InteractiveFly: GeneBrief

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Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. Using a recently-developed glycoproteomic method, this study found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila. Wdp is a single-pass transmembrane protein with leucine-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Wdp modulates the Hedgehog (Hh) pathway. In the wing disc, overexpression of wdp inhibits Hh signaling, which is dependent on its CS chains and the LRR motifs. wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. RNAi knockdown and mutant clone analyses showed that loss of wdp leads to the upregulation of Hh signaling. Altogether, this study demonstrates a novel role of CSPGs in regulating Hh signaling (Takemura, 2020).

Spatial and temporal regulation of growth factor signaling pathways is essential to proper development and disease prevention. Cell surface signaling events, such as ligand-receptor interactions, are often modulated by proteoglycans. Proteoglycans are carbohydrate-modified proteins that are found on the cell surface and in the extracellular matrix. They are composed of a core protein and one or more glycosaminoglycans (GAGs) covalently attached to specific serine residues on the core protein. GAGs are long, unbranched, and highly sulfated polysaccharide chains consisting of a repeating disaccharide unit. Based on the composition of the disaccharide units, proteoglycans are classified into several types, including heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) (Takemura, 2020).

HSPGs function as co-receptors by interacting with a wide variety of ligands to modulate signaling activities. Drosophila offers a powerful model system to study the functions of HSPGs in vivo because of its sophisticated molecular genetic tools and minimal genetic redundancy in genes encoding core proteins and HS synthesizing/modifying enzymes. In vivo studies using the Drosophila model have shown that HSPGs orchestrate information from multiple ligands in a complex extracellular milieu and sculpt the signal response landscape in a tissue. However, the molecular mechanisms of co-receptor activities of HSPGs still remain a fundamental question. Previous studies predict that there are unidentified molecules involved in the molecular recognition events on the cell surface (Takemura, 2020).

In addition to HS, Drosophila produces CS, another type of GAG. CSPGs are well known as major structural components of the extracellular matrix. CSPGs have also been shown to modulate signaling pathways, including Hedgehog (Hh), Wnt, and fibroblast growth factor signaling. Given the structural similarities between CS and HS, CSPGs may have modulatory, supportive, and/or complementary functions to HSPGs. However, the mechanisms by which CSPGs function as a co-receptor are unknown. In contrast to a large number of studies on HSPGs, very few CSPGs have been identified and analyzed in Drosophila. Unlike HSPGs, CSPG core proteins are not well conserved between species. Therefore, the identification of CSPGs cannot rely on the sequence homology to mammalian counterparts (Takemura, 2020).

Recently, a glycoproteomic method was developed to identify novel proteoglycans. Briefly, this method includes trypsinization of protein samples, followed by enrichment of glycopeptides using strong anion exchange (SAX) chromatography. After enzymatic digestion of HS/CS chains, the glycopeptides bearing a linkage glycan structure common to HS and CS chains are identified using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). This method has successfully identified novel CSPGs in humans (Takemura, 2020).

To study the function of CSPGs in signaling, the glycoproteomic method was used to identify previously unrecognized CSPGs in Drosophila. This study found that Windpipe (Wdp) is a novel CSPG and affects Hh signaling. Overexpression of wdp inhibits Hh signaling in the wing disc. This inhibitory effect of Wdp on Hh signaling is dependent on its CS chains and LRR motifs. Consistent with the overexpression analysis, loss of wdp increases Hh signaling: wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. This study highlights a novel function of CSPGs in cell signaling (Takemura, 2020).

Glycoproteomic analysis identified Wdp as a novel CSPG. Apart from Wdp, no additional novel core proteins were found in this study. However, some previously established core proteins were also identified, which were found with both CS- and / or HS- modifications. In a recent glycoproteomic study of C. elegans, 15 novel chondroitin core proteins were identfied, in addition to the 9 previously established. The reason for this discrepancy with the regard to the number of identified core proteins in the two model organisms is unclear, but it may suggest that optimization of sample preparation is necessary for identifying additional CSPGs in Drosophila (Takemura, 2020).

Although Wdp was found modified with CS in both wild-type and ttv backgrounds, general assessment of spectral intensities suggest that Wdp was present in higher abundance in the ttv samples. Earlier studies in Zebrafish, mammalian cells, and C. elegans indicated that reduced HS sulfation results in increased CS sulfation. Thus, it is not surprising to see a compensatory increase of CS synthesis in a strain lacking HS polymerase (ttv). It should be noted that Wdp modified with HS was not detected in wild-type flies, although this variant was explicitly looked for (Takemura, 2020).

Genetic analyses of Wdp showed that it acts as a negative modulator of Hh signaling in a CS- and LRR motif-dependent manner. It has also been reported that Wdp negatively regulates JAK-STAT signaling and controls adult midgut homeostasis and regeneration (Ren, 2015). The authors showed that Wdp interacts with the Dome receptor and promotes its endocytosis and lysosomal degradation. Although the mechanism by which Wdp regulates Hh signaling is not known, it is possible that Wdp modulates these two pathways via a similar mechanism: by controlling the stability of cell surface components of the pathways. Hh signaling is controlled by two key membrane proteins-Ptc and Smo. In the absence of Hh, Ptc inhibits the phosphorylation of Smo, which is internalized and degraded. In the presence of Hh, restriction of Ptc on Smo is relieved, allowing Smo to accumulate on the cell surface and activate Hh signaling. Preliminary observation showed that knockdown of wdp increases Smo protein levels. Thus, Wdp may downregulate Hh signaling by affecting Smo levels (e.g. disrupting Smo translocation to the cell membrane or the stability of Smo on the cell surface). However, this does not exclude other possibilities for Wdp action, such as sequestering the ligand, inhibiting Ptc in its Smo phosphorylation/activation, and competing with a HSPG co-receptor. In mice, sulfated CS is necessary for Indian hedgehog (Ihh) signaling in the developing growth plate. Although Ihh and Sonic hedgehog (Shh) have been shown to bind to CS, the molecular mechanisms of CSPG function in Hh signaling remain to be elucidated (Takemura, 2020).

It is worth noting that both JAK-STAT and Hh signaling, the two pathways negatively controlled by Wdp, are also regulated by HSPGs. Dally-like, a glypican family of HSPGs, positively regulates Hh signaling. In the developing ovary, Dally upregulates the JAK-STAT pathway. Given the importance of precise dosage control of oncogenic pathways, such as JAK-STAT and Hh signaling, this dual proteoglycan system could play an important role in fine-tuning of the signaling output in order to prevent cancer formation. In vertebrates, HSPGs and CSPGs show opposing effects in neural systems. For example, axon growth is typically promoted by HSPGs but inhibited by CSPGs. The current findings suggest that such competing effects of HSPGs and CSPGs may be a general mechanism for the precise control of signaling cascades and pattern formation (Takemura, 2020).

In addition to its functions in signaling, Wdp may play other roles. This study found that overexpression of wdp results in massive apoptosis in the wing disc, independent of Hh signaling inhibition. Since CSPGs are well known for structural functions, an excess amount of Wdp may affect the epithelial integrity of the wing disc, leading to subsequent apoptosis. The observation that Wdp is enriched on the basal side of the wing disc and adult midgut cells suggests that Wdp may interact with components of the basement membrane, which surrounds these organs (Takemura, 2020).

Previous studies also reported that wdp is associated with aggressive behaviors in Drosophila species. wdp is upregulated in the head of socially isolated male flies, which exhibit more aggressive behaviors than males raised in groups. Also, wdp expression is slightly higher in the brain of Drosophila prolongata, which is more aggressive compared to its closely-related species. Since CSPGs are important in neuronal patterning, it is interesting to define the molecular mechanisms by which Wdp affects Drosophila behavior. In mammals, there is a class of CSPG molecules with LRR motifs (small leucine-rich proteoglycans, or SLRPs). A number of SLRP members are known as causative genes of human genetic disorders. Although Wdp does not have cysteine-rich regions that are commonly found in mammalian SLRPs, MARRVEL (ver 1.1) reports that wdp is a potential Drosophila ortholog of the human NYX gene (nyctalopin), a member of SLRPs (DIPOT score 1). Mutations in NYX cause X-linked congenital stationary night blindness. Further studies on Wdp will provide a novel insight into the function of these disease-related human counterparts (Takemura, 2020).

Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation

The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. This study has identified windpipe (wdp) as a novel component of the JAK/STAT pathway. Wdp was positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity resulted in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibited JAK/STAT signaling activity. Importantly, Wdp interacted with the receptor Domeless (Dome), and promoted its internalization for subsequent lysosomal degradation. Together, these data led the study to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis (Ren, 2015).

This study has provided evidence that the LRR protein Wdp is a novel component of the JAK/STAT pathway that acts in a negative feedback manner to modulate JAK/STAT signaling activity and control intestinal homeostasis. In vivo and in vitro data indicate that wdp expression levels are positively regulated by JAK/STAT signaling. Loss of wdp disrupts midgut homeostasis under both physiological and damage conditions. Conversely, ectopic expression of Wdp leads to the reduction of JAK/STAT signaling activity. Mechanistically, it was shown that Wdp can interact with Dome, and promote Dome internalization and lysosomal degradation, thereby reducing JAK/STAT signaling activity (Ren, 2015).

Midgut homeostasis is tightly controlled by different signaling pathways. During tissue damage, JAK/STAT, EGFR, JNK and Hippo signaling pathways are required for ISC proliferation and midgut regeneration. On the other hand, other signaling pathways, such as BMP signaling, may negatively regulate intestinal homeostasis after injury, although there exists some controversy about the function of BMP signaling during Drosophila intestinal development. However, the mechanism of how ISC activity returns to quiescence after injury remains largely unknown. This study demonstrates that Wdp controls intestinal homeostasis through interfering with JAK/STAT signaling activity to avoid tissue hyperplasia (Ren, 2015).

The data indicate that loss of Wdp disrupts midgut homeostasis under normal conditions and potentiates tissue regeneration under damage conditions. The proliferation rate of ISCs mutant for wdp is increased, while the differentiation of EC and ee cells is not inhibited. In addition, ectopic Wdp expression suppressed the damage induced tissue regeneration. The data further demonstrate that Wdp controls intestinal homeostasis through interfering with JAK/STAT signaling activity. First, Wdp acts as a JAK/STAT downstream target and its expression levels are positively regulated by JAK/STAT signaling. Second, Wdp functions in a negative feedback loop to modulate JAK/STAT signaling activity. It is interesting to note that JAK/STAT signaling is mainly activated in ISCs and EBs. However, it was found that Wdp expression levels seem higher in ECs compared with progenitor cells. One explanation is that low levels of Wdp in progenitors may guarantee high levels of JAK/STAT signaling, while high levels of Wdp in ECs may serve to reduce Dome levels thereby making ECs insensitive to Upd ligands. Consistent with this view, previous work showed that Dome is mainly expressed in the progenitors but not in their progeny. Moreover, it was found Wdp knock down using EC specific Myo1A^ts also leads to the disruption of midgut homeostasis and the presence of 10xSTAT GFP in putative EC cells, suggesting that JAK/STAT signaling is activated upon wdp knockdown in ECs. On the other hand, it was found Wdp expression was reduced but not totally eliminated in JAK/STAT signaling deficient cells, suggesting that the basal level of Wdp in intestines (especially in ECs) may also be regulated by other regulatory mechanisms or signaling pathways. Further experiments are needed to clarify this issue (Ren, 2015).

It’s important to mention that Wdp expression could be induced under injury conditions, such as DSS or bleomycin treatment. Consistent with the results, two recent studies also identified wdp as an upregulated gene upon Ecc15 and Pseudomonas entomophila (P.e) infection through their microarray data respectively. These stress conditions are also associated with the activation of JAK/STAT signaling. Therefore, their findings are consistent with the view that Wdp can be induced by the JAK/STAT pathway and then restrict its signaling activity in restoring intestinal homeostasis after tissue damage (Ren, 2015).

It was further demonstrated the regulation of Wdp to JAK/STAT signaling in eye discs and S2 cells. 10xSTAT GFP activity was decreased in eye discs overexpressing Wdp while increased in wdp mutant eye discs. Similarly, a reduction of 10xSTAT luciferase activity was also observed in S2 cells transfected with Wdp. Thus, it is proposed that Wdp is also likely to modulate JAK/STAT signaling activity for proper development of other tissues (Ren, 2015).

Taken together, it is concluded that Wdp is involved in controlling intestinal homeostasis through interfering with JAK/STAT signaling in a negative feedback manner (Ren, 2015).

Previously, several studies have addressed the roles of endocytosis in regulating JAK/STAT signal pathway. The Noselli lab found blocking internalization led to an inhibition of JAK/STAT signaling activity, while the Zeidler group reported the opposite results. Moreover, several recent studies demonstrate that loss of ept/tsg101 or Rabex-5, two endocytic tumor suppressor genes, also induced JAK/STAT signaling activation and tissue overgrowth. Yet, the regulatory mechanism of how Dome receptors are internalized remains largely unknown. This study demonstrates that Wdp promotes Dome endocytosis and subsequent lysosomal degradation. First, in S2 cells Wdp ectopic expression induces the formation of Dome endocytotic vesicles which were colocalized with the early endosome marker and lysosome marker. Second, it was found Wdp expression can also promote Dome endocytosis in wing and eye imaginal discs. Furthermore, the decreased Dome levels caused by Wdp expression can be suppressed by CQ treatment. All of these data argue that Wdp acts to promote Dome endocytosis from the cell membrane, first into the early endosomes, and finally into the lysosomes for degradation. Previous work are mainly about Dome receptors undergo ligands induced endocytosis, while this work showd that Wdp is able to promote Dome internalization in a Upd independent manner. Coimmnoprecipitation data indicate Wdp can interact with Dome. Moreover, Dome-GFP is aggregated on the cell membrane before they are internalized in the presence of Wdp. Therefore, one possible mechanism is that Wdp interacts with Dome, induces the aggregation of Dome on the cell membrane and then promotes Dome endocytosis. Further experiments are needed to define the detailed mechanism (Ren, 2015).

On the basis of these findings, the following model is proposed (see Model for the function of Wdp): Wdp regulates intestinal homeostasis through its modulation of JAK/STAT signaling. Under physical conditions, low levels of Wdp in progenitors are needed to maintain proper levels of JAK/STAT signaling activity, while high levels of Wdp in ECs reduce Dome levels to ensure these cells are insensitive to JAK/STAT signaling. When midgut epithelium is damaged by environmental challenges, high levels of JAK/STAT signaling activity are induced to replenish the damaged midgut. Then Wdp expression is highly induced in the intestines to reduce Dome levels, thereby switching off the overactivated JAK/STAT signaling. Through this way, ISC proliferative rate returns to normal levels to avoid tissue hyperplasia. While other mechanisms or regulators are likely to be involved in regulating intestinal homeostasis, the data suggest that Wdp is one of the key regulators in this process through interfering with JAK/STAT signaling activity (Ren, 2015).

Drosophila windpipe codes for a leucine-rich repeat protein expressed in the developing trachea

The embryonic tracheal system of Drosophila provides an important model for understanding the process of epithelial branching morphogenesis. This study reports the sequence and expression analysis of a novel tracheal gene, named windpipe (wdp). wdp is identical to the predicted gene CG3413 and encodes a transmembrane, leucine-rich repeat family member. wdp transcripts appear abruptly at stage 15 and are restricted to primary tracheal branches that give rise to secondary branches (Huff, 2002).

Search PubMed for articles about Drosophila Windpipe

Huff, J. L., Kingsley, K. L., Miller, J. M. and Hoshizaki, D. K. (2002). Drosophila windpipe codes for a leucine-rich repeat protein expressed in the developing trachea. Mech Dev 111(1-2): 173-176. PubMed ID: 11804792

Ren, W., Zhang, Y., Li, M., Wu, L., Wang, G., Baeg, G.H., You, J., Li, Z. and Lin, X. (2015). Windpipe controls Drosophila intestinal homeostasis by regulating JAK/STAT pathway via promoting receptor endocytosis and lysosomal degradation. PLoS Genet 11: e1005180. PubMed ID: 25923769

Takemura, M., Noborn, F., Nilsson, J., Bowden, N., Nakato, E., Baker, S., Su, T. Y., Larson, G. and Nakato, H. (2020). Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila. Mol Biol Cell: mbcE19060327. PubMed ID: 32049582

date revised: 12 January 2022

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