InteractiveFly: GeneBrief

papilin: Biological Overview | References

Differentiation of prohaemocytes, the precursors of Drosophila blood cells (haemocytes), and the release of haemocytes from the lymph gland, a major larval haematopoietic organ, are vital responses to wasp infestation or tissue degeneration. Although cells and extracellular matrix (ECM) in the lymph gland are known to play a crucial role in haemocyte differentiation, the underlying mechanisms remain unclear. This study shows that the matrix glycoprotein papilin (Ppn) is essential for maintaining the prohaemocyte population in lymph glands. In Ppn-depleted larvae, haemocyte differentiation increased with a reduction in the prohaemocyte-containing medullary zone, and lymph gland lobes dispersed prematurely. Ppn was synthesised by plasmatocytes, forming lamellae mainly in the medullary zone. Microbial infection or wasp infestation disrupted the Ppn meshwork within lymph glands. Ppn colocalised with collagen, laminin, nidogen and perlecan. Ppn depletion disrupted the ECM structure, including perlecan organisation. Phenotypes caused by Ppn depletion were partially rescued by perlecan overexpression or inactivation of the epidermal growth factor receptor pathway. Thus, Ppn is crucial for maintaining lymph gland architecture and regulating haemocyte differentiation, highlighting an intricate interaction between the ECM and signalling pathways in haematopoiesis (Lee, 2025).

A key aspect of haematopoiesis is balancing the maintenance and differentiation of haematopoietic stem and progenitor cells (HSPCs), akin to other developmental processes. In the bone marrow, HSPCs require proper interactions with neighbouring cells and the extracellular matrix (ECM) for growth, survival and developmental competence (Gao et al., 2018; Mercier and Leonard, 2011; Morrison and Scadden, 2014; Wei and Frenette, 2018). The ECM comprises fibrous proteins, proteoglycans, glycosaminoglycans and glycoproteins, such as collagens, laminins, heparan sulphate and thrombospondins. These are essential components of the stem cell niche and are recognised by various cell surface receptors such as integrins and selectins (Domingues et al., 2017; Klamer and Voermans, 2014; Papy-Garcia and Albanese, 2017). ECM proteins can bind to multiple receptors, and a single receptor can recognise various ECM proteins, facilitating a diverse range of signalling outcomes. Furthermore, each specific interaction may activate distinct signalling cascades, depending on the particular ECM protein and receptor involved. This results in highly flexible and context-specific cellular responses (reviewed by Lee-Thedieck et al., 2022). However, understanding these complex interactions remains challenging due to their combinatorial nature and the diverse composition of niche microenvironments (Lee, 2025).

Drosophila has a robust yet relatively simple immune system, highly conserved with that of mammals. In larvae, haematopoiesis occurs in the lymph gland near the dorsal aorta, consisting of a pair of anterior-most primary lobes and a variable number of smaller posterior lobes. The primary lobes are organised into three distinct zones: the posterior signalling centre (PSC), the medullary zone (MZ) and the cortical zone (CZ). The PSC, which expresses Knot (also known as Collier), Antennapedia (Antp) and Hedgehog (Hh), serves as the haematopoietic niche, and instructs prohaemocytes to differentiate and disperse during wasp parasitisation. The MZ, which expresses Drosophila E-cadherin (DE-cad; Shotgun), Thioester-containing protein 4 (TepIV; Tep4) and Domeless (Dome), contains prohaemocytes that are initially active in cell proliferation but become less proliferative as larvae reach the mid-third instar stage. The CZ contains differentiated haemocytes derived from the MZ. Between the MZ and CZ lies a relatively small group of transitional cells known as intermediate progenitors in a less-well characterised intermediate zone (IZ). These cells can be specifically labelled using chromator-GAL4 or Nplp2-GAL4. The posterior lobes contain prohaemocytes and function as reservoirs. Prohaemocytes in the MZ are composed of multiple cell subtypes, distinguished by subtly different gene expression patterns. Despite this complexity, they remain relatively simple as a group, making them an excellent model for dissecting dynamic interactions during haematopoiesis (Lee, 2025).

Drosophila has three major types of haemocytes. Plasmatocytes, which are small, round and phagocytic, and make up 95% of the total haemocyte population. They uniquely express cell-surface molecules such as Eater and NimC1. Plasmatocytes are responsible for engulfing microbes, apoptotic cells and cellular debris; secreting ECMs to remodel tissues; and attaching to foreign surfaces, such as invading wasp eggs or degenerating self-tissues. Crystal cells, slightly larger in size, are involved in blood clotting and oxygen transport, comprising 5% of total haemocytes. These cells express prophenoloxidase A1 (ProPOA1; PPO1), Lozenge (Lz) and Hindsight (Hnt; Pebbled). Lastly, lamellocytes are large, flat cells that normally appear only during metamorphosis or infection, when they encapsulate foreign or abnormal surfaces of large particles. Lamellocytes can be visualised by the expression of the cell-surface antigen Atilla (L1) or by high expression levels of the integrin βPS subunit Myospheroid (Mys) (Lee, 2025).

Shortly after parasitoid wasp infestation, Drosophila lymph glands exhibit increased proliferation. Lamellocytes are massively induced, and, ultimately, all haemocytes are released into the haemolymph through the dispersal of the lymph gland. Eggs are initially recognised by haemocytes in an unidentified manner, leading to plasmatocyte migration, adhesion and spreading, and the formation of septate junctions. Lamellocytes then bind to the first layer of plasmatocytes, forming multilayered capsules, and the eggs are eventually melanized by prophenoloxidases supplied by the lamellocytes and crystal cells. These responses can also target degenerating self-tissues, leading to the formation of melanotic masses, effectively sequestering them (Lee, 2025).

The maintenance and differentiation of prohaemocytes in the MZ are regulated by multiple signals from various sources. Maintenance signals include autonomous signals within the MZ from a subset of prohaemocytes expressing Knot, Wingless signalling and its downstream target DE-cad, and feedback signals from differentiating haemocytes in the CZ. Notably, ECM molecules in the lymph glands, such as collagen IV, laminin, Trol (the Drosophila perlecan), nidogen (Ndg) and tiggrin, appear to moderate differentiation based on their distribution patterns and the loss-of-function phenotypes of related genes. Induction signals for prohaemocyte differentiation have also been identified, including PSC-derived reactive oxygen species, which activate both Toll and epidermal growth factor receptor (EGFR) pathways, leading to prohaemocyte differentiation. These signals are triggered upon wasp infestation (Lee, 2025).

papilin (Ppn) was first identified as a large, sulphated glycoprotein from the culture media of Drosophila Kc cells. Ppn contains multiple domains, including TSR and Kunitz, and its structural organisation is well conserved from nematodes to humans. Ppn is among the most abundant proteins in the basement membrane, highlighting its role as a core component of this structure. Knockdown of Ppn results in defects in muscle development and leads to lethality during embryogenesis and the first instar stage. In Caenorhabditis elegans, Ppn facilitates collagen removal during basement membrane expansion and tissue growth in the gonad, and it mediates axon-dendrite adhesion and dendrite patterning. Drosophila Ppn inhibits procollagen N-proteinase, an ADAMTS metalloproteinase, in vitro. However, its role in cell differentiation or immune defence remains unclear (Lee, 2025).

This study isolated the Ppn gene using an RNA interference (RNAi)-based genetic screen and explored its potential roles in Drosophila larval lymph glands. Ppn forms a network of interconnected lamellae and septa primarily in the MZ, where it supports ECM structure and suppresses prohaemocyte differentiation by inhibiting EGFR signalling and interacting with perlecan. During bacterial infection or wasp infestation, this Ppn structure collapsed, correlating with prohaemocyte differentiation and lymph gland dispersal. These findings emphasise an essential role of Ppn in maintaining the prohaemocyte population, which is crucial for effective pathogen defence, and support the broader observation that niche ECM is vital for progenitor maintenance (Lee, 2025).

The composition of the haematopoietic niche is crucial for maintaining an adequate supply of progenitor cells and preventing their depletion. This study found that the Drosophila ECM glycoprotein Ppn forms lamellae and septa that associate more intimately with progenitor cells in the MZ and less prominently in the CZ, which is reminiscent of previous reports on basement membrane distribution in this organ. Ppn colocalises with collagen, laminin, nidogen and perlecan, and also forms an outer sheath encasing the lobe. The three-dimensional Ppn meshwork is disrupted at the onset of metamorphosis or wasp infestation, similar to what has been observed in lymph gland ECMs. The phenotypes displayed by Ppn-deficient larval lymph glands share many aspects with those lacking Vkg, Trol, Ndg or the accessory protein tiggrin, particularly the precocious differentiation of plasmatocytes, supporting the idea that lymph gland ECMs maintain the prohaemocyte pool (Lee, 2025).

How does Ppn regulate prohaemocyte differentiation? Ppn and the other ECM components may collectively inhibit this process via cellular receptors such as integrins on prohaemocytes. Integrin βPS is highly expressed in the MZ, and its depletion in the MZ, but not the CZ, selectively leads to prohaemocyte differentiation in the lymph glands. Upon immune challenge or developmental signals, Ppn may be disrupted in an instructive manner, triggering the differentiation pathway. Alternatively, Ppn might function as a meshwork of latches that passively block haemocyte differentiation. In this scenario, the balance between inducers and latches would determine the state of cell differentiation. The frequent presence of Ppn lamellae near local differentiation sites during wasp infestation supports this hypothesis. In this case, differentiated haemocytes may secrete proteinase to degrade matrix proteins, including Ppn (Lee, 2025).

The current results indicate that larval Ppn is primarily synthesised by a subset of plasmatocytes, and possibly by some cells in the IZ. Since Ppn lamellae are mainly observed in the MZ, it suggests that Ppn synthesised by plasmatocytes is trapped in the MZ, forming lamellae and septa, and even accumulating on fat bodies to create collagen IV intercellular concentration-like deposits. This contrasts with the general finding that both haemocytes and fat bodies contribute to Drosophila larval basement membranes. However, it aligns with findings that the de novo formation of the embryonic basement membrane is largely contributed to by embryonic haemocytes rather than fat bodies. Similarly, migrating embryonic haemocytes produce laminins as part of their migratory trails, reinforcing directional and effective migration. Ppn forms prominent lamellae in the MZ, possibly due to the selective presence of its cellular receptors in that area. Lymph glands have small cell populations that are currently not well characterised, and identifying the subgroup that supplies Ppn and examining its regulation during haematopoiesis will be interesting (Lee, 2025).

Ppn is required for the proper organisation of Trol but not vice versa. Ppn is also required for the normal distribution of the other three major components of the basement membrane to varying degrees. However, the possibility of interdependence among these components has not been tested. During de novo synthesis of the basement membrane, a structural hierarchy exists among its major components, with laminin typically acting as the initiator and collagen IV as the most abundant protein. Perlecan, nidogen and other associated components confer or modulate the properties of the meshwork. For example, Drosophila Trol and C. elegans Ppn facilitate collagen IV removal during tissue growth, allowing for basement membrane expansion. In Drosophila, nidogen serves as a link between laminin and collagen IV networks. Trol is also known to inhibit fibroblast growth factor ligands and promote the Hh signalling pathway, thereby maintaining the prohaemocyte population. In this context, Ppn may be crucial as a key regulator in both development and pathogen defence, particularly in dynamic changes to basement membrane properties and cell signalling (Lee, 2025).

The sharp increase in Ppn levels in trol knockdown lymph glands is intriguing. This change could result from plasmatocyte expansion, or it may arise from an interplay between Ppn, Trol and other ECM components. Given that both Trol and Ppn interact closely with collagen, Ppn may have increased binding opportunities following Trol depletion. Alternatively, this interaction could be mediated through a shared receptor. Further studies are required to clarify this mechanism (Lee, 2025).

This study also discovered that depleting Ppn derepresses the activation of the EGFR signalling pathway in the lymph glands, revealing a molecular mechanism underlying the Ppn loss-of-function phenotype. Wasp infestation triggers reactive oxygen species production and the secretion of one of the four known EGFR ligands, Spitz (Spi), in the PSC, which in turn activates the EGFR pathway in the MZ. Ppn may play a role in sequestering EGFR ligand(s), creating a reservoir in the MZ. Attempts to rescue the Ppn knockdown phenotype by removing spi were unsuccessful, suggesting that the underlying mechanism may be more complex, involving multiple ligands or acting downstream of the receptor level (Lee, 2025).

It has been reported that Ppn and other ECM proteins are released from freshly collected haemocytes that strongly adhere to foreign surfaces. This study focused on the role of Ppn in haematopoietic organs. Given the formation of melanotic masses in Ppn knockdown larvae, it is plausible that Ppn may also play a role in self/non-self discrimination, potentially serving as a self-tolerance tag. Future research should explore its potential functions in peripheral tissues (Lee, 2025).

Hemocyte-secreted papilin bearing mucin-type O-glycans regulates peripodial stalk formation via epidermal JAK/STAT signaling in Drosophila

Protein glycosylation is an essential post-translational modification. In evolutionarily conserved mucin-type O-glycosylation, the most common O-glycan, T antigen, is synthesized by core 1 beta1,3-galactosyltransferase 1 (C1GalT1). Loss of C1GalT1 leads to developmental defects across organisms. Previously work found that Drosophila C1GalT1 mutants exhibit malformed legs, but the underlying mechanism was unclear. This study identify a glycan-mediated inter-tissue signaling mechanism wherein embryonic hemocytes regulate leg morphogenesis. T antigen-modified papilin (Ppn), an extracellular matrix (ECM) protein secreted by embryonic hemocytes, suppresses JAK/STAT signaling in the epidermis surrounding Keilin's organ. This repression is essential for proper tubulogenesis of the peripodial stalk anchoring the leg disc and ensuring its correct positioning during development. Disrupted mucin-type O-glycosylation impairs Ppn secretion and causes mislocalized leg discs and morphogenetic defects. These findings identify Ppn carrying mucin-type O-glycan as long-range modulators of epithelial signaling and underscore the role of immune-like cells in coordinating organogenesis via ECM (Fuwa, 2026).

Tubulogenesis is a fundamental developmental process critical for shaping epithelial architecture across organs. Although stalk-like epithelial tubes connecting imaginal discs to the epidermis have long been observed in Drosophila, their developmental origin, molecular regulation, and physiological importance have remained unexplored. This study has defined the peripodial stalk as a regulated epithelial tube whose formation is essential for proper leg development. The findings uncover a previously unrecognized morphogenetic mechanism in which mucin-type O-glycans synthesized by dC1GalT1 in embryonic hemocytes modulate the local signaling environment of epidermis to control stalk formation (Fuwa, 2026).

The failure of stalk formation in dC1GalT1 mutants leads to the mislocalization of the third leg discs and subsequent leg malformation. Importantly, tissue-specific rescue experiments revealed that the expression of dC1GalT1 in embryonic hemocytes—but not in the leg tissue itself—is sufficient to restore both stalk morphogenesis and leg development. These findings establish that hemocyte-derived glycoconjugates have an unexpected non-cell-autonomous role in guiding epithelial tube formation in adjacent tissues. By visualizing cytoskeletal dynamics and junctional markers during early disc development, this study has delineated the stepwise process of disc formation. Convergent extension contributes to the stalk elongation, allowing a proposal that the constriction of the disc cell cluster is required for initial stalk formation. The disruption of dC1GalT1 impaired constriction of the disc cell cluster beneath the KO, which is likely to indicate failure of this epithelial remodeling process. Mechanistically, it was discovered that epidermal JAK/STAT signaling is aberrantly upregulated in dC1GalT1 mutants and can be restored to normal levels by hemocyte-specific dC1GalT1 expression. Furthermore, a slight reduction in JAK/STAT activity partially rescued stalk formation in dC1GalT1 mutants. oth forced activation and inactivation of epidermal JAK/STAT activity in WT were sufficient to disrupt stalk formation. Therefore, these data identify moderate JAK/STAT activity as a critical target of glycan-mediated regulation during stalk morphogenesis. Lastly, through a focused RNAi screen, it was identified that Ppn, an ECM protein with a mucin-like domain, is an essential mediator of this pathway. Hemocyte-specific knockdown of Ppn phenocopied dC1GalT1 mutants, displaying short stalks, JAK/STAT hyperactivation, and failure of disc cluster constriction. Overexpression of dC1GalT1 did not rescue the short stalk phenotype caused by the loss of Ppn, positioning Ppn downstream of dC1GalT1 in the pathway. Moreover, Ppn was robustly expressed in embryonic hemocytes and partially colocalized with T antigen. Biochemical assays confirmed that secreted Ppn carries mucin-type O-glycans, including T antigen. Furthermore, loss of dC1GalT1 impaired the secretion of Ppn from hemocyte-derived cells. These findings suggest that dC1GalT1-mediated glycosylation of Ppn facilitates its secretion from hemocytes and enables it to function as an ECM regulator that modulates epidermal signaling (Fuwa, 2026).

This work identifies a glyco-regulatory axis in which embryonic blood cells secrete a glycosylated ECM protein (Ppn) that tunes local signaling pathways in neighboring epithelial tissues. This represents a previously unappreciated mechanism by which inter-tissue glycan signaling shapes organ morphogenesis. Mucin-type O-glycans function was identified as non-cell-autonomous modulators of JAK/STAT signaling in the context of tubulogenesis (Fuwa, 2026).

During metamorphosis, mature third leg discs form sac-like structures comprising a folded columnar epithelium and an overlying squamous peripodial epithelium. The columnar epithelium ultimately gives rise to most of the adult leg structure. At the early pupal stage, the leg disc formed by the columnar epithelium everts through the opened stalk, and then elongates into the space of the pupal case. Ultrastructural analyses revealed that the third leg discs in dC1GalT1 mutants were ectopically located between the ventral muscles and the cuticle, and appeared compressed in contrast to WT larvae, where the discs are freely exposed to the body cavity. This misplacement may mechanically prevent disc eversion and appendage elongation, leading to structural failure of the leg. Indeed, some dC1GalT1 mutants displayed partially buried or entirely missing third legs, strongly supporting this model. dC1GalT1 mutant larvae exhibited short stalks anchoring other imaginal discs, as well as those of the third leg discs. However, the positioning of discs other than the third leg discs remained normal, probably because the stalks anchoring them are inherently shorter than those of the third leg discs. In contrast, the positioning of third leg discs is strongly influenced by their stalk length (Fuwa, 2026).

Stalk elongation seems to involve convergent extension—a conserved morphogenetic mechanism in which the cells intercalate along a narrowing axis, driving tissue elongation. The mechanism of convergent extension has been well-characterized in germband extension, where it is driven by myosin-II-dependent apical contraction and basolateral protrusions. The disc cell cluster failed to constrict beneath the KO in dC1GalT1 mutants, and knockdown of the gene encoding myosin light chain (sqh) recapitulated this phenotype, indicating defective convergent extension. Notably, JAK/STAT signaling is known to regulate convergent extension in other contexts, including gut elongation and germband extension. Thus, these findings position JAK/STAT signaling as a key modulator of epithelial remodeling during stalk tubulogenesis and highlight mucin-type O-glycans as upstream regulators of this pathway (Fuwa, 2026).

Ppn is well known as a heavily glycosylated ECM protein with a Ser/Thr-rich domain that carries glycosaminoglycans. Impaired synthesis of glycosaminoglycans in hemocytes did not disrupt stalk formation, suggesting that glycosaminoglycans on Ppn may be dispensable for its secretion from hemocytes and its bioactivity for stalk formation. Biochemical analysis demonstrated that Ppn also carries mucin-type O-glycans, including T antigen, in Drosophila. This finding is consistent with previous reports indicating that the human ortholog papilin is modified by mucin-type O-glycans. Expression analysis confirmed that Ppn is highly expressed in embryonic hemocytes, coinciding with the developmental window in which dC1GalT1 function is required for stalk formation. Observed expression of Ppn is consistent with previous studies showing that hemocytes mainly produce Ppn during embryonic stages, whereas the lymph gland mainly produces Ppn during larval stages. This study also found that dC1GalT1 expression during embryonic stages, but not larval stages, is required for stalk formation. Together, these data support a model in which glycosylated Ppn secreted from embryonic hemocytes modulates local signaling and tissue architecture (Fuwa, 2026).

Further analysis revealed that defective synthesis of mucin-type core 1 glycans impaired Ppn secretion from hemocyte-derived cells, suggesting that core 1 glycans on Ppn are required for its secretion. These data are consistent with previous studies showing that loss of GALNT genes (which encode the polypeptide N-acetylgalactosaminyltransferases responsible for the initiation of mucin-type \(O\)-glycosylation) results in the impaired secretion of ECM components, including Ppn. The data also showed that loss of dC1GalT1 does not increase intracellular Ppn levels, despite impaired Ppn secretion. This raises the possibility that hypoglycosylation of core 1 glycans may destabilize Ppn, thereby promoting its intracellular degradation. Therefore, it is proposed that impaired Ppn secretion due to hypoglycosylation is the main cause of the stalk defects in dC1GalT1 mutants. Drosophila is known to possess 10 GALNT paralogs. According to DGET (Drosophila Gene Expression Tool: https://www.flyrnai.org/tools/dget/web/, five of these paralogs—Pgant1, Pgant3, Pgant5, Pgant6, and Pgant7—are moderately expressed in Kc167 cells. These paralogs may therefore contribute to the mucin-type O-glycosylation of Ppn in embryonic hemocytes (Fuwa, 2026).

Interestingly, dGlcAT-P—the enzyme responsible for glucuronylation of T antigen—has been previously shown to regulate neuromuscular junctions and VNC morphology, and dGlcAT-P mutant phenotypes are reminiscent of those observed in dC1GalT1 mutants. The VNC phenotype in dGlcAT-P mutants is rescued by hemocyte-specific expression of dGlcAT-P, indicating that glucuronylated T antigen from hemocytes has a key role in VNC formation. Furthermore, Ppn mutants also exhibit VNC elongation, suggesting that Ppn may act as a functional carrier of this glycan. It is therefore plausible that the short stalk phenotype of dC1GalT1 mutants may result from loss of glucuronylated T antigen, highlighting the importance of specific glycan structures for Ppn secretion. Future analysis will reveal whether the glucuronylated T antigen is involved in Ppn secretion and stalk formation (Fuwa, 2026).

This analysis revealed that the forced expression of hST6GALNAC1 (human sialyltransferase) in hemocytes partially rescued stalk formation in dC1GalT1 mutants, indicating that the synthesis of sialylated Tn antigen (Siaα2,6GalNAcα1-Ser/Thr), instead of core 1 glycans, may facilitate Ppn secretion, leading to stalk elongation. This raises the possibility that sialylated Tn antigen possesses bioactivity similar to that of core 1 glycans, including glucuronylated T antigen. Given that sialylated Tn antigen carries a negative charge, similar to glucuronylated T antigen, the negative charge from the terminal monosaccharide might contribute, at least in part, to Ppn stability and secretion (Fuwa, 2026).

Ppn localizes in the basement membrane (BM) of multiple Drosophila tissues, including the VNC, gut, malpighian tube, and proventriculus. Hemocytes contribute to the BM formation of the embryonic VNC by secreting Ppn that is incorporated into the VNC BM. Although the domain structure of Ppn is well conserved from nematodes to humans, only Drosophila Ppn contains a Ser/Thr-rich domain. Drosophila Ppn also includes a papilin cassette with one thrombospondin type-1 repeat (TSR) domain, a cysteine-rich spacer domain, and several partial TSR domains at the amino-end, homologous to ADAMTS-like proteins but lacking proteolytic function. Notably, Drosophila Ppn can bind and inhibit the procollagen-processing activity of vertebrate ADAMTS2 in vitro, and C. elegans Ppn regulates collagen remodeling by modulating the access of ADAMTS proteases to the BM. These observations suggest that Ppn functions as a regulator of BM composition by controlling the localization and activity of collagen-modifying enzymes during development. Ppn also colocalizes with major ECM components—namely, collagen IV, laminin, nidogen, and Trol (Drosophila perlecan)—and is required for their proper organization in Drosophila larval lymph glands. In addition, it negatively regulates EGFR signaling in the lymph gland via interaction with perlecan, indicating that Ppn may contribute to the sequestration of EGFR ligands. In the context of leg disc development, it is proposed that hemocyte-secreted glycosylated Ppn contributes to BM organization of epidermal cells around the KO, thereby limiting JAK/STAT signaling by sequestering its ligands and promoting stalk tubulogenesis. In contrast, defective mucin-type O-glycosylation of Ppn impairs its secretion from hemocytes, leading to excessive ligand exposure caused by epidermal BM deformation, which in turn inhibits stalk tubulogenesis. Consistent with this hypothesis, a previous study demonstrated that dC1GalT1 mutants exhibit BM deformation on larval muscles, as revealed by ultrastructural analysis (Fuwa, 2026).

Taken together, these findings establish a glycan-mediated inter-tissue signaling axis in which mucin-type O-glycans, synthesized by dC1GalT1 in embryonic hemocytes and presented on the ECM protein Ppn, regulate epithelial morphogenesis by modulating the BM environment. By limiting epidermal JAK/STAT signaling around the KO, glycosylated Ppn promotes the formation of the peripodial stalk—a previously overlooked epithelial structure essential for proper leg disc positioning and adult leg morphogenesis. This work expands the functional scope of mucin-type O-glycans beyond cell-autonomous glycoprotein modification, positioning them as long-range modulators of signaling and tissue architecture. Such principles may extend to vertebrate systems, where glycan-modified ECM components are increasingly recognized as key regulators of morphogenesis, regeneration, and disease (Fuwa, 2026).

Although PNA lectin blotting indicated that Ppn was modified by T antigen, this was not directly confirmed by mass spectrometry. Biochemical analysis also revealed that loss of dC1GalT1 impaired extracellular Ppn levels in the culture medium of Kc167 cells, suggesting reduced secretion of Ppn. However, it cannot be excluded that Ppn undergoes extracellular degradation after secretion due to its destabilization. Furthermore, it is not currently clear whether Ppn secretion from hemocytes is impaired in vivo (Fuwa, 2026).

Lastly, it was found that the hemocyte-specific overexpression of hST6GALNAC1 partially rescued stalk formation in dC1GalT1 mutants. However, synthesis of sialylated Tn antigen in the flies was not demonstrated, although the expression of hST6GALNAC1 was confirmed (Fuwa, 2026).

Interplay between MIG-6/papilin and TGF-beta signaling promotes extracellular matrix remodeling and modulates the maintenance of neuronal architecture

Neuronal architecture laid out during embryogenesis persists lifelong, ensuring normal nervous system function. However, the mechanisms underlying the long-term maintenance of neuronal organization remain largely unknown. Previous work uncovered that the conserved extracellular matrix protein MIG-6/papilin impacts collagen IV remodeling and neuronal maintenance, such that disruption of MIG-6/papilin leads to a collagen IV fibrotic state and altered tissue biomechanics, thereby stabilizing neuronal architecture. This study combine incisive molecular genetics and in vivo quantitative imaging to determine how this mig-6-dependent fibrotic phenotype is modulated, by investigating the implication of the TGF-beta pathway, which is well known to regulate fibrosis in mammals. The findings highlight a mechanism whereby the interplay between MIG-6/papilin and the TGF-beta pathway regulates ECM composition and neuronal maintenance, with MIG-6/papilin acting as a positive regulator of TGF-beta signaling. This work provides key insights into the molecular basis of sustaining neuronal architecture and offers a foundation for understanding age-related neurodegenerative and fibrotic conditions (Nadour, 2025b).

Remodeling of extracellular matrix collagen IV by MIG-6/papilin regulates neuronal architecture

Neuronal architecture established embryonically must persist lifelong to ensure normal brain function. However, little is understood about the mechanisms behind the long-term maintenance of neuronal organization. To uncover maintenance mechanisms, we performed a suppressor screen in sax-7/L1CAM mutants, which exhibit progressive disorganization with age. We identified the conserved extracellular matrix protein MIG-6/papilin as a key regulator of neuronal maintenance. Combining incisive molecular genetics, structural predictions, in vivo quantitative imaging, and cutting-edge Brillouin microscopy, we show that MIG-6/papilin remodels extracellular matrix collagen IV, working in concert with the secreted enzymes MIG-17/ADAMTS and PXN-2/peroxidasin. This remodeling impacts tissue biomechanics and ensures neuronal stability, even under increased mechanical stress. Our findings highlight an extracellular mechanism by which MIG-6/papilin supports the integrity of neuronal architecture throughout life. This work provides critical insights into the molecular basis of sustaining neuronal architecture and offers a foundation for understanding age-related and neurodegenerative disorders (Nadour, 2025a).

papilin, a novel component of basement membranes, in relation to ADAMTS metalloproteases and ECM development

papilins are homologous, secreted extracellular matrix proteins which share a common order of protein domains. They occur widely, from nematodes to man, and can differ in the number of repeats of a given type of domain. Within one species the number of repeats can vary by differential RNA splicing. A distinctly conserved cassette of domains at the amino-end of papilins is homologous with a cassette of protein domains at the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteases. papilins primarily occur in basement membranes. papilins interact with several extracellular matrix components and ADAMTS enzymes. papilins are essential for embryonic development of Drosophila melanogaster and Caenorhabditis elegans (Fessler, 2004).

Alternative splicing of papilin and the diversity of Drosophila extracellular matrix during embryonic morphogenesis

papilins are extracellular matrix proteins that share a particular, common order of types of protein domains. They occur widely, from nematodes to man, and can differ in the number of repeats of a given type of domain. Protein variety is increased by differential splicing of pre-mRNA. Drosophila, which has a compact genome, expresses three splice variants of papilin during embryogenesis in developmentally defined patterns. These isoforms have different numbers of Kunitz and IgC2 domains. The papilin isoforms are expressed in specific cell types and contribute to different extracellular matrices in gastrulation folds, early mesoderm, heart formation, basement membranes, and elaboration of the excorporeal peritrophic membrane that lines the gut. This finding indicates an unexpectedly broad spectrum of different pericellular matrices in Drosophila embryos. Such papilin-containing matrices have developmental as well as functional significance, as it was previously showen that both suppression of papilin synthesis and ectopic overexpression lethally disrupt organogenesis (Kramerova, 2003).

papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases

papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis (Kramerova, 2000).

papilin: a Drosophila proteoglycan-like sulfated glycoprotein from basement membranes

A sulfated glycoprotein was isolated from the culture media of Drosophila Kc cells and named papilin. Affinity purified antibodies against this protein localized it primarily to the basement membranes of embryos. The antibodies cross-reacted with another material which was not sulfated and appeared to be the core protein of papilin, which is proteoglycan-like. After reduction, papilin electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad band of about 900,000 apparent molecular weight and the core protein as a narrow band of approximately 400,000. The core protein was formed by some cell lines and by other cells on incubation with 1 mM 4-methylumbelliferyl xyloside, which inhibited formation of the proteoglycan-like form. The buoyant density of papilin in CsCl/4 M guanidine hydrochloride is 1.4 g/ml, that of the core protein is much less. papilin forms oligomers linked by disulfide bridges, as shown by sodium dodecyl sulfate-agarose gel electrophoresis and electron microscopy. The protomer is a 225 +/- 15-nm thread which is disulfide-linked into a loop with fine, protruding thread ends. Oligomers form clover-leaf-like structures. The protein contains 22% combined serine and threonine residues and 25% combined aspartic and glutamic residues. 10 g of polypeptide has attached 6.4 g of glucosamine, 3.1 g of galactosamine, 6.1 g of uronic acid, and 2.7 g of neutral sugars. There are about 80 O-linked carbohydrate chains/core protein molecule. Sulfate is attached to these chains. The O-linkage is through an unidentified neutral sugar. papilin is largely resistant to common glycosidases and several proteases. The degree of sulfation varies with the sulfate concentration of the incubation medium. This proteoglycan-like glycoprotein differs substantially from corresponding proteoglycans found in vertebrate basement membranes, in contrast to Drosophila basement membrane laminin and collagen IV which have been conserved evolutionarily (Campbell, 1987).

RNAi-mediated knockdown of papilin gene affects the egg hatching in Nilaparvata lugens

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Owing to the rapid adaptation of BPH to many pesticides and resistant varieties, identifying putative gene targets for developing RNA interference (RNAi)-based pest management strategies has received much attention for this pest. The glucoprotein papilin is the most abundant component in the basement membranes of many organisms, and its function is closely linked to development. In this study, a papilin homologous gene was identified in BPH (NlPpn). Quantitative Real-time PCR analysis showed that the transcript of NlPpn was highly accumulated in the egg stage. RNAi of NlPpn in newly emerged BPH females caused nonhatching phenotypes of their eggs, which may be a consequence of the maldevelopment of their embryos. Moreover, the transcriptomic analysis identified 583 differentially expressed genes between eggs from the dsGFP- and dsNlPpn-treated insects. Among them, the 'structural constituent of cuticle' cluster ranked first among the top 15 enriched GO terms. Consistently, ultrastructural analysis unveiled that dsNlPpn-treated eggs displayed a discrete and distorted serosal endocuticle lamellar structure. Furthermore, the hatchability of BPH eggs was also successfully reduced by the topical application of NlPpn-dsRNA-layered double hydroxide nanosheets onto the adults. These findings demonstrate that NlPpn is essential to maintaining the regular structure of the serosal cuticle and the embryonic development in BPH, indicating NlPpn could be a potential target for pest control during the egg stage (Zhang, 2024).

The role of ADAMTS-2, collagen type-1, TIMP-3 and papilin levels of uterosacral and cardinal ligaments in the etiopathogenesis of pelvic organ prolapse among women without stress urinary incontinence

To investigate the potential role of 'a disintegrin-like and metalloproteinase with thrombospondin type motifs-2 (ADAMTS-2), collagen type-1, tissue inhibitor of metalloproteinases-3 (TIMP-3) and papilin' levels in the uterosacral ligament (USL) and cardinal ligament (CL) of the uterus on the etiopathogenesis of pelvic organ prolapse (POP) among postmenopausal women without stress urinary incontinence (SUI). A total of 45 postmenopausal women, 22 diagnosed as POP stage III-IV and 23 age- and body mass index (BMI)-matched controls referred for hysterectomy due to POP or benign gynecological disease, respectively, were recruited prospectively for this study. The biopsies of the USL and CL were obtained during hysterectomy. ADAMTS-2, collagen type-1, TIMP-3 and papilin levels were determined by enzyme-linked immunosorbent assay (ELISA) method after tissue homogenization. Patients were excluded who smoked or presented with SUI. There were no differences in terms of demographic features including age, BMI, obesity, duration of menopause, gravidity, parity, delivery modes and family history for POP between the POP and non-POP groups. Significant differences in the levels of ADAMTS-2, collagen type-1, TIMP-3 and papilin of USL were noted among the groups. Females with POP had lower levels of ADAMTS-2, collagen type-1, TIMP-3 and papilin in the USL compared to non-POP females. All investigated markers in the CL were also decreased in the POP group, but this relationship was not statistically significant. When age, duration of menopause, gravidity, parity and obesity were taken as covariates, only the USL papilin levels were negatively predictive for the development of POP. ADAMTS-2, collagen type-1, TIMP-3 and papilin levels of the USL play essential roles in the etiopathogenesis of POP among postmenopausal women without SUI. Moreover, significantly decreased USL papilin levels in females with POP suggest the importance of the USL and the impact of papilin on the development of POP (Tola, 2018).

C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV

The gonad arms of C. elegans hermaphrodites acquire invariant shapes by guided migrations of distal tip cells (DTCs), which occur in three phases that differ in the direction and basement membrane substrata used for movement. This study found that mig-6 encodes long (MIG-6L) and short (MIG-6S) isoforms of the extracellular matrix protein papilin, each required for distinct aspects of DTC migration. Both MIG-6 isoforms have a predicted N-terminal papilin cassette, lagrin repeats and C-terminal Kunitz-type serine proteinase inhibitory domains. Mutations affecting MIG-6L specifically and cell-autonomously decrease the rate of post-embryonic DTC migration, mimicking a post-embryonic collagen IV deficit. MIG-6S has two separable functions - one in embryogenesis and one in the second phase of DTC migration. Genetic data suggest that MIG-6S functions in the same pathway as the MIG-17/ADAMTS metalloproteinase for guiding phase 2 DTC migrations, and MIG-17 is abnormally localized in mig-6 class-s mutants. Genetic data also suggest that MIG-6S and non-fibrillar network collagen IV play antagonistic roles to ensure normal phase 2 DTC guidance (Kawano, 2009).

Search PubMed for articles about Drosophila papilin

Campbell, A. G., Fessler, L. I., Salo, T., Fessler, J. H. (1987). papilin: a Drosophila proteoglycan-like sulfated glycoprotein from basement membranes. J Biol Chem, 262(36):17605-17612 PubMed ID: 3320045

Fessler, J. H., Kramerova, I., Kramerov, A., Chen, Y., Fessler, L. I. (2004). papilin, a novel component of basement membranes, in relation to ADAMTS metalloproteases and ECM development. Int J Biochem Cell Biol, 36(6):1079-1084 PubMed ID: 15094122

Fuwa, T. J., Itoh, K., Ichimiya, T., Akimoto, Y., Nishihara, S. (2026). Hemocyte-secreted papilin bearing mucin-type O-glycans regulates peripodial stalk formation via epidermal JAK/STAT signaling in Drosophila. iScience, 29(3):115054 PubMed ID: 41816305

Kawano, T., Zheng, H., Merz, D. C., Kohara, Y., Tamai, K. K., Nishiwaki, K., Culotti, J. G. (2009). C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV. Development, 136(9):1433-1442 PubMed ID: 19297413

Kramerova, I. A., Kawaguchi, N., Fessler, L. I., Nelson, R. E., Chen, Y., Kramerov, A. A., Kusche-Gullberg, M., Kramer, J. M., Ackley, B. D., Sieron, A. L., Prockop, D. J., Fessler, J. H. (2000). papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases. Development, 127(24):5475-5485 PubMed ID: 11076767

Kramerova, I. A., Kramerov, A. A., Fessler, J. H. (2003). Alternative splicing of papilin and the diversity of Drosophila extracellular matrix during embryonic morphogenesis. Dev Dyn, 226(4):634-642 PubMed ID: 12666201

Lee, J. I., Park, S., Park, H., Lee, Y., Park, J., Lee, D., Kim, M. J., Choe, K. M. (2025). The matrix glycoprotein papilin maintains the haematopoietic progenitor pool in Drosophila lymph glands Development, 152(7) PubMed ID: 40094323

Nadour, M., Leatis, R., Biard, M., Frebault, N., Rivollet, L., St-Louis, P., Blanchette, C. R., Thackeray, A., Perrat, P., Bevilacqua, C., Prevedel, R., Cappadocia, L., Rapti, G., Doitsidou, M., Benard, C. Y. (2025a). Remodeling of extracellular matrix collagen IV by MIG-6/papilin regulates neuronal architecture. Res Sq, PubMed ID: 39989960

Nadour, M., Valette, R. L. R., Frebault, N., Fontaine, V., Rivollet, L., Benard, C. Y. (2025b). Interplay between MIG-6/papilin and TGF-beta signaling promotes extracellular matrix remodeling and modulates the maintenance of neuronal architecture. bioRxiv, PubMed ID: 40654875

Tola, E. N., Koroglu, N., Yildirim, G. Y., Koca, H. B. (2018). The role of ADAMTS-2, collagen type-1, TIMP-3 and papilin levels of uterosacral and cardinal ligaments in the etiopathogenesis of pelvic organ prolapse among women without stress urinary incontinence. Eur J Obstet Gynecol Reprod Biol, 231:158-163 PubMed ID: 30388611

Zhang, C., Zhang, J. Y., Wang, N., Abou El-Ela, A. S., Shi, Z. Y., You, Y. Z., Ali, S. A., Zhou, W. W., Zhu, Z. R. (2024). RNAi-mediated knockdown of papilin gene affects the egg hatching in Nilaparvata lugens. Pest Manag Sci, 80(9):4779-4789 PubMed ID: 38837578

date revised: 20 May 2026

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