986857

Norose, K., Lo, W. K., Clark, J. I., Sage, E. H. and Howe, C. C. (2000). Lenses of SPARC-null mice exhibit an abnormal cell surface-basement membrane interface. Exp. Eye Res. 71: 295-307. PubMed ID: 10973738

Olofsson, B., and Page, D.T. (2005). Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity. Dev. Biol. 279, 233-243. PubMed ID: 15708571

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

Portela, M., et al. (2010). Drosophila SPARC is a self-protective signal expressed by loser cells during cell competition. Dev. Cell 19(4): 562-73. PubMed ID: 20951347

Pöschl, E., et al. (2004). Collagen IV is essential for basement membrane stability but dispensable for initiation of its assembly during early development. Development 131: 1619-1628. PubMed ID: 14998921

Pottgiesser, J., Maurer, P., Mayer, U., Nischt, R., Mann, K., Timpl, R., Krieg, T. and Engel, J. (1994). Changes in calcium and collagen IV binding caused by mutations in the EF hand and other domains of extracellular matrix protein BM-40 (SPARC, osteonectin). J. Mol. Biol. 238: 563-574. PubMed ID: 8176746

Rentz, T. J., Poobalarahi, F., Bornstein, P., Sage, E. H. and Bradshaw, A. D. (2007). SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts. J. Biol. Chem. 282: 22062-22071. PubMed ID: 17522057

Rosenblatt, S., Bassuk, J. A., Alpers, C. E., Sage, E. H., Timpl, R. and Preissner, K. T. (1997). Differential modulation of cell adhesion by interaction between adhesive and counter-adhesive proteins: characterization of the binding of vitronectin to osteonectin (BM40, SPARC). Biochem. J. 324: 311-319. PubMed ID: 9164872

Sage, H., Vernon, R. B., Decker, J., Funk, S. and Iruela-Arispe, M. L. (1989). Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J. Histochem. Cytochem. 37: 819-829. PubMed ID: 2723400

Sangaletti, S., Stoppacciaro, A., Guiducci, C., Torrisi, M. R. and Colombo, M. P. (2003). Leukocyte, rather than tumor-produced SPARC, determines stroma and collagen type IV deposition in mammary carcinoma. J. Exp. Med. 198: 1475-1485. PubMed ID: 14610043

Shahab, J., Baratta, C., Scuric, B., Godt, D., Venken, K. J. and Ringuette, M. J. (2014). Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster. Dev Dyn [Epub ahead of print]. PubMed ID: 25529377

Yan, Q., Clark, J. I., Wight, T. N. and Sage, E. H. (2002). Alterations in the lens capsule contribute to cataractogenesis in SPARC-null mice. J. Cell Sci. 115: 2747-2756. PubMed ID: 12077365

Yan, Q., Perdue, N., Blake, D. and Sage, E. H. (2005). Absence of SPARC in murine lens epithelium leads to increased deposition of laminin-1 in lens capsule. Invest. Ophthalmol. Vis. Sci. 46: 4652-4660. PubMed ID: 16303962

Yurchenco, P. D., Amenta, P. S. and Patton, B. L. (2004). Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol. 22: 521-538. PubMed ID: 14996432

date revised: 25 March 2017

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