Feedback regulation of cytoneme-mediated transport shapes a tissue-specific FGF morphogen gradient
Diverse tissue shapes and patterns are created by conserved paracrine signaling proteins such as Transforming Growth Factor β ( TGFβ ) , Hedgehog ( Hh ) , Wingless ( Wg/WNT ) , Epidermal Growth Factor ( EGF ) and Fibroblast growth factor ( FGF ) . Irrespective of the diversity of the functions and forms created , a universal mode by which these signals coordinate responses in a large population of cells involves the generation of concentration gradients . For instance , morphogen gradients induce concentration-dependent gene activities that regulate tissue growth and patterning ( reviewed in ( Bier and De Robertis , 2015; Christian , 2012; Restrepo et al . , 2014; Rogers and Schier , 2011; Wolpert , 2016 ) ) . Similarly , growth factor and chemokine gradients are thought to control cell migration during vasculogenesis , neurogenesis , wound healing , and immune responses ( Cai and Montell , 2014; Majumdar et al . , 2014 ) . Despite advances in our understanding of signal transduction pathways , how signals disperse and how the dispersion mechanism is dynamically modulated to shape gradients in three-dimensional tissue structures are poorly understood . Moreover , the formation of signal gradients is unexplored in most morphogenetic contexts , so we do not know how dispersion of the signals through extracellular space can generate the required diversity in gradient shapes and contours for a multitude of tissue architectures . Several models primarily based on free/restricted/facilitated extracellular diffusion of signals were proposed previously to explain the establishment of signal gradients , but the proposed mechanisms are highly controversial ( Majumdar et al . , 2014; Müller et al . , 2013; Wolpert , 2016 ) . Recent advancements in live imaging technology facilitated the discovery of a radically different mechanism of signal dispersion , where signals move directly from producing to recipient cells through actin-based specialized filopodia or cytonemes ( Bischoff et al . , 2013; Eom and Parichy , 2017; Roy et al . , 2011b; Roy et al . , 2014; Sanders et al . , 2013; Stanganello and Scholpp , 2016 ) . Cytonemes and cytoneme-like projections are now known to be required for cell-cell communication mediated by many families of signaling proteins ( Kornberg , 2014 ) . For instance , a cytoneme-dependent mechanism was shown to be essential for generating concentration-dependent signaling responses to the Hh and Dpp morphogens ( Bischoff et al . , 2013; Chen et al . , 2017; Roy et al . , 2014 ) . However , how local cytoneme-mediated cell-to-cell communication might create long-range signal gradients to regulate tissue morphogenesis was unknown . In this study , we aimed to address these fundamental questions by characterizing gradient formation of a Drosophila FGF family protein , Branchless ( Bnl ) . In Drosophila , Bnl is the primary signal that guides the branching morphogenesis of tracheal epithelial tubes ( Sutherland et al . , 1996 ) . At the third instar larval stage , Bnl produced from a restricted group of wing imaginal disc cells induces budding and growth of a wing disc-associated tracheal branch , the air-sac primordium ( ASP ) , from the disc-associated transverse connective ( TC ) ( Figure 1A ) ( Sato and Kornberg , 2002 ) . ASP tracheoblast cells are precursors of adult air-sac , an organ that is analogous to the vertebrate lung . Traditionally , all paracrine signals such as FGF family proteins are thought to function as diffusible signals and form a gradient by passive extracellular dispersion . In accordance with the same line of thinking , one hypothesis predicts that Drosophila Bnl diffuses from its source to form an extracellular gradient and that the gradient guides directional migration of the tracheal branches such as the ASP ( Horowitz and Simons , 2008; Ochoa-Espinosa and Affolter , 2012 ) ( Figure 1A’ ) . However , whether Bnl forms a gradient and if so , how Bnl is transported and modulated in the extracellular space to form a gradient remain unexplored . Moreover , all tracheal cells express the Bnl receptor , Breathless ( Btl ) , and develop under the guidance of the same Bnl signal , but different embryonic and larval tracheal branches adopt different developmental stage-specific morphologies ( Sato and Kornberg , 2002; Sutherland et al . , 1996 ) . We do not understand whether and how the shape of a Bnl gradient can dynamically adapt to diverse tissue morphologies . To investigate these important gaps in knowledge , we chose to focus on the Drosophila larval ASP because of its unique features . In this system , the wing disc bnl-source is spatially separated from the recipient ASP epithelium ( Figure 1A” ) . Bnl signals exclusively to the ASP because the wing disc cells and myoblasts located between the disc and the ASP do not express its receptor , Btl . Moreover , the two additional Drosophila FGF family proteins , Pyramus and Thisbe , do not share receptors with Bnl ( Sato and Kornberg , 2002; Stathopoulos et al . , 2004 ) . This system where there is a clear inter-organ communication path and a single Bnl-specific receptor exclusively expressed in the ASP is ideal for an unbiased interpretation of the extracellular route of Bnl transport and gradient formation . Genome-edited Bnl:GFP and Btl:Cherry constructs generated in this study revealed Bnl distribution with high sensitivity and precision , allowing direct visualization of an endogenous signal and signaling gradient . We showed that Bnl moves target-specifically from the producing cells to the ASP through cytonemes and forms a long-range concentration gradient by dynamically adopting the recipient ASP-specific shapes and contours . We also demonstrated that Bnl functions as a morphogen and , most importantly , we uncovered a novel self-regulatory mechanism of cytoneme-mediated signaling by which the Bnl morphogen gradient forms . Thus , these findings showed an example of morphogenesis in which cytoneme-dependent signaling can provide precision and adaptability in shaping long-range positional gradients and tissue architectures in space and time .
To visualize Bnl dispersion without affecting normal tracheal morphogenesis , we employed the CRISPR/Cas9-based genome-editing technique to generate flies that harbor an in-frame insertion of a superfolder-GFP ( sfGFP ) sequence within the third coding exon of the bnl gene ( Figure 1B; Figure 1—figure supplement 1A–C; Materials and methods ) . Animals that harbored the bnl:gfpendo knock-in allele were homozygous viable , had normal tissue morphology , and the expected gene expression patterns ( Du et al . , 2017; Sato and Kornberg , 2002 ) . Under sensitive confocal microscopy ( see Materials and methods ) , endogenous Bnl:GFP molecules were visualized as fluorescent puncta ( Figure 1C , C’ ) . The distribution patterns of these Bnl:GFP puncta revealed several unexpected features . In the wing disc source cells that were marked by CherryCAAX expression , Bnl:GFP puncta were faint , hardly detectable , and homogeneously distributed ( Figure 1C–D’ ) . As a paracrine signal , Bnl was expected to disperse and form a continuous gradient surrounding the wing disc source ( Figure 1A’ ) . However , all of the detectable bright fluorescent Bnl:GFP puncta outside of the source cells were asymmetrically distributed only in the recipient ASP cells ( Figure 1C–D’ ) . Thus , although Bnl:GFP moved from the wing disc source to the ASP , the punctate signal did not localize in the non-specific wing disc cells surrounding the signal source or in the myoblast cells located between the disc source and the ASP ( Videos 1 and 2 ) . To ensure that the GFP-marked puncta in the ASP were actual Bnl molecules , we performed an immunohistochemistry assay ( IHC ) with αBnl antibody on the homozygous bnl:gfpendo larval tissues . The Bnl antibody recognized all of the Bnl:GFP puncta in the larval ASP , confirming that they represent the Bnl protein ( Figure 1—figure supplement 2A , A’ ) . To further validate whether the Bnl:GFP distribution in the ASP represented a functional distribution of the ligand , we knocked-down bnl:gfpendo expression in the wing disc source . Since Bnl is essential for tracheal/ASP growth ( Sutherland et al . , 1996; Sato and Kornberg , 2002 ) , loss of a functional Bnl:GFP distribution was expected to abrogate ASP development . As expected , overexpression of bnlRNAi from the disc bnl-source in the bnl:gfpendo larvae knocked down both Bnl:GFP expression and ASP growth in most samples , indicating that the tagged signaling protein is functional ( Figure 1—figure supplement 2B–C ) . However , due to incomplete RNAi-mediated knock-down of bnl:gfpendo , a few samples still contained low levels of Bnl:GFP . Importantly , these samples also had small , stunted ASPs with a shallow range of Bnl:GFP distribution . This correlation between Bnl:GFP levels/distribution and ASP growth suggested that the levels and distribution of Bnl:GFP in the ASP have a significant developmental role ( Figure 1—figure supplement 2B–C ) . Contrary to what was predicted earlier ( Figure 1A’; ( Horowitz and Simons , 2008; Ochoa-Espinosa and Affolter , 2012 ) ) , our imaging results ( Figure 1C–D’ ) suggested that a pre-existing Bnl:GFP gradient surrounding the wing disc signal source was not formed . Instead , the Bnl:GFP puncta appeared to distribute in a graded pattern along the distal-to-proximal ( D-P ) axis of the recipient ASP ( Figure 1D , D’ ) . Measurement of Bnl:GFP intensities along the longest D-P axis of the late third instar larval ASPs revealed a long-range exponential gradient spanning ~100–120 μm in range . The distal ASP tip , which is nearest to the disc bnl-source , received the highest levels of Bnl:GFP ( Figure 1E ) . The signal concentration decreased exponentially toward the proximal ASP stalk as the distance from the source increased . Thus , although Bnl:GFP is produced in the wing disc , its gradient is formed in the recipient ASP . The Bnl:GFP gradient shapes were variable , and developed concomitantly with the growth of the recipient ASP ( Figure 1F ) . In the early third instar larval stage ( 18–26 hr after third instar larval molt ) , the ASP was hardly detectable . At this stage , only a few TC cells proximal to the bnl-source had received Bnl:GFP . In the later stages , as the ASP budded from the TC and then elongated , the Bnl:GFP gradient shapes adopted the variable shapes of the recipient ASP . These results suggested that the shape of the Bnl:GFP gradient is adaptable and its formation is coordinated with growth of the recipient ASP . We further examined the relationship of the slope of the Bnl:GFP gradient with the length of the D-P axis of the recipient ASP . In the shorter early third instar larval ASPs ( ~35–40 hr after third instar larval molt ) , Bnl:GFP formed relatively steeper and narrower gradients ( slope of a straight line joining Cmax to C1/2max =~3 . 14 ) compared to the larger/longer late-stage ASPs ( slope =~0 . 71 ) ( Figure 1E , G ) . A strong negative correlation of the gradient slopes with the length of the ASP D-P axis was also observed in a population of randomly harvested wandering third instar larval ASPs , which showed natural developmental variations in size ( Figure 1H ) . These results showed that the range and slope of the Bnl:GFP gradients are dependent on the ASP morphology and that the gradient did not pre-exist , but instead developed dynamically in coordination with the recipient tissue morphogenesis . Although 2D Z-projected images were sufficient to visualize the gradient in the X-Y plane , the ASP epithelium has a complex three-dimensional tubular morphology . As shown in the cross-section diagram of the ASP and wing disc in Figure 1A” , the relative distance of the ASP cells from the disc source can be measured along the D-P axis as well as in the upper and lower layers across the vertical Z-axis . Across the Z-axis , most of the cells in the upper ASP layer ( ventral face ) were situated away from the underlying bnl-source . In spite of the physical distances , Bnl:GFP distribution was not restricted to the ASP cells that were immediately juxtaposed to the bnl-source . Instead , Bnl:GFP distributed in both layers formed a continuous long-range gradient , adopting the complex 3D tubular contour of the epithelium ( ~300 μm long , equivalent to 30 cells in just a single medial cross-section ) ( Figure 1I–I” ) . When we probed for the expression of a Bnl-signaling target gene pntP1 , an ETS family transcription factor ( Sutherland et al . , 1996 ) , overlapping signal and signaling gradients were revealed ( Figure 1I–J , Figure 1—figure supplement 2D–E” ) . Interestingly , in a digitally straightened continuous epithelium , the signal and signaling gradients had a bilateral symmetric distribution , where the positions with highest concentrations corresponded to the ASP cells nearest to the disc bnl-source ( Figure 1I’ , I” , J ) . Tubular contours of the ASP epithelium were variable among different samples , but irrespective of the differences in 3D morphologies , the Bnl:GFP distribution consistently adopted the recipient ASP-specific contours ( Figure 1—figure supplement 2D–E” ) . Collectively , these results showed that formation of the Bnl gradient depends on the ASP growth , and that the shapes of the gradient can dynamically adapt to the changing morphologies of the growing tissues . A trachea-specific Bnl:GFP gradient might reflect a receptor-bound signal distribution . To examine this possibility , we created animals harboring a btl:cherryendo knock-in allele using genome-editing technology ( Figure 2A; Figure 2—figure supplement 1A–C; Materials and methods ) . As expected , Btl:Cherry expressed at physiological levels marked all tracheal cells , but formed a concentration gradient along the D-P axis of the ASP ( Figure 2B; Figure 2—figure supplement 1D ) . This observation is consistent with an earlier report describing activation of btl transcription by Bnl signaling ( Ohshiro et al . , 2002 ) . In trans-heterozygous btl:cherryendo/bnl:gfpendo animals , all the Bnl:GFP puncta in the ASP colocalized with Btl:Cherry and the receptor gradient coincided with that of the ligand gradient ( Figure 2C–D; Figure 2—figure supplement 1E , E’ ) . Most of the colocalized receptor-ligand puncta were compartmentalized apically in early- ( αRab5 IF; ~76% of total puncta/ASP examined ) and late- ( αRab7 IF; ~90% of total puncta/ASP ) endosomes , but not in recycling endosomes or lysosomes ( Figure 2E–E”’; Figure 2—figure supplement 2A–C ) . These results suggested that the branch-specific Bnl:GFP gradient is receptor-bound , and mostly intracellular . Notably , a small number of receptor-ligand puncta were found at the basal side of ASP cells ( arrows in Figure 2C , E ) . They were located mostly at the distal ASP tip cells and did not colocalize with any of the endosomal markers we examined ( Figure 2E ) . These puncta on the exposed basal side of the ASP might represent Btl:Cherry-bound Bnl:GFP molecules prior to their receptor-mediated endocytosis . Bnl molecules were expected to move through the extracellular space from the wing disc source to the ASP . To clearly visualize the extracellular distribution pattern of post-secretory Bnl:GFP prior to their endocytosis in the ASP , we employed a standard detergent-free immunofluorescence ( EIF ) protocol ( see Materials and methods ) . This protocol was specifically developed for detecting extracellular dispersion profiles of morphogens ( Schwank et al . , 2011; Strigini and Cohen , 2000 ) . An αGFP EIF assay identified a distinct steady-state surface-bound extracellular/externalized pool of Bnl:GFP molecules ( Figure 3A; henceforth referred as Bnl:GFPex ) . Interestingly , even though we used a highly sensitive Gallium arsenide phosphide detector for imaging ( see Materials and methods ) , endogenous levels of EIF-stained Bnl:GFPex puncta appeared to emit very weak GFP-fluorescence , which was rapidly quenched during high magnification ( 40X ) imaging . However , this property of GFP-tagged molecules enabled us to estimate the levels of externalized/extracellular Bnl:GFP ( Bnl:GFPex ) , distinguishing them from the brightly fluorescent but non-EIF stained intracellular Bnl:GFP puncta in the ASP ( henceforth referred as Bnl:GFPin ) ( Figure 3A ) . Most importantly , this experiment showed that although the EIF-stained Bnl:GFPex molecules represented secreted signals in the extracellular space , they did not disperse randomly in all directions . Instead , they were asymmetrically localized and only detected on the surfaces of signal producing cells and of the recipient ASP ( Figure 3A , A’ ) . Bnl:GFPex were highly enriched on the ASP and appeared to form a gradient on the recipient surface , in exactly the same way as the endocytosed Bnl:GFPin molecules ( Figure 3A” ) . In contrast to the asymmetric Bnl:GFPex distribution , a secreted-GFP construct containing a sfGFP tag and an N-terminal signal peptide derived from the Bnl sequence ( secGFP; see Materials and methods ) randomly dispersed in the extracellular space surrounding the disc bnl-source ( Figure 3B–B”; Figure 3—figure supplement 1A ) . These results showed that Bnl:GFP moved target-specifically in polarized fashion . The polarized distribution pattern of Bnl:GFPex was consistent with a potential cytoneme-mediated direct transport of the molecules . Previous studies showed that ASP cells project receptor-containing cytonemes to establish direct membrane contacts with the wing disc Bnl source . These cytonemes were shown to be essential for induction of pMAPK signaling in the ASP ( Roy et al . , 2014 ) . However , Bnl transport through cytonemes was never visualized before . To examine if Bnl:GFPex molecules localized on the surface of the ASP cytonemes , we carried out EIF assays with either αGFP or αBnl antibody on CD8:Cherry-marked ASPs of bnl:gfpendo homozygous larvae . These assays showed that the EIF-stained Bnl:GFPex puncta were highly enriched on cytonemes emanating from the distal ASP tip ( N > 50 ASP , Figure 3C–E; Figure 3—figure supplement 1B–B” ) . However , as observed earlier , many of these puncta were poorly fluorescent under standard confocal fluorescence imaging conditions . Therefore , to clearly visualize the endogenous Bnl:GFP molecules on cytonemes , we employed an enhanced gain super-resolution detection method ( see Materials and methods ) . This imaging method revealed that the surfaces of the ASP and ASP cytonemes were densely populated with a high concentration of Bnl:GFP molecules , which appeared as small sub-resolution-sized puncta of <200 nm in diameter ( henceforth referred as nanopuncta due to their sub-resolution size; arrowheads in Figure 3F–F” , Figure 3—figure supplement 2A–A”’ ) . These nanopuncta were also enriched on the source cells , especially around the bouton-like sites of contact between the cytonemes and the source ( Figure 3F ) . An αGFP EIF staining assay in combination with super-resolution imaging showed that these Bnl:GFP nanopuncta included both surface-bound molecules ( EIF-stained ) and intracellular molecules ( non-EIF stained ) ( Figure 3—figure supplement 2B , B’ ) . Occasionally , several nanopuncta aggregated to form bright punctal clusters on the cytonemes , the fluorescence of which was likely to be easily detectable under our standard confocal fluorescence imaging conditions ( arrows in Figure 3F’ , F” ) . Although the molecular and cellular nature of the nanopuncta and their clusters on the cytonemes are unclear , these results provided substantial evidence for a selective distribution of Bnl:GFP molecules along the surface of cytonemes . Native , untagged Bnl could also be detected on cytoneme surfaces using the αBnl antibody . The αBnl EIF assay showed that a high concentration of externalized Bnl ( Bnlex ) molecules selectively decorate actin-rich ASP cytonemes and the ASP surface of w- wild-type larvae ( Figure 3G ) . The distribution of Bnlex was asymmetrical , exactly as observed for Bnl:GFP on CD8:Cherry-marked ASPs ( Figure 3C ) . These results suggested that Bnl molecules , irrespective of the presence or absence of fluorescent tags , specifically localize on the surface of the ASP cytonemes for inter-organ transport . Since efficient inter-tissue movement of Bnl molecules along the surface of the recipient cytonemes might require receptor binding , we imaged ASP cytonemes in btl:cherryendo larvae . Densely packed Btl:Cherry molecules were visualized on the surface of the ASP cytonemes ( Figure 3—figure supplement 3A ) . An αBnl-EIF assay in btl:cherryendo larvae showed that the native Bnlex puncta colocalized with the Btl:Cherry puncta on the cytonemes and on ASP surfaces ( Figure 3H ) . Receptor-bound Bnl:GFP puncta were also observed on cytonemes during live imaging ( Figure 3—figure supplement 3B–C’; N = 30 ) . Thus , Bnl is transported by cytonemes from the source to the ASP in a receptor-bound state . Cytonemes exchange signals by establishing direct physical contacts between the source and recipient cells ( Chen et al . , 2017; González-Méndez et al . , 2017; Huang and Kornberg , 2015; Roy et al . , 2014 ) . These signaling contacts between the ASP and the bnl source were originally discovered using a membrane GFP-reconstitution method ( GFP reconstitution across synaptic partners , GRASP ) ( Huang and Kornberg , 2015; Roy et al . , 2014 ) . As shown in Video 3 , an improved GRASP technique , ‘Synaptobrevin-GFP-reconstitution-across-synaptic-partners ( syb-GRASP ) ’ revealed multiple functional contact sites between each of the ASP cytonemes and the bnl-source . To estimate the contact-dependency of the cytoneme-mediated Bnl reception , we combined the syb-GRASP experiment with the αBnl EIF assay . This experiment showed that the actin-rich ( phalloidin-stained ) ASP cytonemes that contacted the disc bnl-source received a significantly higher number of Bnlex puncta in comparison to the non-contacting cytonemes ( Figure 3I , I’ ) . These observations , together with the previous reports on the effect of loss of cytoneme contacts on pMAPK signaling in the ASP ( Roy et al . , 2014 ) , provided substantial evidence that cytoneme-mediated Bnl exchange is contact-dependent . Molecular reconstitution of split GFP between ASP cytonemes and source cells suggested that the signaling contacts between the cytoneme and source cell membranes juxtapose within a 20 nm gap distance ( Kornberg and Roy , 2014 ) . To directly visualize and resolve Bnl exchange at these cytoneme contacts , an αBnl EIF assay was combined with super-resolution imaging ( see Materials and methods ) of the tissues that had both the source and recipient ASP cells marked with distinct membrane-tagged proteins ( Figure 3J ) . The images revealed that ASP cytonemes traverse along the surface of the source cells , with each cytoneme establishing contacts with multiple signal-producing cells . Although the producing cells occupy a large area , the externalized Bnl proteins were specifically enriched at the cytoneme contact sites . This observation suggests that the producing cells selectively release Bnl at these contact sites . These ASP cytonemes also localized Bnlex puncta on their shafts , suggesting that the signal might be transported from the contact sites to the ASP along the surface of the cytonemes . A similar retrograde movement of Dpp was previously observed through ASP cytonemes ( Roy et al . , 2014 ) . To examine whether cytoneme-mediated reception of Bnl:GFP is essential for generating the long-range Bnl:GFP gradient in the ASP , we generated mosaics of marked cytoneme-deficient mutant clones within the ASP of bnl:gfpendo larvae by knocking down several known cytoneme-regulator genes such as diaphanous ( dia ) , a formin homolog , neuroglian ( nrg ) , an L1-cell adhesion molecule ( Roy et al . , 2014 ) , and singed ( sn ) , a Fascin homolog and a regulator of tracheal filopodia ( Okenve-Ramos and Llimargas , 2014 ) . To assess Bnl reception in the mutant clones , we compared Bnl:GFP levels within the mutant area ( inside of the clone ) with that in the wild-type ( WT ) neighboring areas at an equivalent position ( outside of the clone ) in the same ASP . The ratios of Bnl:GFP concentration outside to inside of the marked wild-type ( WT ) control clones were ~1 , suggesting no significant differences in the signal uptake within the marked ( inside ) and unmarked ( outside of the clone ) WT areas ( Figure 4A , E ) . However , knockdown of cytoneme-regulator genes in clones of cells significantly decreased Bnl:GFP levels in the mutant cells , but not in the neighboring WT cells ( Figure 4B–E; Figure 4—figure supplement 1A ) . Consequently , the ratio of Bnl:GFP concentration outside ( WT area ) to inside ( cytoneme deficient ) of the mutant clones increased significantly compared to the WT control condition ( Figure 4E ) . These results showed that cytonemes are essential for the ASP cells to receive Bnl:GFP from the wing disc source . The distribution profile of Bnl:GFP in btl-LOF clones further suggested that cytonemes are the only major routes for Bnl gradient formation . As illustrated in Figure 4F , F’ , a cytoneme-mediated mechanism of dispersion , which is receptor-dependent , would create cell-autonomous loss of receptor-bound Bnl:GFPex on the surface of a btl-LOF clone . This predicted distribution pattern is different from that of a passive extracellular Bnl:GFP diffusion , which would be expected to result in unperturbed levels of the extracellular EIF-stained Bnl:GFPex on the receptor-LOF clone . Indeed , RNAi-mediated knockdown of btl in clones of ASP cells led to a significant reduction of Bnl:GFPex on the mutant clonal areas compared to the WT neighboring cells ( Figure 4G , G’ ) . Clearly , the distribution pattern of Bnl:GFPex was consistent with a cytoneme-dependent mechanism of gradient formation . Note that an autonomous reduction in intracellular Bnl:GFP ( non-EIF stained ) was also observed in the mutant clones , but such reduction in intracellular Bnl:GFP levels was expected irrespective of the mechanism of signal dispersion , and did not affect our conclusions . Furthermore , in the ASP stalk , where normal levels of Btl are low , generation of btl overexpressing ( btl-GOF , gain of function ) clones induced extension of long polarized cytonemes that projected toward the underlying disc bnl-source . These cytonemes were enriched with EIF-stained Bnl:GFPex on their surfaces ( Figure 4H , H’ ) and the mutant clones had a higher concentration of Bnl:GFPex than their neighbors ( Figure 4—figure supplement 1B ) . This result provided direct evidence that the induction of cytonemes in ASP cells increased cell-autonomous contact-dependent Bnl:GFP uptake . How might cytoneme-mediated local signaling in each ASP cell generate a long-range signal gradient across the entire ASP epithelium ? One possibility is that tracheal cells , relative to their distances from the bnl-source , produce different numbers of Bnl-specific cytonemes and/or establish variable numbers of signaling contacts with the disc bnl-source . To examine this possibility , we live imaged randomly generated CD8:GFP-marked clones in different positions of the ASP and analyzed the orientation and number of cytonemes with different lengths they extend ( Figure 5A–A” ) . In addition , to compare the frequency of signaling contacts among cytonemes emanating from different regions of the ASP , we devised a mosaic analysis method for generation of clonal syb-GRASP . We expressed syb-GFP1-10 in the bnl-source and induced small ( ~1–4 cells ) randomly localized CD8:RFP-marked clones across the ASP epithelium that also expressed CD4:GFP11 ( Figure 5D , D’; see Materials and methods ) . In this technique , when cytonemes emanating from the clones in different parts of the ASP established contact with the disc bnl-source , GFP was reconstituted at their contact sites . For assessing the steady-state number of contacts established by different parts of the ASP with the signal source , ASPs containing only 1–2 small clones were considered . These experiments showed that the stalk region of a third instar larval ASP , beyond 7 cell-distances along the D-P axis ( ~12 cells long ) from the ASP tip ( Figure 1A” ) , produced only randomly oriented short cytonemes . These cytonemes rarely contacted the bnl-source ( Figures 5A , B , C , D–D” ) . This region also received lower levels of Bnl:GFP ( Figure 1A” , E ) . The number of long polarized cytonemes that oriented toward the bnl-source and established direct contacts with the source increased gradually in the mid ( 4th-7th cells from the ASP tip along the D-P axis ) and the distal tip cells ( 1–3 cells from the tip along the D-P axis ) ( Figure 5A’ , A” , B , C , D' , D"; Figure 5—figure supplement 1A–A’’ ) . Thus , long oriented cytonemes that could establish contacts with the Bnl source were produced in a graded fashion across the length of the ASP . A similar gradient in the number of signaling contacts was also observed from cytonemes produced across the vertical axis of the tubular tissue . In a syb-GRASP experiment between the disc and ASP , ASP cells in the lower layer that is proximal to the bnl-source had a higher frequency of cytoneme contacts compared to the upper cell layer ( Figure 5—figure supplement 1B; Videos 3 , 4 and 5 ) . These results suggested that differential levels of signal uptake through a graded number of cytonemes give rise to the tissue-specific shapes of the Bnl:GFP gradient . How does a steady state pattern of cytonemes across the ASP epithelium develop ? The gradient of numbers of Bnl-receiving cytonemes correlated with the spatial gradient of Bnl ( Figures 5A–B;1E , I; 2D ) . Thus , the zones of variable numbers of ASP cytonemes might reflect differential target gene activities in response to the Bnl gradient . Therefore , we examined the correlation of the Bnl:GFP gradient with the spatial expression domains of several known Bnl target genes , including pntP1 and sprouty ( sty ) ( Hacohen et al . , 1998; Ohshiro et al . , 2002 ) . We also examined expression of the Ets family transcriptional repressor yan , which is suppressed by Ras/MAPK signaling ( Ohshiro et al . , 2002 ) , and of cut , a homeodomain transcription repressor . Although cut is not known to be a target of Ras/MAPK signaling , this gene was shown to be expressed in the larval spiracular branch and in the ASP ( Ohshiro et al . , 2002; Pitsouli and Perrimon , 2013; Rao et al . , 2015 ) . The D-P axis of a third instar larval ASP consists of ~12 cells . We found that ASP cells within a three-cell diameter domain from the distal tip along the D-P axis , which received the highest concentration of Bnl:GFP , expressed sty ( Figure 6A , E; Figure 6—figure supplement 1A; Figure 6—figure supplement 2A , B ) . A broader zone of ~6–7 cell-diameters from the tip along the D-P axis received high-to-moderate levels of Bnl:GFP and induced pntP1 ( Figure 6B , E; Figure 6—figure supplement 1A; Figure 6—figure supplement 2C , D ) . In contrast , the proximal stalk of the ASP from the 7th to 12th cell away from the tip along the D-P axis received negligible levels of Bnl:GFP . In this ASP domain , cut and yan expression gradually increased with increasing distance from the source ( Figure 6C–E; Figure 6—figure supplement 1A; Figure 6—figure supplement 2E-H ) . Thus , the gradients of cut and yan expression had the exact opposite pattern as the pntP1 and Bnl:GFP gradients . This concentration-dependent activity was also observed across the Z-axis ( Figure 1A” ) . ASP cells in the lower Z-sections that are proximal to the underlying bnl-source expressed higher levels of sty and pntP1 relative to the upper layer cells situated away from the disc source ( Figure 6F; Figure 6—figure supplement 1B , C ) . Thus , across the recipient ASP epithelium , high levels of Bnl:GFP correlated with sty expression , and gradually decreasing levels of Bnl:GFP further away from the source correlated with the expression zones of pntP1 , cut , and yan , respectively ( Figure 6E , F ) . Similar spatial patterning in the gene-expression zones of sty and different ETS family proteins is known to be induced by the vertebrate FGF8 morphogen in mouse neuroectoderm ( Toyoda et al . , 2010 ) . Previously Bnl was considered to be a chemoattractant ( Lebreton and Casanova , 2016; Ochoa-Espinosa and Affolter , 2012 ) , which can only elicit a binary signaling response . In contrast , our results suggested that Bnl activated multiple target genes at its various concentrations similar to a morphogen . To further examine whether the target genes were differentially expressed in response to different Bnl concentrations , we overexpressed cDNA-derived Bnl constructs either from the wing disc bnl-source or from small clones of cells within the recipient ASP . Remarkably , even while highly expressed from the wing disc source , Bnl:GFP moved only target-specifically from the disc source to the ASP ( Figure 6H , H’ ) . In comparison to the normal range of endogenous Bnl:GFP distribution and Bnl ( pMAPK ) signaling ( Figure 6G ) , the spatial range of the overexpressed signal and the signaling expanded to all parts of the ASP , including the stalk ( Figure 6H ) . The expanded range of signal led to retraction of the Cut and Yan expressing zone from within the stalk to only the farthest region in the TC , which received little or no Bnl:GFP from the disc source ( Figure 6H–I ) . Moreover , ectopic clones of either Bnl:GFP or Bnl in the ASP stalk non-autonomously induced sty and pntP1 and suppressed cut in the surrounding cells ( Figure 6J , K , L; Figure 6—figure supplement 1D ) . These results showed that Bnl acts as a morphogen to induce concentration-dependent expression of different genes . It was previously reported that PntP1 and Yan antagonize each other’s expression ( Ohshiro et al . , 2002 ) . To examine whether Cut and PntP1 also antagonize each other , we induced ectopic pntP1 and cut mutant clones in the ASP . Reduction of PntP1 levels in the mutant pntP1 LOF ( pntP1 RNAi ) clones in the ASP tip autonomously upregulated Cut expression within the clones , whereas overexpression of PntP1 in the ectopic pntP1 GOF clones in the ASP stalk suppressed cut in the mutant cells ( Figure 6M , N ) . On the other hand , ectopic cut GOF clones in the ASP tip suppressed pntP1 and cut LOF clones ( cut RNAi ) in the proximal ASP stalk induced pntP1 within the mutant cells ( Figure 6O , P ) . Thus , PntP1 and Cut reciprocally inhibit each other’s expression . Notably , although PntP1 is a known inducer of btl , pntP1 GOF/LOF clones showed only cell-autonomous effects on cut . The btl GOF clones in the ASP showed a similar autonomous signaling effect ( Figure 6—figure supplement 1E ) . In contrast , each Bnl:GFP GOF clone in the ASP induced non-autonomous effects and organized the surrounding cells to form a new branch ( N = 35; Figure 6K , L ) . All these results provided evidence that Bnl acts as a classical morphogen to induce sty at a high level and pntP1 , cut , and yan gradually at lower levels in the ASP ( Figure 6E , F ) . To examine whether the different target genes of Bnl signaling may regulate cytoneme formation , we focused on pntP1 and cut , the two transcription factors that formed counteracting gradients from two opposing poles of the ASP and antagonized each other’s expression ( Figures 6B , C , E , M–P ) . When we induced ectopic cut GOF clones in the distal tip/mid-ASP regions , the mutant cells produced significantly fewer long and oriented cytonemes compared to the wt clones at an equivalent position ( Figure 7A , A’ versus Figure 5A’’ , C ) . This cytoneme deficiency in the cut GOF clones correlated with the autonomous reduction of Bnl:GFP uptake in the mutant clones from the mid/tip of the ASP ( Figure 7A–B’ ) . On the other hand , the orientation and number of cytonemes from the ASP stalk cells , which generally had high levels of Cut and produced randomly oriented short cytonemes , remained unaffected by cut GOF ( Figure 7A’ ) . Thus , high levels of Cut negatively regulate the formation of cytonemes that orient specifically toward the bnl-source . In addition to this finding , Cut was also known to be a suppressor of btl in the larval spiracular branch ( Pitsouli and Perrimon , 2013 ) . Therefore , high levels of Cut elicit negative feedback on Bnl signaling by suppressing both the Bnl-receiving cytonemes and Btl synthesis ( Figure 8A , A’ ) . A similar inhibitory effect on cytonemes was observed for Yan , which was a known antagonist for PntP1 and embryonic tracheal filopodia formation ( Okenve-Ramos and Llimargas , 2014 ) ( Figure 7—supplement figure 1A-B’; Figure 8A’ ) . Collectively , these results showed that cut and yan , the two genes that are expressed in the ASP stalk in response to lower Bnl levels , suppress cytoneme-formation and Btl expression . Generally , high-to-mid levels of Bnl signaling induced pntP1 in the distal tip-mid ASP region ( Figure 6B , E ) . When ectopic pntP1 GOF clones were generated in the ASP stalk , the mutant cells extended significantly more long ( >30 μm ) cytonemes oriented toward the underlying disc bnl-source than the WT clones ( Figure 7C , C’ , compared to Figure 5A , C ) . In contrast , pntP1-LOF clones at the ASP tip extended a significantly fewer number of long oriented cytonemes from the tip cells than the WT clones ( Figure 7D , D’ , compared to Figure 5A’’ , C ) . Loss of cytonemes in the pntP1 LOF clones correlated with the suppression of Bnl:GFP uptake in the mutant ASP cells , and an increase in the number of cytonemes in the pntP1 GOF cells increased the levels of Bnl:GFP in them ( Figure 7E–H ) . An αGFP EIF assay revealed that the long cytonemes projecting from the pntP1 GOF clones in the ASP stalk were enriched in EIF-stained Bnl:GFPex , suggesting that the cells received signal by extending cytonemes ( Figure 7G , H ) . In addition , PntP1 was known to induce btl transcription in embryonic trachea ( Ohshiro et al . , 2002 ) . When we generated small ( 1–4 cells ) pntP1 GOF clones in the stalk of the ASP in btl:cherryendo larvae , the PntP1 overexpression increased the levels of Btl:Cherry in the clones . Strikingly , the newly synthesized Btl:Cherry molecules also formed local concentration gradients within each of the pntP1 GOF clones ( Figure 7I ) . These results showed that PntP1 elicits positive-feedback on Btl synthesis and Bnl-receiving cytoneme production ( Figure 8A’ ) . Interestingly , PntP1 and Cut ( Figure 6M–P ) or PntP1 and Yan ( Ohshiro et al . , 2002 ) can reciprocally inhibit each other’s expression . Therefore , a ratio of the levels of PntP1 to Cut or to Yan could determine the number of cytonemes and Btl receptors in a cell . Based on our results , we propose that the counteracting activities of PntP1 and Cut/Yan and their expression from two opposite poles in response to the high-to-low Bnl levels in the ASP establish a steady-state gradient of numbers of Btl-containing cytonemes . Signaling through these cytonemes would self-generate the shape of a robust signal and signaling gradient ( Figure 8A’ ) .
Prior to this study , branching morphogenesis of tissues such as vertebrate lung or vasculature was proposed to be induced by exogenous , diffusible signal gradients . For instance , a mesenchymal FGF10 gradient induces branching in the vertebrate lung , and a Vascular Endothelial Growth Factor/VEGF gradient induces vascular sprouting ( Affolter et al . , 2009; Spurlin and Nelson , 2017 ) . Although the paracrine functions of these signals were well characterized , our demonstration and detailed characterization of the Bnl gradient represents the first example of an inductive gradient for branching morphogenesis . In contrast to the traditional idea of a preexisting diffusible gradient , we showed that the shapes of the inductive Bnl gradient are dynamic , adaptable , and form only in a receptor-bound state within the recipient tissue . Moreover , we showed that the Bnl gradient self-generates its variable recipient branch-specific shapes by regulating its cytoneme-mediated transport . Self-generation of tissue-specific gradients may be a common and essential strategy for sustaining the robust , yet variable gradient shapes in dynamically migrating tissues . Consistent with this view , the chemokine Cxcl12a also forms a self-generating gradient to regulate dynamic migration of the Zebrafish lateral line ( Donà et al . , 2013 ) . Notably , inductive signals such as Bnl or VEGF were previously thought to induce chemotactic migration by inducing binary signaling responses in the recipient cells ( Ochoa-Espinosa and Affolter , 2012 ) . Therefore , identification of an organizer-like morphogenetic role of Bnl brings a critical shift to our understanding of branching morphogenesis . Several earlier reports , including biophysical analyses of vertebrate FGF8 and its distribution patterns in tissues , proposed a free or restricted diffusion-based dispersion mechanism for gradient formation ( Bökel and Brand , 2013; Toyoda et al . , 2010; Yu et al . , 2009 ) . An alternative model proposed that the FGF8 morphogen gradient is also formed by gradual decay of fgf8 mRNA and its translation in a growing vertebrate embryo ( Dubrulle and Pourquié , 2004 ) . In contrast , all of our genetic analyses and direct imaging results show that the Bnl gradient is formed by cytoneme-mediated direct transport . An earlier study showed that ASP cells project Btl-containing cytonemes to establish contact with the disc Bnl source , and fail to induce pMAPK signaling without these cytoneme-mediated contacts ( Roy et al . , 2014 ) . In this study we extended those initial findings . By generating endo-tagged Bnl:GFP and Btl:Cherry constructs expressed at physiological levels , utilizing an EIF assay to detect externalized Bnl molecules , and by applying high resolution visualization techniques , we obtained reliable and unbiased evidence for Bnl dispersion via cytonemes . We showed that ASP cytonemes receive Bnl molecules by establishing direct membrane synapses with the signal producing cells . Super-resolution imaging of membrane-marked ASP and source cells documented these synaptic sites and showed a selective enrichment of externalized Bnl molecules only at these signaling contacts ( Figure 3J ) . At this point , we do not know why and how Bnl is released from producing cells only at cytoneme-source membrane contact sites . Understanding the mechanisms underlying this process is an important new direction for future investigations . Antibody-based EIF analysis under detergent-free conditions and super-resolution imaging of endogenously expressed Bnl:GFP showed that at a steady state , a high concentration of the signal moves along the surface of the cytonemes in receptor-bound forms ( Figure 3C–H ) . It is likely that receptor-bound Bnl ligands move along the surface of the cytonemes by an energy-dependent motor transport and are endocytosed when they reach the ASP cell body . Super-resolution imaging also revealed a previously uncharacterized sub-resolution distribution of Bnl:GFP nanopuncta ( Figure 3F–F’’; Figure 3—supplement figure 2 ) , suggesting the existence of novel cellular and molecular events that regulate the cytoneme-mediated signaling process . Each of these individual cellular and molecular mechanisms would influence the levels of Bnl signaling in a cell and thereby would contribute to the essential feedback mechanisms by which the cytoneme and the morphogen gradients are formed . Contact-dependent Bnl exchange implies that the ASP cells can interpret the direction from where ( and from whom ) they receive the Bnl signal , in addition to its levels . We do not know how ASP cytonemes find the bnl-source and establish the signaling contacts . A recent study had shown an important regulatory role of the components of the extracellular matrix , which interact with the signal-specific cytonemes and stabilize their directionality ( Huang and Kornberg , 2016 ) . Another possible mechanism could be that the cells in the disc Bnl source extend Bnl-containing cytonemes to establish an initial contact with the recipient cytonemes to provide them the necessary pathfinding information . In fact , Hh is known to be delivered by the source cytonemes to the recipient cytonemes in Drosophila wing disc and vertebrate limb buds ( Chen et al . , 2017; González-Méndez et al . , 2017; Sanders et al . , 2013 ) . However , the imaging conditions used in this study could not detect Bnl-containing source cytonemes . Future live imaging analyses are required to elucidate the mechanisms of cytoneme pathfinding . How morphogen gradients are produced in tissues is a long-standing central question . Based on our experimental results , we propose a model for the mechanism by which cytonemes can create and maintain a morphogen gradient ( Figure 8A’ ) . We uncovered that ASP epithelial cells extend a graded number of cytonemes that reach out and contact the disc bnl source to receive the signal ( Figure 5A–D’’ ) . Recipient cells close to the source have many long polarized cytonemes , but their numbers gradually reduced as the distance from the source increases . Clonal GFP-reconstitution analyses suggested that the number of cytoneme-contacts an ASP cell makes is inversely proportional to its distance from the signal source ( Figure 5D–D” ) . Since reception of Bnl is contact-dependent ( Figure 3I , I’ ) , these features of patterned cytoneme formation in the recipient ASP explain how cytoneme-mediated signal reception can generate the recipient tissue-specific shapes of the gradient . These observations led us to address a very important question: how are the cytoneme patterns developed and maintained in the tissue ? Answering this question was expected to provide the basis by which cytonemes create and regulate gradient shapes and tissue patterns . A key finding providing insight into this question came from our analysis of the spatial domains of targets of Bnl signaling in the ASP . One of the target genes , pntP1 , is induced by high Bnl levels in the distal region of the ASP and positively regulates the formation of cytonemes and Btl synthesis ( Figure 6B , E ) . On the other hand , surprisingly , the target gene cut , which is induced by gradually lower levels of Bnl in the ASP stalk region away from the source , negatively controls cytoneme formation ( Figure 6C , E; 7A , A’; 8A' ) and Btl synthesis ( Pitsouli and Perrimon , 2013 ) . Moreover , the transcription factors Cut and PntP1 feedback-inhibit each other’s expression to maintain their zones of expression in response to Bnl levels ( Figure 6M–P ) . The consequence is a regulatory loop that controls the generation and reinforcement of different numbers of cytonemes and levels of Bnl signaling , making the contours of the Bnl gradient robust , precisely tissue-specific , and self-sustaining . The roles of these target genes might also explain how variable shapes of Bnl gradients are sculpted in coordination with the growth and development of recipient tissues . One current morphogen model proposes that the signal gradients are generated and scaled to the size of the recipient tissue by the ratio of two diffusible molecules , a morphogen and a contractor/expander , which emanate from opposite poles of the tissue and elicit non-autonomous global feedback to control each other’s distribution ( Barkai and Shilo , 2009; Ben-Zvi et al . , 2011; Restrepo and Basler , 2011; Shilo and Barkai , 2017 ) . Although Cut and PntP1 are intracellular transcription factors that function cell autonomously , their counteracting feedback activities , regulating where and when signal-specific cytonemes are made as a function of the levels of signal received , can self-generate gradients adopting any recipient-specific shape ( Figures 1F; 8A' ) . This mechanism implies an interesting systemic feedback where the gradient shape is a result of the growth and patterning of the recipient tissue , which it controls . However , this mechanism also implies that to initiate such a self-sustaining gradient , initial physical contact of a part of the recipient epithelium with the signal source is required . In fact , each tracheal cell is capable of inducing either PntP1 or Cut/Yan expression irrespective of its position within the recipient epithelium ( Figure 8A ) . Thus , an ectopic new bnl source near a pre-existing tracheal branch can initiate a local gradient of cytoneme contacts leading to polarized signal and signaling gradient formation as well as neo-branch organization ( Figure 8B ) . In agreement with this view , spatiotemporal expression of Bnl surrounding the embryonic tracheal placode is known to induce new branches ( Sutherland et al . , 1996 ) . Moreover , if the Bnl response genes are not the same or are not regulated in the same manner at all sites and at all times , different patterns of cytoneme formation can be generated leading to changes in spatial patterns of signal distribution and tissue morphology . Given the commonality of fundamental signaling events mediated by conserved signaling proteins , feedback regulation of cytoneme-mediated transport may offer an explanation for why signal gradients are so precise , yet adaptable and for how diverse tissue morphologies can result from just one signal transduction pathway .
The bnl:gfpendo and btl:cherryendo alleles were generated following CRISPR/Cas9 based genome editing method described earlier ( Du et al . , 2017 ) ( Figures 1B and 2A; Figure 1—figure supplement 1A–C; Figure 2—figure supplement 1A–C ) . The bnl:gfpendo knock-in allele expressed a chimeric Bnl:GFP protein containing a superfolder-GFP ( sfGFP ) fused in-frame at ~53 amino acid downstream to the conserved FGF domain ( 247 to 376 amino-acid ) ( Figure 1B ) . Due to its high solubility and efficient folding kinetics in extracellular environment ( Pédelacq et al . , 2006 ) , we selected sfGFP for tagging Bnl . The btl:cherryendo knock-in allele expressed Btl:Cherry containing in-frame C-terminal fusion of mCherry ( Figure 2A ) . Multiple gRNA binding sites with variable efficiency were selected for independent parallel genome-editing experiments to achieve the highest probability of obtaining the intended chimeras and identify the most consistent patterns of Bnl:GFP and Btl:Cherry distribution in tissues among various lines . The gRNA expression constructs were cloned by ligating the annealed complementary oligos ( Key resources table ) as described in ( Du et al . , 2017 ) . The gRNAs ( PAM sequence underlined ) used for generating bnl:gfpendo and btl:cherryendo were: BnlGFP_gRNA-1: GGGCCAATCGCGTCAAGCACGG BnlGFP_gRNA-2: ATATTAGCAGTAGCCTTAGCGG BnlGFP_gRNA-3: CATCACGGAGACGGTGCCGCAGG BtlCherry_gRNA-1: AGGTGTACTGATATCTAAGCGG BtlCherry_gRNA-2: CGGCATCGAAAGGTCCAGATAGG Replacement donors , pDonor-bnl:GFP , and pDonor-btl:Cherry were designed and generated following ( Du et al . , 2017 ) . For efficient ‘Homology-Directed Repair’ ( HDR ) , these constructs contained the tag sequence flanked by ~1 . 5 to 1 . 7 kb long gene-specific 5’- and 3’- homology arms ( Figure 1—figure supplement 1A; Figure 2—figure supplement 1A; Key resources table ) . Both 5’- and 3’- homology arms were PCR-amplified from the genomic DNA from nos-cas9 parent fly , sequence verified , and were fused together with the sfgfp or mCherry sequence by overlap extension PCR . To avoid retargeting of the gRNA to the engineered locus , synonymous mutations were introduced into or near the PAM sequence of the homology arms via the primers used for their amplification ( Key resources table ) . The clones were fully sequenced before being used for genomic replacement . Transgenic flies harboring gRNA vectors at the attP40 landing site ( y1 v1 nos-phiC31; attP40 host ) were generated following ( Kondo and Ueda , 2013 ) . Males harboring a specific U6:3-gRNA were crossed to nos-Cas9 ( nos-Cas9 ZH2A , BL# 54591 ) females to obtain nos-cas9/+; U6:3-gRNA/+embryos . For each genome-editing experiment , the corresponding replacement donor plasmid was injected into the germline of these embryos . A step-wise crossing strategy , as described earlier ( Du et al . , 2017 ) , was followed to obtain the G0-F2 progenies and establish individual fly lines for screening . The correct ‘ends-out’ HDR was screened by a PCR based strategy ( Du et al . , 2017 ) , followed by the analyses of tissue-specific expression patterns of the tagged-genes under a confocal microscope ( Figure 1—figure supplement 1A–C; Figure 2—figure supplement 1A–C ) . Amplification products obtained using PCR primers fwd1-rev2 and fwd2-rev1 confirmed knock-in positive lines , whereas amplification by the M13F and rev3 primers indicated unintended ‘ends-in’ HDR . Finally , a correct sized PCR product obtained using primers fwd1-rev1 confirmed the ends-out HDR ( Figure 1—figure supplement 1A–B’’’; Figure 2—figure supplement 1A–B’’’ ) . All GFP or mCherry positive , sequence verified lines were outcrossed to remove unintended mutations ( if any ) , and any putative non-specific off-target sites were further sequence confirmed to be free of genome-editing . The homozygous btl:cherryendo , and bnl:gfpendo fly lines with accurate genomic sequences had a normal tissue morphology , and expected expression/distribution patterns ( Figure 1C–D’ , Figure 1—figure supplement 1C; Figure 2B; Figure 2—figure supplement 1C ) . We used one BnlGFP_gRNA-3 ( c26-9 ) , and a Btl:Cherry_gRNA-1 ( a21-10 ) line for all analyses . For secGFP , the N-terminal 32 amino-acid ( 96 nucleotides ) long signal peptide sequence of Bnl was cloned in-frame upstream of a sfgfp sequence into pUC19 using Gibson Assembly ( Key resources table ) . The product was sub-cloned into the BglII , Acc651 sites of the pUAST for P-element mediated germline transformation in Drosophila . A transgenic fly was generated that harbored an overexpression UAS-Bnl:GFP construct derived from bnl cDNA ( DGRC ) . All crosses were incubated at 25°C unless specified otherwise . For nsybGRASP labeling of cytoneme contacts ( Figure 3I , I’; Videos 3 and 5 ) : btl-Gal4 , UAS-CD8:Cherry; bnl-LexA/TM6 virgin females were crossed to lexO-nsyb:GFP1-10 , UAS-CD4:GFP11 males . Flip-out clones of specific genotype were generated by 8–15 min 37°C heat shock of the early 3rd instar larvae followed by 25°C incubation until they reached the late 3rd instar stages ( ~24 hr ) . A standard immunostaining protocol was used following ( Du et al . , 2017; Roy et al . , 2014 ) . To detect the surface exposed/localized Bnl:GFPex or Bnlex , standard detergent-free live immunostaining protocol ( EIF ) was used following ( Schwank et al . , 2011; Strigini and Cohen , 2000 ) , except the live tissues were incubated with αBnl or αGFP antibody in optimized WM1 medium ( Du et al . , 2017 ) . Alexa Fluor-conjugated secondary antibodies ( 1:1000 from Molecular Probes ) were used for immunofluorescence detection . Affinity-purified rabbit anti-Bnl antibody was generated from YenZym Antibodies , LLC against a peptide containing amino acid residues from 699 to 717 region of the Bnl protein . For live imaging , wing imaginal discs and their associated trachea were prepared following ( Roy et al . , 2014 ) . The imaging systems used in this study included Leica SP5X confocal microscope equipped with an ultrasensitive Hybrid Detector , an Andor spinning disc confocal equipped with iXon 897 EMCCD camera , Perkin Elmer UltraVIEW Vox with an EMCCD camera , and a ZeissLSM 800 with Airyscan . Super-resolution images of the Airyscan detector resolved poorly fluorescent and densely populated Bnl:GFP nano-puncta on the ASP/disc cell surface ( Figure 3F–F’’; Figure 3—figure supplement 2 ) . The images were processed and analyzed with Fiji . For 3D-rendering , Andor iQ3 software was used . To visualize the faint signals , brightness/intensities of the images were digitally increased following ( Roy et al . , 2014; Roy et al . , 2011b ) . For most images , maximum intensity projections of at least 30 optical sections were shown . The Intensity profiles of Bnl:GFP , Btl:Cherry , pntP1 , cut , sty and yan gradients were derived from the maximum intensity projections along the D-P axis of the ASPs . For Figure 1I–I’’ , Figure 1—figure supplement 2D–E” , the intensity profiles were derived across the entire digitally straightened epithelium following ( Long et al . , 2009 ) . A ~ 40 pixels wide line was fitted following the curved tracheal/ASP epithelium . For quantification of Bnl:GFP and Bnl:GFPex uptake in mutant clones , total GFP or EIF ( red , anti-GFP ) intensities were measured from selected areas in mutant clones and neighboring wt cells and normalized with the selected area ( Figure 4; Figure 4—figure supplement 1; Figure 7B , B’ , E–H ) . For Figure 5A–C , Figure 7A , A’ , C–D’ , Figure 7—figure supplement 1 , cytonemes were manually counted and were grouped by length ( <15 μm , 15–30 μm , and >30 μm ) as reported in ( Roy et al . , 2011b ) . Cytonemes emanating from each clone were captured over ~30–40 μm Z-sections with 0 . 35 μm step size . The R program was used to generate R plots from at least five preparations of each genotype to analyze cytoneme orientation and number . Statistical significance was determined with a two-tailed t-test ( Figure 3I'; 4E , G’; 7B’ , E’ ) , or a one-way ANOVA ( Figure 5B , D’’ ) followed by Tukey honestly significant different ( HSD ) test . For all the numerical data analyses , the amount of variation from the mean is indicated by the standard deviation .
Gradients of signaling proteins are essential for inducing tissue morphogenesis . However , mechanisms of gradient formation remain controversial . Here we characterized the distribution of fluorescently-tagged signaling proteins , FGF and FGFR , expressed at physiological levels from the genomic knock-in alleles in Drosophila . FGF produced in the larval wing imaginal-disc moves to the air-sac-primordium ( ASP ) through FGFR-containing cytonemes that extend from the ASP to contact the wing-disc source . The number of FGF-receiving cytonemes extended by ASP cells decreases gradually with increasing distance from the source , generating a recipient-specific FGF gradient . Acting as a morphogen in the ASP , FGF activates concentration-dependent gene expression , inducing pointed-P1 at higher and cut at lower levels . The transcription-factors Pointed-P1 and Cut antagonize each other and differentially regulate formation of FGFR-containing cytonemes , creating regions with higher-to-lower numbers of FGF-receiving cytonemes . These results reveal a robust mechanism where morphogens self-generate precise tissue-specific gradient contours through feedback regulation of cytoneme-mediated dispersion .
When an embryo develops , its cells must work together and ‘talk’ with each other so they can build the tissues and organs of the body . A cell can communicate with its neighbors by producing a signal , also known as a morphogen , which will tell the receiving cells what to do . Once outside the cell , a morphogen spreads through the surrounding tissue and forms a gradient: there is more of the molecule closer to the signaling cells and less further away . The cells that receive the message respond differently depending on how much morphogen they get , and therefore on where they are placed in the embryo . How morphogens move in tissues to create gradients is still poorly understood . One hypothesis is that , once released , they spread passively through the space between cells . Instead , recent research has shown that some morphogens travel through long , thin cellular extensions known as cytonemes . These structures directly connect the cells that produce a morphogen with the ones that receive the molecule . Yet , it is still unclear how cytonemes can help to form gradients . Du et al . aimed to resolve this question by following a morphogen called Branchless as it traveled through fruit fly embryos . Branchless is important for sculpting the embryonic airway tissue into a delicate network of branched tubes which supply oxygen to the cells of an adult fly . However , no one knew how cells communicate Branchless , whether or not Branchless formed a gradient , and if it did , how this gradient was created to set up the plan to form airway tubes . It was assumed that the molecule would diffuse passively to reach airway cells – but this is not what the experiments by Du et al . showed . To directly observe how Branchless moves among cells , insects were genetically engineered to produce Branchless molecules attached to a fluorescent ‘tag’ . Microscopy experiments using these flies revealed that Branchless did not diffuse passively; instead , airway cells used cytonemes to ‘reach’ towards the cells that produced the molecule , collecting the signal directly from its source . The gradient was created because the airway cells near the cells that make Branchless had more cytonemes , and therefore received more of the molecule compared to the cells that were placed further away . Genetic analysis of the airway tissue showed that Branchless acts as a morphogen to switch on different genes in the receiving cells placed in different locations . The target genes activated by the gradient instruct the receiving cells on how many cytonemes need to be extended , which helps the gradient to maintain itself over time . Du et al . demonstrate for the first time how cytonemes can relay a signal to establish a gradient in a developing tissue . Dissecting how cells exchange information to create an organism could help to understand how this communication fails and leads to disorders .