Abstract
Secreted exosomal miRNAs mediate inter-organ/tissue communication by downregulating gene expression, thereby modulating developmental and physiological functions. However, the source, route, and function have not been formally established for specific miRNAs. Here, we show that glial miR-274 non-cell autonomously modulates the growth of synaptic boutons and tracheal branches. Whereas precursor miR-274 was expressed in glia, mature miR-274 was secreted. miR-274 secretion to circulating hemolymph was detected in exosomes, a process requiring ESCRT components in exosome biogenesis and Rab11 and Syx1A in exosome release. miR-274 downregulated Sprouty to activate MAPK in synaptic boutons and tracheal branches, thereby promoting their growth. Expression of miR-274 solely in glia of a mir-274 null mutant reset normal levels of Sprouty and MAPK, and hemolymphatic exosomal miR-274. mir-274 mutant larvae were hypersensitive to hypoxia, which was suppressed by increasing tracheal branches. Thus, glia-derived miR-274 coordinates growth of synaptic boutons and tracheal branches to modulate larval hypoxia responses.
Introduction
Cells communicate at multiple levels during development, from short-to long-range, between the same or different types of cells, and between different tissues/organs in the body. Long-range communication requires transport of signals, leading to coordinated growth and differentiation in multicellular organisms. Several mechanisms for transporting long-range signals from source to target have been identified, including transport by extracellular vesicles (EVs) derived from ligand-expressing cells (1, 2). These EVs originate from at least two sources; direct shedding of plasma membranes to form microvesicles, and secretion of intraluminal vesicles (exosomes) from multivesicular bodies (MVBs). Exosomal transportation has been better characterized due to the consistent size of the vesicles (30-100 nm in diameter), easy detection in the circulatory system, and well-characterized cargoes (3). Furthermore, the physiological functions and diseases associated with secreted exosomes have been studied in greater detail (4).
Secreted exosomes host a major type of cargo, i.e., non-coding microRNAs (miRNAs), which can functionally inhibit protein expression in target cells (3). In animals, miRNAs are small RNAs of ~22 nucleotides, which possess a seed region of typically 2-7 nucleotides at their 5’ ends that binds to sequences of the target mRNAs to promote mRNA degradation or translational repression (5). Although cell-autonomous functions of miRNAs have been amply reported, non-cell-autonomous functions have only been recently discovered. Circulating miRNAs can target gene expression in distant tissues. For example, during the formation of immune synapses, exosomal miR-335 is transferred from T cells to antigen-presenting cells to downregulate SOX-4 mRNA translation (6). Exosomal miR-451 and miR-21 are transferred from glioblastoma to microglia to downregulate c-Myc expression (7). Adipocyte-derived exosomal miR-99b targets Fgf21 in hepatic cells to downregulate mRNA and protein expression (8). In Drosophila, epithelial cells express bantam miRNA to regulate neuronal growth non-cell-autonomously (9). miRNAs have also been isolated from the circulating hemolymph of Drosophila (10), suggesting that some Drosophila miRNAs are carried by exosomes in the circulatory system to function systematically or in specific target cells. However, mechanistic details—such as the sources of exosomal miRNAs, their presence in circulating hemolymph, and their direct target genes in recipient tissues, as well as functional modulation of recipient tissues and relevant physiological functions—have not been established for a specific miRNA, especially in a model organism that would greatly facilitate a precise mechanistic understanding at the genetic level.
During vertebrate development, nerve and blood vessel formation share many cellular processes, including cone-like growth tips, branching patterns and ramifying networks (11, 12). Pairs of signals and receptors such as Slit and Robo, Netrin and Unc5/DCC co-receptor, and Ephrin and Eph, which were initially identified as being involved in axon outgrowth and patterning, have since been shown to be involved in similar processes in vasculogenesis (11, 12). Expression of vascular endothelial growth factor (VEGF), which plays critical roles in angioblast migration and vessel ingression, is spatiotemporally regulated in the neural tube during embryonic development (13). Although VEGF167 and the axon guidance signal Sema3A function separately in early vessel and nerve formation, both signals function through the shared receptor neurophilin-1 (14). During post-developmental stages, neuronal activity of the nervous system and oxygen delivery are also prominently coupled, forming the neurovascular units (15). Given the extreme sensitivity of the nervous system to alterations of ions, nutrients and potentially harmful molecules in the vascular system, an interface between both systems is necessary. Astrocytes, the major type of glia in the mammalian brain are structurally and functionally coupled to neuronal synapses and vascular endothelial cells to directly regulate their activities and communication (16–20). The insect trachea, the prototypical vascular system, allows oxygen delivery to the inner parts of the animal body. Nerves, glial sheath, and tracheal branches have been described for the larval brains and adult NMJs of Drosophila (21–23). Synapse organization and activity of larval NMJs, as well as their glial interactions, have also been well characterized (23–25).
In this study, we explored larval Drosophila neuromuscular junctions (NMJs) where axonal terminals branch out to form synaptic boutons with muscle membranes. We show that tracheal terminal branches are also highly arborized at the NMJs, making them an ideal system for studying coordinated nervous and vascular development. We screened a collection of miRNA knockout mutants and identified the mir-274 mutant as having defects in both synaptic and tracheal growth. By fluorescent in situ hybridization (FISH), we showed that the miR-274 precursor was expressed in glia and the mature form was ubiquitously detected. Consistently, miR-274 was required in glia for synaptic and tracheal growth. Glial expression of miR-274 could be detected in the hemolymph of the larval circulatory system. Indeed, miR-274 was secreted as an exosomal cargo as shown by genetic analysis and biochemical fractionation. We identified a miR-274 target sprouty (sty) that includes a targeting site in the 3’UTR of the sty transcript and showed that glial miR-274 expression induced Sty downregulation and MAPK activation in synaptic boutons and tracheal branches. Intriguingly, the mir-274 mutant that had fewer tracheal branches were hypersensitive to hypoxia, and both phenotypes were suppressed sty loss-of-function mutations. Thus, we demonstrate the non-cell-autonomous developmental role of glial miR-274, which coordinates the growth of synaptic boutons and tracheal branches and is required for normal responses to hypoxia.
Results
miR-274 is required in glia to modulate synapse and tracheal growth
By immunostaining synaptic, glial and tracheal structures, we show that glial processes wrap around incoming motor axons, and envelopment ends before terminal branching at muscle 6/7 (Figures S1A & A’, arrowheads). Axonal terminal branches form bouton-like structures that innervate muscles to form functional synapses, and multiple tracheal branches terminate near these synaptic boutons (Figure S1A”, empty arrowheads). Ultrastructure analysis by transmission electron microscopy shows that glial processes enwrap axonal processes close to tracheal branches (Figure S1B, arrows). Synaptic boutons wrapped in the subsynaptic reticulum (SSR) are also visible (Figure S1B, arrowhead). These observations suggest that the glia-synapse-trachea organization might represent functional units and its formation might be developmentally regulated.
To investigate whether these structures are developmentally co-regulated, we screened a collection of miRNA knockout mutants (26). We identified mir-274KO as displaying reduced growth of both synaptic boutons and tracheal branches (Figure 1A). Quantification revealed that larvae homozygous for mir-274KO exhibited a ~40% reduction in the numbers of synaptic boutons and tracheal terminal branches compared to both wild-type w1118 and mir-274KO/+ larvae (Figure 1D). We also detected reduced numbers of synaptic boutons and tracheal branches for the trans-heterozygous mir-274KO/mir-2746-3 mutant (Figures 1A and 1D), confirming that the lack of miR-274 activity accounts for growth defects in both systems. This reduction in synaptic bouton number was not limited to NMJs of muscle 6/7, as we also observed bouton reduction at NMJs of muscle 4 in the mir-274KO mutant (Figures S1C and S1D). Likewise, tracheal branching was also reduced in the dorsal region of the mir-274KO mutant (Figures S1C and S1D). These data suggest that larvae lacking miR-274 fail to develop complete sets of synaptic boutons and tracheal branches.
To examine whether specific types of cells require miR-274 for growth of synaptic boutons and tracheal branches, we employed the UAS-decoy-mir-274 transgene (see Experimental Procedures) driven by cell-type specific GAL4 drivers to inhibit miR-274 functions. Neuronal elav-GAL4, glial repo-GAL4, and tracheal btl-GAL4 were individually crossed to UAS-decoy-mir-274 to analyze synaptic bouton and tracheal branch phenotypes. Surprisingly, although miR-274 inhibition by neuronal and tracheal drivers had no obvious phenotypic impact (Figures S1E-H), glial depletion caused significant reductions in synaptic bouton and tracheal branch numbers (Figures 1B and 1E). Whereas the glial processes at NMJs of muscle 6/7 presented a normal morphology in the mir-274KO mutant, the hemolymph brain barrier (HBB, which is mainly composed of glia) was defective (Figure S1I), confirming the findings of a previous study (26). However, the reductions in synaptic boutons and tracheal branches are not secondary to the defective HBB, as an intact HBB was retained in glial expression of UAS-decoy-mir-274 (Figure S1I) while synaptic boutons and tracheal branches were reduced (Figures 1B and 1E). Thus, it seems that glial inhibition of miR-274 is sufficient to compromise synapse and tracheal growth.
Furthermore, we performed a glial rescue experiment. In the homozygous mir-274KO mutant carrying either repo-GAL4 or UAS-mir-274 alone, the numbers of synaptic boutons and tracheal branches were fewer than the numbers in the heterozygous mutants carrying either repo-GAL4 or UAS-mir-274 alone (Figures 1C and 1F). However, in the homozygous mir-274KO mutant carrying both repo-GAL4 or UAS-mir-274, the numbers of synaptic boutons and tracheal branches were comparable to those in the heterozygous mutants (Figures 1C and 1F). These results strongly support that glia-expressed miR-274 promotes growth of synaptic boutons and tracheal branches.
Expression of miR-274 precursor and mature forms
To characterize miR-274 expression, we performed FISH experiments using probes complementary to the loop or the stem sequence to detect the precursor or the mature forms of miR-274 (Figure 2A), respectively, in dissected larval fillets (Figure 2B). In control, we only detected low background or non-specific signals in larval brain using scrambled probes (Figure 2C). However, the loop probe for detecting the miR-274 precursor presented prominent signals in the brain (Figures 2D and 2D’). These signals were localized in glia labeled with repo-GAL4-driven mCD8-GFP and occasionally strong nuclear signals were detected (Figure 2D’, arrowhead). In contrast, low background signals were detected in synaptic boutons and tracheal branches (Figures 2F, 2F’, 2H and 2H’). We then employed the stem probe to detect mature miR-274 (Figure 2A). Interestingly, we observed strong and ubiquitous signals, i.e., not restricted to specific cells in the brain (Figure 2E and 2E’). These signals were also detected in muscle cells and within synaptic boutons (Figures 2G and 2G’), as well as in tracheal soma and branches (Figures 2I and 2I’). Signals of mature miR-274 were not detected in the mir-274KO mutant control (Figure 3G). Taken together, these results are consistent with the idea that the miR-274 precursor is mainly synthesized in glia and that the mature form is detected in muscles, synaptic boutons and tracheal cells.
Exosomal secretion of miR-274 from glia
To examine how miR-274 is secreted, we first examined whether miR-274 could be secreted from S2 cells. Indeed, we detected miR-274 in S2 cell extracts (Figure 3A). Significant levels of miR-274 could also be detected in the medium used to culture S2 cells, but not in the medium in which S2 cells were not cultured, indicating that miR-274 could be secreted from S2 cells into the medium (Figure 3A). To examine whether secreted miRNA is through secretory exosomes, we fractionated and pelleted the S2 cell culture medium to enrich for exosomes. Similar to whole cell extracts, the fractionated exosomes were enriched with miR-274 (Figure 3B). As a positive control, the exosomal markers TSG101, Rab11 and Syntaxin (27, 28) were detected in the exosomal fraction, and not detected in the exosome-depleted supernatant (Figure 3C). As negative controls, Ephrin mRNA that is not present in the secreted exosomes (29) was only detected in whole cell extracts and not in the exosome fraction or in the exosome-depleted supernatant (Figure 3B).
As miRNAs could be transported by circulating exosomes, we then examined whether miR-274 could be detected in the larval hemolymph. Significant amounts of miR-274 were detected in the hemolymph of wild-type larvae, which were absent in the hemolymph isolated from the mir-274KO mutant (Figure 3D). We fractionated and pelleted exosomes from the hemolymph and found that fractionated exosomes were enriched with miR-274 (Figure 3D). The exosomal fraction isolated from the mir-274KO hemolymph did not contain miR-274 (Figure 3D). The typical exosomal markers TSG101, Rab11 and Syntaxin were detected in exosomal fractions derived from both hemolymphs of wild-type control and mutant larvae but not in the exosome-depleted supernatants (Figure 3D). Thus, miR-274 could be secreted into larval hemolymph and S2 cell culture medium as circulating exosomes.
With the detection of miR-274 in the exosomes of hemolymphs, we would like to detect whether glia could secret miR-274 carried by exosomes to the hemolymph. As mir-274KO is a deletion allele, no miR-274 could be detected in whole larval lysates, hemolymphs and hemolymph-derived exosomal fractions in the homozygous mir-274KO mutant (mir-274KO/mir-274KO; repo-GAL4, Figure 3E). In contrast, miR-274 was detected in all three of these preparations from the homozygous mir-274KO larvae carrying both repo-GAL4 and UAS-mir-274 (mir-274KO/mir-274KO; repo>mir-274, Figure 3E). Thus, glia could secrete miR-274 as an exosomal cargo in the hemolymph. We performed the same sets of experiments for neuronal and tracheal miR-274 expression in the mir-274KO mutant. miR-274 was only detected in whole larval lysates, but not in the isolated hemolymphs or exosomal fractions (Figures 3E). Quantification of miR-274 levels by absolute qPCR (Experimental Procedures) in hemolymph-derived exosomal fractions showed a three-fold increase in mir-274KO/mir-274KO; repo>mir-274 as compared to mir-274KO/mir-274KO; repo-GAL4 whereas elav-GAL4- and btl-GAL4-driven expressions were comparable to respective GAL4 driver controls (Figure 3F). Therefore, glia is the major source to release exosomal miR-274 into the hemolymph.
To further show that glial expression of miR-274 could reach synaptic boutons and tracheal branches for function, we performed the FISH experiment with the mature miR-274 probe. In the control of the mir-274KO null mutant carrying only repo-GAL4 (mir-274KO/mir-274KO; repo-GAL4), low background FISH signals appeared to be homogeneously throughout different tissues, including synaptic boutons, muscle cells, and tracheal cells (Figure 3G). This confirms the specificity of the mature probe in detecting miR-274. In glial expression of miR-274 in the mir-274KO null mutant (mir-274KO/mir-274KO; repo>mir-274), we detected strong and punctate signals in synaptic boutons, muscle cells, and tracheal cells (Figure 3G). We also examined neuronal expression of UAS-mir-274 in the mir-274KO mutant, which showed strong miR-274 FISH signals in synaptic boutons (Figure S2A). However, only background signals were detected in tracheal cells (Figure S2A). Likewise, miR-274 FISH signals were detected only in tracheal cells and not in synaptic boutons of mir-274KO mutants upon tracheal UAS-mir-274 expression (Figure S2D). Taken together, these results suggest that miR-274 expressed in glia could be secreted and detected in the target sites like synaptic boutons and tracheal cells.
Exosomal secretion of miR-274 from glia requires ESCRT components, Rab11, and Syx1A
Exosomal release requires Rab11 in MVB transportation and Syntaxin 1A (Syx1A) in membrane fusion with the plasma membrane (30). To show that miR-274-carrying exosomes are secreted through the process, we expressed Rab11RNAi or Syx1ARNAi in glia by repo-GAL4. We observed dramatically reduced miR-274 transcript levels in exosomes isolated from the hemolymph compared to the driver control (Figure 4A). To further confirm the absence of miR-274 by inhibiting glial exosomal secretion, we performed FISH with the mature miR-274 probe. In the repo-GAL4 control, miR-274 signals delineated bouton morphology and were strongly punctate in muscle and tracheal cells (Figure 4B). However, these miR-274 signals were not detected upon glial knockdown of Rab11 or Syx1A (Figure 4B). The failure to detect miR-274 in the hemolymphs and target sites in Rab11 and Syx1A knockdowns is consistent with the idea that the exosomal release pathway is essential for miR-274-carried exosomes to be released from glia. We then examined whether the growth of synaptic boutons and tracheal branches, the target sites of miR-274, were affected in the inhibition of exosomal release. As expected, glial knockdowns of Rab11 or Syx1A caused reduced growth of synaptic boutons and tracheal branches (Figures 4C and 4D). Thus, just like the lack of miR-274 in glia, the lack of Rab11 or Syx1A in glia causes synapse and tracheal undergrowth. Cargo-carrying exosomes are assembled through serial actions of the ESCRT complexes, which promote membrane invagination and formation of intraluminal vesicles in MVBs (27). To confirm that miR-274 is packaged as exosomal cargo, we examined whether disruption of ESCRT complex components in glia could affect the level of circulating miR-274. We chose to knock down TSG101 of the ESCRT-I complex and Shrb of the ESCRT-III complex as RNAi knockdown of TSG101 or Shrb in glia resulted in efficient suppression of synapse and tracheal growth (Figures S3B and S3C). When we specifically expressed TSG101RNAi or shrbRNAi in glia by repo-GAL4, we observed reduced levels of miR-274 in exosomes isolated form the hemolymph, as compared to the driver control (Figure S3A). Thus, the glial ESCRT complex is also involved in the presence of miR-274-carrying exosomes in the hemolymph. Taken together, these results strongly suggest that miR-274 is secreted as an exosomal cargo to be released into the hemolymph to modulate synapse and tracheal growth.
sprouty as a target gene for miR-274 regulation
To understand how miR-274 regulates synapse and tracheal growth, we searched for genes that harbor miR-274 target sites and exhibited upregulated expression in the mir-274KO larvae (Figure S4A). From among the resulting candidate genes (Figure S4B), we chose sprouty (sty) for further study since Sty plays a critical role in feedback inhibition of receptor tyrosine kinase (RTK) signaling during tracheal branching and synaptic bouton formation (31–34). The 3’UTR of sty mRNA contains a target site for miR-274 recognition. We then generated two luciferase reporter transgenes with either precise or mismatched miR-274 sequences targeting the sty 3’UTR (Figure 5A). As expected, the precise miR-274 targeting sequence downregulated reporter activity (relative to the vector control) when it was co-transfected with miR-274 (Figure 5A). The mismatched reporter was not downregulated upon miR-274 co-transfection. Thus, sty mRNA levels might be regulated by miR-274 through its 3’UTR targeting sequence. We then addressed whether miR-274 regulates sty mRNA expression in vivo. Indeed, higher sty transcript levels were detected in mir-274KO larvae compared to the levels in wild-type control (Figure 5B), consistent with miR-274 having a role in downregulating sty expression.
We further confirmed that Sty is regulated by miR-274 in synaptic boutons and tracheal branches by performing immunostaining. We first detected Sty expression in synaptic boutons and tracheal branches of the wild-type control. Levels of Sty in both these sites were enhanced relative to the control in the mir-274KO mutant (Figure 5C), which is supported by quantifications of Sty immunofluorescence intensities (Figure 5F). Sty expression was also upregulated in muscle cells, suggesting that miR-274 might exert systemic regulation in multiple tissues (Figure 5C, also see Discussion). As a negative regulator, Sty inhibits several downstream components in RTK signaling, leading to downregulation of MAPK activity and inhibition of tissue growth (32–34). We further examined RTK/MAPK signaling activity by immunostaining for diphosphorylated-ERK (dpERK) (34). Levels of dpERK in synaptic boutons and tracheal cells of mir-274KO were greatly reduced as compared to wild-type control (Figures 5D and 5G). Downregulation of dpERK levels is dependent on Sty, as elimination of one copy of sty in mir-274KO (mir-274KO/mir-274KO; sty226/+ or mir-274KO/mir-274KO; styΔ5/+) restored dpERK levels in tracheal cells and synaptic boutons to levels comparable to the control (Figures 5D and 5G). Restoration of dpERK levels was also detected in muscle (Figure 5D). We then tested whether miR-274 negatively regulates Sty expression to modulate synaptic and tracheal growth. Indeed, reducing the sty gene dosage in the miR-274 mutant suppressed both growth phenotypes (Figures 5E and 5H). These data support that miR-274 inhibits Sty expression, which leads to MAPK activation to promote the growth of synaptic boutons and tracheal branches.
Glia-derived exosomal miR-274 targets Sty within synaptic boutons and trachea to modulate their growth
To understand whether glia-derived miR-274 regulates Sty and dpERK levels within synaptic boutons and tracheal cells, we first performed immunostaining in repo>decoy-mir-274 larvae that had reduced synaptic and tracheal growth (Figures 1B and 1F). Similarly, trapping miR-274 in glia induced higher Sty levels in synaptic boutons and tracheal branches compared to repo-GAL4 (Figures 6A and 6E). We also detected reduced levels of dpERK at these two sites (Figures 6B and 6F). In glia rescue larvae (mir-274KO/mir-274KO; repo>mir-274), as expected, we found reduced Sty and increased dpERK levels upon glial rescue (Figures 6C, 6D and 6G) and accompanied with increased numbers of synaptic boutons and tracheal branches (Figures 1C and 1F). These results strongly support that glia-expressed miR-274 reaches target sites to downregulate Sty expression and to promote growth of synaptic boutons and tracheal branches.
Finally, we showed that disruption of the exosomal biogenesis, transportation and release by glial knockdowns of Rab11, Syx1A, TSG101 and Shrb also caused Sty upregulation and dpERK downregulation in synaptic boutons and tracheal cells (Figures S5A-H). Taken together, these results suggest that glia-derived miR-274 downregulates Sty and upregulates dpERK levels in synaptic boutons and tracheal cells.
miR-274 modulates larval hypoxia response
The Drosophila trachea is a highly branched network with open ends and air-filled terminal branches. It functions in gas exchange similarly to mammalian circulatory systems (35, 36). Oxygen tension is important for inducing tracheal terminal branching (37). We postulated that miR-274 might play a physiological role in glia-modulated tracheal branching during hypoxia. To test this possibility, we assayed the hypoxia escape response for the mir-274KO mutant (see Behavior assay in Materials and Methods) (38). When exposed to hypoxia (1% O2), only about 20% of control larvae (w1118 and Canton S) had responded strongly within 5 min by fleeing the food source, and this percentage increased to almost 40% by 10 min and to close to 50% by 15 min (Figure 7A). In contrast, almost 50% of mir-274KO mutants exhibited a strong hypoxia response (fleeing the food source) by 5 min and about 60% by 10 and 15 min (Figure 7A). No significant differences were found when we assayed these three genotypes under normoxia as a control, with almost all larvae (> 95%) staying in the food source (Figure 7B). Moreover, we confirmed that the hypoxia-induced response is not due to differences in larval locomotion, as we observed comparable crawling lengths among mir-274KO mutants and controls (Figure S6A). We performed additional control experiments to show that mir-274KO larvae are indeed more responsive to lower oxygen levels. First, mir-274KO mutants still exhibited a significantly different hypoxia response compared to control larvae in a ten-fold-diluted food source, suggesting that the enhanced exploratory behavior of mutant flies is not due to differences in evaluating nutrition (Figure S6B). Second, feeding motivation toward nutritious (yeast) or non-nutritious (grape juice) foods, as evaluated by counting mouth hook contractions, revealed almost identical results under both fed and starved conditions (Figure S6C). Finally, the difference we had observed between mir-274KO and control larvae was still significant when we conducted the assay in 10% oxygen, suggesting that the mutant larvae exhibited hypersensitivity towards reduced oxygen levels (Figure S6D).
We performed rescue experiments to examine whether glia-expressed miR-274 is required for normal larval hypoxia responses. Homozygous mir-274KO mutants carrying both repo-GAL4 and UAS-mir-274 transgenes showed a significantly reduced hypoxia response compared to homozygous mir-274KO mutants carrying either the repo-GAL4 or UAS-mir-274 transgenes (Figure 7C). As glial rescue larvae displayed a normal level of tracheal branching (Figures 1C and 1F), our results suggest that reduced tracheal branching might contribute to the altered hypoxia escape response. To examine whether miR-274 functions through Sty downregulation to affect this response, as for developmental regulation of tracheal branching, we performed genetic suppression of Sty by mutation. Indeed, we found that introducing a sty mutant allele in homozygous mir-274KO mutants almost completely suppressed the enhanced hypoxia escape response (Figure 7D). Since tracheal branching was also restored by the sty mutant alleles in the absence of miR-274 (Figures 5E and 5H), this result supports that tracheal branching is linked to the hypoxia escape response.
Discussion
Here, we report that glia-derived miR-274 non-cell-autonomously regulates synaptic and tracheal growth during development. miR-274 is expressed in glia and regulates Sty expression and MAPK signaling in synapses and trachea to control their growth. Exosome-borne miR-274 derived from glia was present in the circulatory hemolymph, which requires glial Rab11, Syx1A, and ESCRT complex components. We further show that miR-274 is present in synapses and trachea following specific glial expression of miR-274 in the mir-274KO mutant. Glial expression of miR-274 also restored synapse and tracheal growth through Sty downregulation. We observed that mir-274KO larvae with compromised tracheal branching were hypersensitive to hypoxia, which could be suppressed by reducing the sty gene dosage to reestablish the tracheal system. We propose that developmentally regulated neuron-trachea coupling via glial-secreted miR-274 is synchronized post-developmentally to physiological demands (Figure 7E).
Circulating miR-274
Extracellular miRNAs detected in the blood serum and other body fluids are highly stable and resistant to RNase treatments, making them ideal signaling molecules for long-distance communication among tissues and organs (39–42). In Drosophila, miRNAs have been isolated from the circulating hemolymph (10), suggesting that Drosophila miRNAs are also carried by exosomes in the circulatory system to function systematically for tissue and organ interactions. Formation of cargo-carrying exosomes requires the ESCRT complexes, which facilitate membrane invagination and form the intraluminal vesicles within MVBs (27). Rab11 and Syx1A in neurons effectively block exosome secretion from presynaptic boutons by disrupting the exosome transportation and release machinery (30). We detected miR-274 in exosomal fractions of larval hemolymph (Figure 3D), even when we contrived to specifically express miR-274 solely in glia (Figure 3E). Furthermore, Rab11, Syx1A and partially the ESCRT components in glia were required to release miR-274 into the hemolymph (Figures 4A and S3A) and in target tissues (Figure 4B). These results support that glia-expressed miR-274 is carried by exosomes in the larval circulatory system.
Circulating exosomes carry diverse molecules including proteins and RNAs. Although some studies have suggested that extracellular miRNAs might be “cellular by-products” disposed of by apoptotic cells (42), our genetic data provide strong evidence of a non-cell-autonomous developmental role for glial miR-274. Though miR-274 may cell-autonomously regulate secreted factors in glia to execute its function indirectly, our findings support an active and direct role for miR-274 at target sites. In addition to confirming the presence of miR-274 at target tissues following glia-only expression (Figure 3G), we observed that expression of Sty that carries a miR-274 target site at its 3’UTR was suppressed in target sites upon glia-only miR-274 expression (Figure 6C). Circulating miR-274 in the hemolymph could potentially target multiple tissues given that we detected miR-274 as well as regulation of Sty and dpERK expression also in muscle cells (for example, see Figures 6C and 6D). The function of miR-274 in muscle cells awaits further study. Accordingly, miR-274 might have a systematic role in multiple tissues that could contribute to coordinating their developmental processes and post-developmental physiology.
Glia specificity of miR-274 secretion
The non-cell-autonomous role of miR-274 appears to be highly cell-type specific. Although expression of pre-miR-274 is highly glia-enriched, perhaps accounting for the majority of specificity, other layers of regulation may confer this specificity. We have shown that miR-274 secretion is highly specific to glia, as glia-expressed miR-274 was detected in synaptic boutons, muscle and tracheal cells, whereas neuron- and trachea-expressed miR-274 was only detected in the respective expressing cells (Figures 3E, S2A and S2D). Interestingly, neuron-expressed miR-274 was also detected in muscle cells (Figure S2A), which might be transported by transverse exosomes crossing the synaptic cleft at NMJs (30). The Wnt/Wg signal is carried by Evenness interrupted (Evi)-positive exosomes from pre- to post-synapses (30). Developmental signals like Hedgehog are also transported over long distances in wing epithelia for cell fate induction (28). The Drosophila retrovirus-like Gag protein Arc1 (dArc1) binds to darc1 mRNA to be sorted into exosomes for transport across synaptic boutons (29). Interestingly, although presynaptic release of Wg/dArc1 and glial miR-274 shares a requirement for Rab11 and Syx1A, they may still exhibit substantially difference. Thus, multiple secretory exosomal pathways carry distinct cargos and function in different tissues of Drosophila.
Exosomes are formed through multiple pathways—such as ubiquitination-dependent and –independent or ESCRT-dependent and –independent pathways—that package different combinations of cargoes (2, 3). We did not detect miR-274 in hemolymph exosomes when it was specifically expressed in neurons or tracheal cells (Figure 3E), perhaps because neurons or tracheal cells do not generate miR-274-bearing exosomes. Furthermore, data from our cell-type-specific rescue experiments clearly indicate that only glial miR-274 expression in a mir-274KO mutant had a profound rescue effect on synapse and tracheal growth (Figures 1C and 1F). In contrast, the minor rescue effects of neuronal and tracheal expressions of miR-274 were restricted to the expressing cells (Figures S2B, S2C, S2E and S2F).
It has been suggested that miRNAs are subjected to cell-type-specific modifications, including uridylation and adenylation that alter miRNA localization, stability or activity (43). Such modifications may further induce packaging of miRNAs into exosomes for secretion in glia. Glia-specific miR-274 release suggests another layer of regulation for exosome-mediated cell-cell communication. Cargo packaging and exosome formation pathways are distinct in different types of cells (2, 3). We observed differential effects of knocking down several ESCRT components in terms of regulating synapse and tracheal growth, which could reflect the existence of heterogeneous populations of exosomes (Figures S3B and S3C). Differential requirements for ESCRT components have also been observed for blocking Hh-borne exosomes from wing-disc epithelial cells (28), as well as in the presynaptic release of Evi-positive exosomes (30). Thus, it would seem that complex regulation of the biogenesis of distinct exosomal populations may underlie different exosome-mediated communications between specific pairs of source and target cells. Different miRNA species have been detected in exosomes isolated from various types of immune, cancer, adipose and glial cells (6–8). Our analysis of the non-cell-autonomous function of miR-274 serves as a foundation for further study of the cell- and tissue-specificity involved in exosome-mediated cell-cell communication.
Glia-modulated growth of trachea branches and hypoxia responses
Similar to mammalian systems, Drosophila glia are linked to neurons and vascular systems in terms of their structure and function. In the larval Drosophila brain, trachea grow alongside glial processes toward the central neuropiles (22). In the peripheral nervous system of adult flies, glial processes are intertwined with synaptic bouton-bearing axonal terminals and tracheal terminal branches to form the functional complex (21). This coupling between tracheal and neuronal processes may ensure efficient oxygen supply to neurons for activity and homeostasis, which is similar to the coupling between the vascular and nervous systems in vertebrates. At NMJs, the gliotransmitters Wnt/Wg and TNF-α regulate synaptic plasticity (44, 45). Glia also function as macrophages, engulfing synaptic debris and shaping neurites after injury (46). Direct ablation of glia throughout development induces tracheal branching, suggesting that tracheal branching is restricted by glia (22). In this study, we further report the co-regulation of both tracheal and nervous systems by glial-derived miR-274, reinforcing the idea of glia-neurovascular coupling in Drosophila. We show that only blockage of miR-274 in glia, not other target tissues, significantly affected the growth of synaptic boutons and trachea.
Here, we chose Sty to investigate miR-274 targeting since Sty is a negative regulator of RTK/Ras/MAPK signaling and is involved in synaptic growth and tracheal branching (32–34). Synaptic bouton numbers are consistently reduced when sty is overexpressed in neurons (32), a phenotype recapitulated in the mir-274KO mutant. Loss-of-function mutations in sty enhanced tracheal branching, with increases ranging from 20-70% depending on different allelic combinations (33). Thus, Sty is sensitive to modulation, such as by miR-274 as shown in our study. It would be of great interest to establish whether and how miR-274 is regulated throughout development and physiology. Nevertheless, direct (and therefore more effective) glial regulation is supported here by the fact that Sty levels in target sites were modulated by miR-274. Such direct modulation in target sites could ensure synchronized growth regulation for both synaptic boutons and tracheal branches.
Recently, miRNAs were also shown to be essential for physiological functions. In Drosophila, miR-iab4/iab8 is expressed in self-righting node neurons (SRNs) and is responsible for larval self-righting behavior. Lack of miR-iab4/iab8 or overexpressing the target gene Ultrabithorax in SRNs inhibits the ability of larvae to right themselves (47). Similarly, astrocyte-specific expression of miR-263b and miR-274 is essential for circadian locomotor activity rhythms (48). Here, we reveal that mir-274KO mutants barely tolerated a sustained low oxygen environment. This behavioral defect was correlated with mir-274KO mutants having fewer tracheal branches as it was rescued by sty genetic suppression to restore tracheal branching. Oxygen may be delivered in the body less efficiently by having fewer tracheal terminal branches, rendering mutants less tolerant to low oxygen levels. Thus, miR-274 seems to ensure a well-developed tracheal system (and perhaps also synaptic boutons), allowing larvae to tolerate hypoxia. Our study highlights a coordinating role for glia in regulating a coupled developmental and physiological process.
Material and methods
Fly stocks
All flies were reared at 25 °C under a 12:12 hr light:dark cycle. Third instar wandering larvae were used for experiments. Mutant flies including mir-274KO (58904), styΔ5 (6382) and sty226 (6383) were obtain from Bloomington Drosophila Stock Center (BDSC). mir-2746-3 was generated by Dr. Yu-Chen Tsai in this paper. The mir-2746-3 allele bears a mutation in the seeding motif. The GAL4/UAS system was used, with repo-GAL4 (BDSC_7415), elav-GAL4 (BDSC_458) and btl-GAL4 (BDSC_8807) used to drive glia, neuron and tracheal expression of transgenes. UAS responders were: UAS-mir-274 (BDSC_59895), UAS-mCD8-GFP (BDSC_5137) and UAS-myr-RFP (BDSC_7119). RNAi flies were obtained from BDSC or Vienna Drosophila Resource Center (VDSC) including: UAS-TSG101RNAi (BDSC_35710), UAS-shrbRNAi (BDSC_38305), UAS-Vps28RNAi (VDRC_105124), UAS-Chmp1RNAi (BDSC_28906), UAS-ALiXRNAi (BDSC_33417), UAS-Rab11RNAi (BDSC_27730) and UAS-Syx1ARNAi (BDSC_25811). The lexA/lexAop system was used, btl-lexA-driven lexAop-CD2-GFP was used to present the pattern of tracheal branches, the gift of Dr. Kornberg (49). The Decoy-mir-274 construct was generated as described (50). In brief, decoy miR-274-binding sites were synthesized by PCR to assemble multiple copies of the Decoy-linker-mir-274 cassette. Three copies of the Decoy-linker-mir-274 cassette were integrated into the pUAST vector (Drosophila Genomics Resource Center) to generate the Decoy-mir-274 plasmid for producing transgenic lines.
Immunostaining, microscopy and image processing
Dissected third instar larval fillets were fixed and stained as described (51). Primary antibodies used in this study were goat anti-HRP-conjugated TRITC (1:100; Jackson Lab), rabbit anti-HRP-conjugated 647 (1:100; Jackson Lab), chicken anti-GFP (1:500; Abcam), mouse anti-Dlg (1:100; Developmental Studies Hybridoma Bank (DSHB)), mouse anti-activated MAP kinase (diphosphorylated ERK-1&2, 1:20; Sigma-Aldrich), and rabbit anti-Sty (1:50; gift of Dr. Krasnow (33)). Muscles were visualized by TRITC-conjugated phalloidin (1:2000; Sigma-Aldrich). Tracheal nuclei were visualized by DAPI (1:5000; Sigma-Aldrich). Goat anti-chicken (Invitrogen) or mouse Alexa Fluor 488-(Jackson Lab), goat anti-rabbit Cy5-(Jackson Lab) and goat anti-mouse Cy5-conjugated (Jackson Lab) secondary antibodies were used at 1:500 dilutions. Z-stack confocal images were obtained by Zeiss LSM510 or LSM 710 microscopy and processed by Imaris 8.4.1 (Bitplane).
Fiji (https://fiji.sc/) was used to quantify the signal intensities of Sty and dpERK in synaptic boutons, tracheal branches or cell nuclei. In brief, fluorescent integrated densities of Sty or dpERK were measured within the regions of interest (ROI), i.e., HRP-labeled synaptic boutons, GFP-labeled tracheal branches, or DAPI-labeled tracheal nuclei. The integrated densities were normalized to background readings of respective images.
For electron microscopy imaging, ultrastructures of dissected third instar larvae were processed as described to reveal glia-neuron-trachea organization (51).
Fluorescent in situ hybridization (FISH)
Wandering larvae were dissected to perform EDC-fixed FISH as described (52–54). To detect precursor miR-274, we designed a 5’-biotin-labeled probe complementary to the loop sequence. To design a probe for mature miR-274 and a scramble probe, we used a 5’-DIG-labeled miRCURY LNA probe complementary (or scrambled) to the stem sequence (Exiqon). After hybridization, samples were blocked in Western blocking (WB):PBT (1:1) solution (Sigma-Aldrich) for 1 hr and incubated overnight with sheep anti-DIG-POD primary antibody (1:1000, for the DIG-labeled probe; Roche) or mouse anti-biotin (1:1000, for the biotin-labeled probe; Abcam) in the WB:PBT solution at 4 °C. Samples with the biotinylated probe were incubated for 2 hr in biotinylated donkey anti-mouse secondary antibody (1:500; Jackson Lab) in WB:PBT solution, washed three times in PBS for 10 min, and then incubated in HRP-labeled avidin-biotin complex (ABC; Vector Laboratories) solution for 1 hr. After washing three times in PBT, each time for 10 min, all samples were incubated in TSA Plus Cyanine5 Evaluation kit (1:50; PerkinElmer) for signal amplification at room temperature for 1 hr with protection from light. Finally, the samples were washed six times in PBT, each time for 5 min. To double-stain proteins of interest, we incubated the samples in 0.1% hydrogen peroxide in PBT for 10 min to eliminate enzymatic activity in the TSA Plus Cyanine5 Evaluation kit, followed by standard immunostaining procedures (51).
Exosome isolation and Western blot
Exosome fractions were isolated from Drosophila S2 cell culture (2 × 106 cells/ml) medium and the hemolymph of 50 to 100 larvae. In brief, the S2 cell culture medium or larval extracellular fluid was centrifuged at 300 g for 5 min, the supernatant was next spun at 2,000 g for 10 min and then at 10,000 g for 30 min to remove large cell debris. Exosomes were then collected following the manufacturer’s instructions for the ExoQuick™kit (System Biosciences). For Western blot analysis, we homogenized exosome fractions in RIPA lysis buffer with protease inhibitor cocktails. Total protein (40 µg) was loaded in each lane of 4-12% Nupage Bis-Tris gels. We used mouse anti-TSG101 (1:1000; Abcam), mouse anti-Syntaxin (1:1000; DSHB), mouse anti-Rab11 (1:1000; BD Biosciences) and horseradish peroxidase conjugated anti-mouse IgG (1:5000; Jackson Lab) for immunostaining.
RT-PCR and real-time PCR
Total RNA was extracted form larvae or exosomal fractions using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. SuperScript™ IV VILO™ Master Mix (ThermoFisher Scientific) was used according to the manufacturer’s instructions to detect mRNAs. To detect miR-274, a SuperScript™ III Reverse Transcriptase (ThermoFisher Scientific) protocol was performed according to the manufacturer’s instructions with the stem-loop RT primer that contains six nucleotides complementary to mature miR-274 3’ sequences. For each reverse transcription reaction, 2 µg RNA was used. Phusion Green High-Fidelity DNA Polymerase (ThermoFisher Scientific) was used to perform RT-PCR with specific primer sets for miR-274 and Ephrin (Figures 4A, 5A and S4). To measure sty mRNA levels, we performed real-time PCR in 96-well plates using the LightCycler® 480 system. All mRNA transcripts were normalized to Rpl19 transcript levels. To measure miR-274 levels (Figures 4B, 5B and S5C), we conducted absolute qPCR by generating 8-point standard curves with miR-274 oligonucleotides in the range of 1 μM to 10−7 μM. Amounts of miR-274 were normalized to each respective GAL4 control group and are shown as “fold change”. All primers were listed in Table 1.
RNA sequencing
Total RNA was extracted from wandering female larvae of w1118 and mir-274KO using a miRNeasy Mini Kit. Libraries were prepared with a TruSeq Stranded mRNA LT Sample Prep Kit for Illumina sequencing. We selected genes in the mir-274KO mutant with expression levels 1.5-fold those of w1118.
Luciferase construct and assay
Oligonucleotides targeting predicted sequences of miR-274 and the mismatch control at the sty 3’-UTR were synthesized and inserted into the NheI/XhoI multiple cloning site of pmirGLO Dual-Luciferase miRNA target expression vector (Promega). Three plasmid constructs were generated: (1) without 3’-UTR sequences; (2) with wild-type 3’-UTR sequences; and (3) with mismatched 3’-UTR sequences. These plasmids (1 µg each) were transfected by the TransIT system (Mirus) into 2×106 S2 cells that were plated in 6-well plates one day before analysis. Dual-luciferase reporter assays (Promega)were performed 48 hr after transfection according to the manufacturer’s instructions and measured by an EnSpire® Multimode Plate Reader.
Behavioral assay
Early- to mid-third instar larvae (AEL 72-96 hr) were used for the following behavioral assays.
Larval crawling assay
Individual larva was transferred to a 15 cm petri dish with 2% agarose for 1 min habituation. Activity was then video-recorded for 1 min using a Canon N90 camera and analyzed by Fiji with the wrMTrck plugin (55).
Feeding motivation assay
Larvae that were either well fed or food-deprived for 2 hr were transferred to a nutritious yeast or non-nutritious grape juice plate. Mouth-hook contractions of individual larvae were counted under dissecting microscopes for 1 min.
Hypoxia escape response
Hypoxia escape behavior was assessed as described (38), but with the following modifications. We used 10 larvae that had burrowed for 10 min into yeast paste applied to a grape juice agar plate. The assay was performed in a plexiglass chamber (20 × 10 × 10 cm) with O2 levels of 21%, 10% or 1% regulated by N2 infusion, which were monitored and controlled by a Proox 110 (BioSpherix) compact oxygen controller. Activity was video-recorded for 20 min using a Canon N90 camera and the number of larvae that moved beyond the yeast paste was counted at 5, 10, and 15 min.
Statistical analysis
Graphpad Prism v6 (Graphpad) was used to perform statistical analyses. All data are expressed as mean ± SEM. The threshold for statistical significance was set at p < 0.05, with * for p < 0.05, ** for p < 0.01, *** for p < 0.001. Two-way ANOVA was used to analyze feeding motivation assay data. One-way ANOVA followed by Tukey post hoc tests were used to analyze data for three or more genotypes. Independent t-tests were used where appropriate.
Acknowledgements
We thank M. Krasnow, T. Kornberg, Y.-W. Chen, Bloomington Stock Center and Vienna Drosophila Resource Center for providing antibodies and fly stocks, the Genomics Core and Bioinformatics-Biology Service Core in Academia Sinica for performing NGS and respective analysis, and the Imaging Core in the Institute of Molecular Biology of Academia Sinica for supporting image processing by Imaris. This work was supported by grants from the Ministry of Science and Technology (MOST) and Academia Sinica to C.T.C and MOST-104-2633-B-029-001, MOST-105-2633-B-029-001 to Y.C.T. The authors declare no biomedical financial interests or potential conflicts of interest.