TGF-b Family Signaling in Drosophilacshperspectives.cshlp.org/content/early/2017/01/27/... · TGF-b...

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TGF-b Family Signaling in Drosophila Ambuj Upadhyay, Lindsay Moss-Taylor, Myung-Jun Kim, Arpan C. Ghosh, and Michael B. O’Connor Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455 Correspondence: [email protected] The transforming growth factor b (TGF-b) family signaling pathway is conserved and ubiq- uitous in animals. In Drosophila, fewer representatives of each signaling component are present compared with vertebrates, simplifying mechanistic study of the pathway. Although there are fewer family members, the TGF-b family pathway still regulates multiple and diverse functions in Drosophila. In this review, we focus ourattention on several of the classic and best-studied functions for TGF-b family signaling in regulating Drosophila de- velopmental processes such as embryonic and imaginal disc patterning, but we also describe several recently discovered roles in regulating hormonal, physiological, neuronal, innate immunity, and tissue homeostatic processes. Core Drosophila TGF-b Family Signaling Components A s in mammals, the Drosophila transforming growth factor b (TGF-b) family signaling pathway has two branches, initiated by different ligands, activins, and bone morphogenetic pro- teins (BMPs) that are defined by sequence con- servation within families and the downstream signal transduction components that they use (Fig. 1). In both branches, canonical signaling begins upon extracellular ligand binding to a heteromeric receptor complex composed of two molecules each of type I and type II trans- membrane serine/threonine kinases. Forma- tion of this ligand–receptor complex results in phosphorylation of the GS domain within the type I receptor by the constitutively active type II receptor kinase. This phosphorylation event ac- tivates the type I receptor kinase and enables it to phosphorylate an appropriate receptor-activat- ed Smad (R-Smad) substrate, either dSmad2 for the activin branch, or Mad for the BMP branch. The phosphorylated R-Smad then forms a com- plex with the Drosophila common-Smad (co- Smad), Medea, and this complex translocates to the nucleus where it can regulate transcrip- tional responses with a variety of cofactors. Some TGF-b signal transduction components, such as the type II receptors Punt and Wishful thinking (Wit) and the co-Smad Medea, are shared between the activin and BMP branches and often act redundantly, whereas others, such as the type I receptors and the R-Smads, are restricted to signaling within a single subfamily branch. Many accessory factors interact with core TGF-b family signaling components and are discussed throughout this review (Table 1). Editors: Rik Derynck and Kohei Miyazono Additional Perspectives on The Biology of the TGF-b Family available at www.cshperspectives.org Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a022152 1 on June 12, 2020 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/ Downloaded from

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TGF-b Family Signaling in Drosophila

Ambuj Upadhyay, Lindsay Moss-Taylor, Myung-Jun Kim, Arpan C. Ghosh,and Michael B. O’Connor

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis,Minnesota 55455

Correspondence: [email protected]

The transforming growth factor b (TGF-b) family signaling pathway is conserved and ubiq-uitous in animals. In Drosophila, fewer representatives of each signaling component arepresent compared with vertebrates, simplifying mechanistic study of the pathway.Although there are fewer family members, the TGF-b family pathway still regulates multipleand diverse functions in Drosophila. In this review, we focus our attention on several of theclassic and best-studied functions for TGF-b family signaling in regulating Drosophila de-velopmental processes such as embryonic and imaginal disc patterning, but we also describeseveral recently discovered roles in regulating hormonal, physiological, neuronal, innateimmunity, and tissue homeostatic processes.

Core Drosophila TGF-b Family SignalingComponents

As in mammals, the Drosophila transforminggrowth factor b (TGF-b) family signaling

pathway has two branches, initiated by differentligands, activins, and bone morphogenetic pro-teins (BMPs) that are defined by sequence con-servation within families and the downstreamsignal transduction components that they use(Fig. 1). In both branches, canonical signalingbegins upon extracellular ligand binding to aheteromeric receptor complex composed oftwo molecules each of type I and type II trans-membrane serine/threonine kinases. Forma-tion of this ligand–receptor complex results inphosphorylation of the GS domain within thetype I receptor by the constitutively active type IIreceptor kinase. This phosphorylation event ac-

tivates the type I receptor kinase and enables it tophosphorylate an appropriate receptor-activat-ed Smad (R-Smad) substrate, either dSmad2 forthe activin branch, or Mad for the BMP branch.The phosphorylated R-Smad then forms a com-plex with the Drosophila common-Smad (co-Smad), Medea, and this complex translocatesto the nucleus where it can regulate transcrip-tional responses with a variety of cofactors.Some TGF-b signal transduction components,such as the type II receptors Punt and Wishfulthinking (Wit) and the co-Smad Medea, areshared between the activin and BMP branchesand often act redundantly, whereas others, suchas the type I receptors and the R-Smads, arerestricted to signaling within a single subfamilybranch. Many accessory factors interact withcore TGF-b family signaling components andare discussed throughout this review (Table 1).

Editors: Rik Derynck and Kohei Miyazono

Additional Perspectives on The Biology of the TGF-b Family available at www.cshperspectives.org

Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved

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In theBMPsignalingbranch, three ligands—Decapentaplegic (Dpp), Glass-bottom boat(Gbb), and Screw (Scw)—signal through twotype I receptors—Thickveins (Tkv) and Saxo-phone (Sax)—and either of two type II recep-tors—Wit or Punt. Although loss-of-functionmutations in either tkv or sax are lethal, tkv over-expression can rescue sax mutants (Brummelet al. 1994), suggesting that it may be the primaryBMP type I receptor, whereas Sax, in most cases,fine-tunes signaling levels. Downstream fromthe receptors, Mad is the primary R-Smad thatappears to transduce all BMP signals in combi-nation with the co-Smad Medea. One inhibitorySmad, Dad, appears to act specifically to modu-late BMP signaling (Kamiya et al. 2008).

Within the activin subfamily, three li-gands—activin-b (Actb), Dawdle (Daw), andMyoglianin (Myo)—signal through the singletype I receptor Baboon (Babo) and either Witor Punt, and this branch uses dSmad2 as the R-Smad transcriptional transducer. In this path-way, each ligand appears to have unique func-tions, but double mutants have not yet beenanalyzed in detail to determine whether theyalso act redundantly. Consistent with theunique functional activities of the three ligands,three isoforms of Babo have been identified.Each differs in the ligand-binding domain andis produced through alternative splicing of asingle exon (Jensen et al. 2009). Signaling assaysin S2 cells show that Daw signals through BaboC

Activin BMP

Myo Actβ Daw

Punt/Wit

MavGbb

Dpp ScwDpp:Scw

BaboA BaboB BaboC

Plasmamembrane

Sax/Tkv

Plasmamembrane

Cross-signalingSmad2

Smad2

Mad

Medea

Mad

Punt/Wit

P

P

Medea

Figure 1. Core transforming growth factor b (TGF-b) family signaling components in Drosophila. Activinbranch: Three ligands signal through the activin-specific type I receptor Babo. Each ligand is thought to havea dedicated receptor isoform, although evidence for Actb-BaboB is lacking. Ligand binding induces formationof a receptor complex of both Babo and either of the type II receptors, Punt or Wishful thinking (Wit).Constitutively active type II receptors phosphorylate Babo, which activates dSmad2. Phosphorylated dSmad2binds to Medea and translocates to the nucleus to regulate the transcriptional response. Bone morphogeneticprotein (BMP) branch: Four ligands signal through shared the BMP-specific type I receptors Tkv and Sax andeither Punt or Wit. The ligands are thought to primarily form homodimers, but there is one example of aheterodimer: Dpp–Scw. Mav is a divergent ligand based on sequence, but signals to activate Mad. Activation oftype II receptors causes phosphorylation of type I receptors and subsequent phosphorylation of Mad, whichthen complexes with Medea to regulate transcription. In the absence of dSmad2, Babo can phosphorylate Madand can also activate the Mad transcriptional response. Myo, Myoglianin; Actb, Activin-b; Daw, Dawdle; Babo,Baboon; Mav, Maverick; Gbb, Glass-bottom boat; Dpp, Decapentaplegic; Scw, Screw; Sax, Saxophone; Tkv,Thickveins; Wit, Wishful thinking.

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(Jensen et al. 2009), whereas subsequent in vivowork indicates that Myo signals preferentiallythrough BaboA (Zhu et al. 2008; Awasaki et al.2011). It remains to be shown if Actb signalsthrough BaboB, and if there is any promiscuityin ligand binding to the different receptor iso-forms. A handful of genes have been shown to beexpressed downstream from dSmad2 in specificcontexts (Zheng et al. 2003; Gibbens et al. 2011;Bai et al. 2013), but only one, Atg8a, is known tobe a direct target, although a fully validateddSmad2 consensus binding sequence has notyet been determined. The seventh ligand, Mav-erick (Mav), is aptly named because it does notclean fit into either pathway by sequence simi-larity, although one report indicates that it sig-nals through Tkv, which would be consistentwith a BMP family member (Fuentes-Medelet al. 2012).

Unlike in vertebrates, only two non-Smadsignaling pathways have been described in Dro-sophila. One involves the activin branch, whereRho-like GTPases and Lim kinase act down-stream from Babo to limit mushroom body

(MB) axon extension (Ng 2008), and the otheris Lim kinase acting downstream from BMPsignaling to regulate neuromuscular junction(NMJ) growth (Eaton and Davis 2005; Piccioliand Littleton 2014).

In addition to non-Smad-dependent signal-ing, one example of cross-pathway Smad acti-vation has also been documented in Drosophila(Fig. 1). In this case, the activin receptor Babocan phosphorylate the BMP transducer Mad incell culture and also in vivo when overexpressedtogether with Mad (Gesualdi and Haerry 2007;Peterson et al. 2012). However, whether this oc-curs under endogenous conditions is unknown.As discussed below, loss of dSmad2 protein doeslead to enhanced Mad activity in vivo, but it isnot clear whether this comes about as a result ofenhanced carboxy-terminal phosphorylation ofMad or through some other modifications thathappen only in the absence of dSmad2 (Peter-son and O’Connor 2013).

As in vertebrates, many accessory factorsregulate ligand production and availabilityand several are highlighted in Table 1. For ex-

Table 1. Transforming growth factor b (TGF-b) family accessory factors in Drosophila

Gene name Symbol Function Interaction

armadillo arm Transcription factor Complexes with Madcrossveinless cv Lipoprotein Modulates Dpp movementcrossveinless 2 cv-2 BMP binding protein Binds extracellular BMPsdally-like protein dlp Heparan sulfate

proteoglycanControls Dpp gradient by binding Dpp

division abnormallydelayed

dally Heparan sulfateproteoglycan

Controls Dpp gradient by binding Dpp

follistatin fs Secreted factor Modulates ligand activitypentagone pent Secreted factor Interacts with heparan sulfate proteoglycans

to control Dpp gradientPlum plum Immunoglobulin

proteinGenetic interaction with Myo

Schnurri shn Transcription factor Complexes with Mad and MedeaShrew srw Secreted factor Role unknownshort gastrulation sog Secreted factor Binds extracellular BMPsTolloid tld Protease Binds extracellular BMPs, cleaves Sogtolkin (also called

tolloid-related)tok, tlr Protease Cleaves extracellular BMP and activin ligands

twisted gastrulation tsg Secreted factor Binds extracellular BMPsYorkie yki Transcription factor Complexes with Mad

Many additional proteins interact with core TGF-b pathway components and modulate pathway activation and output.

Listed in the table are known accessory factors and their interactions with core TGF-b components.

BMP, Bone morphogenic protein.

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ample Drosophila follistatin (fs) can modulateligand activity of both BMP and activin pathwayligands; inhibiting Dpp and Actbwhile enhanc-ing Daw activity (Bickel et al. 2008; Pentek et al.2009). The BMP heterodimer Dpp–Scw bindsto a complex of Short gastrulation and Twistedgastrulation (Sog–Tsg) and, as described below,is required for shuttling of the Dpp–Scw heter-odimer during embryonic dorsal patterning(Shimmi et al. 2005b). Numerous potential cor-eceptors have also been identified.

Differential processing can also dramaticallyaffect the signal range and activity of the ligand.Furin proteases facilitate maturation of the di-sulfide-linked ligand dimer by processing theinactive proprotein at any one of several poten-tial “maturation sites.” For Dpp, three furincleavage sites are processed in distinct ways, pro-ducing ligands with different amino-terminalsequences. These alternatively processed ligandshave different activities, such as long-range orshort-range diffusion, and variable stabilities(Kunnapuu et al. 2009; Sopory et al. 2010).They can also affect interactions with extracellu-lar matrix (ECM) proteins, such as the Dro-sophila glypican Division abnormally delayed(Dally), through inclusion or exclusion of hep-aran sulfate proteoglycan (HSPG)-binding sites(Akiyama et al. 2008). In some cases, additionalcleavage within the upstream prodomain isneeded to fully release the prodomain from themature ligand, and, in the two Drosophila exam-ples, this processing is performed by a Tolloid/BMP-1 type protease (Serpe and O’Connor2006; Kunnapuu et al. 2014). Lastly, there is anexample in which cleavage of the BMP-type li-gand Gbb does not take place at the normal mat-uration site, but instead at an upstream site lead-ing to production of a very long mature ligandthat, surprisingly, has bioactivity. The choice ofmaturation site seems to be tissue context-spe-cific (Akiyama et al. 2012; Fritsch et al. 2012).

BMPs AND EARLY AXIAL PATTERNINGIN Drosophila

The major body plan of most organisms is laidout during early embryonic development.BMPs play important roles in both eggshell pat-

terning and axis formation. Dpp controls pat-terning of the follicle cells that produce the egg-shell surrounding the oocytes (Dobens andRaftery 1998). In particular, Dpp signalingthrough the type I receptor Tkv and the typeII receptor Wit specifies the location of the oper-culum and the respiratory dorsal appendage(Yakoby et al. 2008a,b; Marmion et al. 2013).

Once fertilized, maternal factors guide theinitial subdivision of the embryo along the an-terior/posterior (A/P) and dorsal/ventral (D/V) axes. At the time of cellularization, the prod-ucts of the first zygotically expressed genes takeover and further refine cell fates within eachsubdivision (Lynch et al. 2012). Two primarytissues are specified within the dorsal half ofthe embryo: the dorsal– lateral ectoderm thatgives rise to portions of the epidermis, and thedorsal-most amnioserosa, a short-lived tissuethat coordinates the movements of gastrulation.Classic genetic screens for embryonic lethal mu-tations first identified six zygotic genes—decap-entaplegic (dpp), screw (scw), short gastrulation(sog), twisted gastrulation (tsg), tolloid (tld) andshrew (srw)—that guide the dorsal subdivisioninto these two tissues (Nusslein-Volhard et al.1984; Wieschaus et al. 1984). Among these sixgenes, loss of dpp caused the strongest pheno-type in which all dorsal cells adopted more lat-eral ectoderm fates (Arora and Nusslein-Vol-hard 1992; Ferguson and Anderson 1992;Wharton et al. 1993). The dpp gene encodes afactor with �75% identity to vertebrate BMP-2and -4 (Padgett et al. 1987). As best describedfor vertebrates (Wu and Hill 2009; Wang et al.2014; Brazil et al. 2015), BMPs control a widerange of developmental and physiological pro-cesses in higher eukaryotes. Remarkably, notonly is the sequence of Dpp closely related tovertebrate BMPs, but it was also shown to befunctionally equivalent to its vertebrate homo-logs. Thus, subcutaneous implantation of Dppprotein in rats leads to ectopic bone formation,while a BMP-4 transgene inserted into the Dro-sophila germline was able to partially rescue ear-ly patterning defects of dpp mutant embryos(Padgett et al. 1993; Sampath et al. 1993).

One of the more noteworthy properties ofmany TGF-b-type proteins is their ability to act

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as morphogens, that is, molecules that instructcells to adopt specific fates in a concentration-dependent manner. Injection of dpp mRNAinto early embryos, along with careful pheno-typic analysis of a large number of embryoniclethal dpp mutant alleles, first suggested thatDpp likely had morphogenetic activity withinthe dorsal region, because low levels of Dppactivity specified dorsal ectoderm whereashigh levels specified the formation of amnioser-osa (Ferguson and Anderson 1992; Whartonet al. 1993). Subsequent comparison of the en-dogenous dpp mRNA pattern compared withprotein revealed that the mRNA was uniformlytranscribed throughout the entire dorsal half ofthe embryo, whereas the protein distributionchanged with time (Shimmi et al. 2005a).When first detectable, the protein pattern mir-rors that of the mRNA, but shortly thereafter itsdistribution is concentrated within a domain of8 to 12 cells centered about the dorsal midline(Fig. 2). Using an antibody to detect phosphor-ylated Mad (pMad), the major BMP transcrip-tional transducer in Drosophila, revealed a sim-ilar temporal and spatial refinement in Dppsignal output, that is, the pMad signal is initially

low and broad and is subsequently refined andintensified along the dorsal midline (Fig. 2)(Dorfman and Shilo 2001; Ray and Wharton2001; Ross et al. 2001).

These observations trigger the obviousquestion of what controls the change in Dppprotein distribution and signaling output. Ad-ditional genetic, biochemical, and computa-tional approaches provide a fairly complete pic-ture of the molecular events that facilitate theformation of the embryonic Dpp activity gradi-ent (Fig. 2) (O’Connor et al. 2006). Key to un-derstanding this process was the elucidation ofbiochemical activities of Scw, Tld, Tsg, and Sog,four products encoded by the other previouslymentioned zygotic genes that, when mutated,produce partially ventralized embryos. All fourproteins are secreted and physically interact invarious ways to form both stable and transientcomplexes with Dpp. The scw gene encodes asecond BMP-type ligand that is uniformly ex-pressed in the embryo (Arora et al. 1994), butcan form a disulfide-linked heterodimer withDpp in the dorsal domain (Shimmi et al.2005b). Cell culture signaling assays showedthat the Dpp–Scw heterodimer induces a

pMad (dorsal view)

Early blastoderm

Earlyblastoderm

Late cellularblastoderm

Latecellular blastoderm

DppScwDpp/Scw

SogTsgTld

Figure 2. Drosophila embryo patterning. Wider distribution of pMad (early blastoderm) is converted to arestricted pMad stripe (late blastoderm) through the action of Sog, Tsg, and Tld. The combined activities ofthese factors result in net flux of the Dpp–Scw heterodimer to the dorsal midline. Homodimers stay broadlydistributed as a result of low affinity to Sog.

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much stronger phosphorylation of Mad on aper-mole basis than does either homodimer(Shimmi et al. 2005b). Because wit mutationscause no early embryonic phenotype (Marqueset al. 2002), it is generally thought that Punt isthe only functional type II receptor in the earlyembryo. Further genetic experiments revealedthat, in addition to Punt, both BMP type I re-ceptors Tkv and Sax are required for hetero-dimer-mediated synergistic signaling; however,the molecular mechanism responsible for syn-ergy is not understood (Nguyen et al. 1998).

Another important property of the hetero-dimer is that it is required for redistribution ofDpp during dorsal patterning since, in scw mu-tants, Dpp homodimers do not accumulate atthe dorsal midline (Shimmi et al. 2005b; Wangand Ferguson 2005). The inability to concen-trate the heterodimer at the midline is alsoobserved in sog, tld, and tsg mutant embryosindicating that these gene products facilitateheterodimer movement. Computational mod-eling provided insight into the mechanism andrelied on several key bits of biochemical data(Eldar et al. 2002; Mizutani et al. 2005; Shimmiet al. 2005b). The first was that the secretedfactors Sog and Tsg could form a tripartite com-plex with BMP ligands (Ross et al. 2001). Aswith signaling, the Dpp–Scw heterodimer hasthe highest affinity for binding the Sog–Tsgcomplex (Shimmi et al. 2005b). The second piv-otal observation was that tld encodes an astacin-like protease, related to vertebrate BMP-1 thatcleaves Sog, but only when bound in a complexwith a BMP (Shimell et al. 1991; Marques et al.1997; Peluso et al. 2011). Once again, the het-erodimer forms a higher affinity complex withSog and Tsg than either homodimer, and thiscomplex also provides the best platform to stim-ulate Sog cleavage by the Tld protease (Shimmiet al. 2005b). The key to understanding directedmovement of the heterodimer toward the dorsalmidline is the ventral lateral expression of Sog(Fig. 2). As Sog diffuses from its site of synthesisinto the ventral lateral region, it first encountersTsg, Dpp–Scw, and Tld at the boundary be-tween the dorsal and ventral halves of the em-bryo. The complex of Sog, Tsg, and Dpp–Scwforms at the boundary and can either diffuse or

is processed by Tld, releasing Dpp–Scw for fur-ther binding and diffusion. Although not intu-itively obvious, the computational models showthat, with the correct set of kinetic parameters,this process results in shuttling of the Dpp–Scwcomplex to the dorsal-most region (Eldar et al.2002; Mizutani et al. 2005; Shimmi et al.2005b).

Despite the apparent success of the firstcomputational models, one problem was that,while they all predict shuttling, the shape of theoutput response was not what was actually ob-served. These models predict that pMad wouldfirst accumulate at the dorsal midline and thenwould intensify and “expand outward” awayfrom the midline with time. However, experi-mentally, exactly the opposite was observed: theinitial pMad signal is low and broad and inten-sifies and “contracts inward” toward the mid-line (Fig. 2). This has been referred to as thespatial bistability issue, and studies using mu-tations in Medea, the Drosophila co-Smad nec-essary for transducing all BMP signals, revealedthat the contraction and intensification of thepMad output is due to positive feedback (Wangand Ferguson 2005). It appears that an early,broad, low-level BMP signal induces the tran-scription of unknown component(s) that thenprovide feedback and enhance binding of Dpp–Scw to its receptors. Subsequent models thatincorporate such a feedback scheme were ableto accurately capture the correct temporal andspatial refinement in the pMad pattern (Umuliset al. 2006; Umulis et al. 2010).

Although the exact molecular mechanismfor enhancing BMP binding to its receptors re-mains unknown, the feedback mechanism ap-pears to be surprisingly complex and involvesinduction of additional positive and negativeregulators of Dpp signaling (Gavin-Smythet al. 2013). The positive effector is eiger, whichencodes a tumor necrosis factora homolog thatsignals through c-Jun amino-terminal kinase(JNK) (Igaki et al. 2002; Moreno et al. 2002).The negative effector is crossveinless 2 (CV-2),an extracellular BMP-binding protein that canhave positive or negative influences on BMPsignals depending on context (Serpe et al.2008). In the absence of both Eiger and CV-2,

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the robustness of the BMP signal is compro-mised and the number of fated amnioserosacells is extremely variable. Although the nega-tive effects of CV-2 may be attributed to its abil-ity to bind and sequester BMP ligands, howEiger signaling through Jnk promotes BMP sig-naling is unknown. It is still not clear if thepositive effect of Eiger–Jnk signaling is at thelevel of enhancing BMP binding to its receptor,as the computational modeling requires, or ifother BMP target genes yet to be discoveredare involved in mediating positive feedback.

Although the computational models usingfeedback are likely correct in many aspects(Umulis et al. 2010), it is probable that somesignificant nuances will arise. To illustrate thispoint, consider that in every proposed compu-tational model, the binding affinity of Sog forligand is tightly constrained and needs to be inthe picomolar range for effective ligand shut-tling to take place (Eldar et al. 2002; Mizutaniet al. 2005; Shimmi et al. 2005b). Thus, a po-tential problem arose when the affinity of Chor-din, a vertebrate homolog of Sog, for BMP-2was determined to be in the nanomolar range(Rentzsch et al. 2006). A likely solution to thisproblem was provided when it was determinedthat two type IV collagen-like molecules are alsorequired for proper dorsal patterning (Wanget al. 2008; Sawala et al. 2012). Loss of thesematrix proteins attenuates the formation ofthe strong dorsal-most Dpp–Scw signal. Fur-thermore, it was shown that Dpp–Scw andSog could both bind to collagen and then bereleased in a complex through the action ofTsg binding. Tolloid, also interacts with collagenIV, via its amino-terminal CUB domains, andthis association enhances cleavage of Sog (Win-stanley et al. 2015). Modeling showed that thelocal concentrating effect of the collagen scaf-fold can greatly reduce the effective binding af-finity of the ligand for Sog, thereby solving theproblem presented by the high solution disso-ciation constant (Umulis et al. 2009).

Other refinements to the mechanism arealso likely. For example, the Scw prodomainwas found to require processing at a site distinctfrom the normal maturation site (Kunnapuuet al. 2014). A mutation that disrupts this pro-

cessing results in a dominant-negative effectthat might be brought about by interference ofthe altered heterodimer with the ECM interac-tion or the kinetics of processing and secretionof the heterodimer in vivo. The co-Smad Medeais also susceptible to covalent regulatory modi-fications that impact early dorsal patterning.Sumoylation, for example, promotes export ofMedea from the nucleus, perhaps enabling nu-clei to continuously monitor the presence ofextracellular Dpp–Scw signal to activate targetgene expression for an appropriate duration(Miles et al. 2008). Medea has also been shownto be ubiquitylated (Dupont et al. 2009), andthe level of ubiquitylation affects the activity ofMedea in regulating early dorsal patterning(Stinchfield et al. 2012). Lastly, of all the classicmutants identified that affect dorsal patterning,the role of shrew still remains unknown.

Later Embryonic Roles of Drosophila BMPs

Although the Screw ligand only appears to havea role in early dorsal patterning (Arora et al.1994), dpp transcription and pMad output con-tinue throughout embryogenesis with complexspatial dynamics (Dorfman and Shilo 2001). Inaddition, Gbb, a third Drosophila BMP-like fac-tor that is closely related to the vertebrate BMP-5, -6 and -7, is broadly activated during gastru-lation in mesodermal derivatives includingmuscle and fat body (Wharton et al. 1999). De-spite some potential overlap in expression withDpp, each of these two ligands plays largely in-dependent roles during embryogenesis suggest-ing that heterodimers are not likely to be im-portant at these stages. In addition, there is noevidence for any shuttling of ligands in embryosafter the early blastoderm stage, and no obviousroles for sog, tsg, or tld have been described.Instead, most patterning and cell fate specifica-tion events regulated by Dpp and Gbb in laterembryo development seem to involve short-range, unassisted ligand diffusion. For Gbb, em-bryonic activities include maintenance of thefirst midgut constriction through regulation ofthe homeotic gene antennapedia, and specifica-tion of the gastric ceca precursors at the anteriorend of the midgut (Wharton et al. 1999). Dpp

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controls signaling from the dorsal ectoderm tothe underlying mesoderm at two stages. In earlygastrulating embryos, it induces expression oftinman, which regulates the formation of heartand visceral muscle (Staehling-Hampton andHoffmann 1994; Frasch 1995). Later, duringgermband retraction, Dpp refines the cardiacfield by limiting the number of specified peri-cardial cells (Johnson et al. 2003, 2007). Dppalso regulates foregut morphogenesis (Fussand Hoch 1998; Nakagoshi et al. 1998), pattern-ing of the second midgut constriction (Bienz1994; Szuts and Bienz 2000), establishment ofembryonic imaginal disc placodes (Goto andHayashi 1997), posterior spiracle development(Takaesu et al. 2008), ectodermal cell movementduring dorsal closure (Glise and Noselli 1997;Fernandez et al. 2007; Zahedi et al. 2008), andtracheal cell migration and branching (Vincentet al. 1997; Myat et al. 2005), by activating ex-pression of two zinc finger transcription factorsencoded by knirps and knirps-related (Chenet al. 1998). Interestingly, during Dpp-directeddorsal closure, the range of Dpp signaling isnegatively regulated by mummy, which encodesa UDP-N-acetylglucosame pyrophosphorylase,a component necessary for N- and O-linkedglycan modification of protein and lipids. Onepossibility is that a glycosylated receptor orECM component might bind Dpp and limitits diffusion away from the ectoderm leadingedge cells (Humphreys et al. 2013).

TGF-b FAMILY SIGNALING IN THE WINGIMAGINAL DISC

Drosophila larvae contain 15 distinct sac-liketissues called imaginal discs, which are mitoticand give rise to adult structures. Here, we willprimarily focus on the wing imaginal disc be-cause it is the best characterized system for un-derstanding TGF-b signaling. Growth and pat-terning of imaginal discs is largely controlled byDpp signaling (Hamaratoglu et al. 2014; Re-strepo et al. 2014). In contrast, activin signalsplay a modest role in cell proliferation but notpatterning (Brummel et al. 1999; Hevia and deCelis 2013; Peterson and O’Connor 2013). Inthe wing disc, as in the embryo, Dpp functions

as a morphogen to specify different cell fates.In this tissue, Dpp is secreted from a narrowstrip of cells abutting the A/P boundary, andspreads to both compartments, forming a gra-dient that regulates the expression of several tar-get genes in a concentration-dependent manner(see Fig. 3A) (Lecuit et al. 1996; Nellen et al.1996; Campbell and Tomlinson 1999; Jazwinskaet al. 1999; Minami et al. 1999). The spatiallydefined expression of Dpp target genes governsboth growth and size, as well as patterning of theadult wing veins. Below, we will focus on howthe Dpp gradient is formed, interpreted, andtranslated to produce a functional wing.

Models of Dpp Gradient Formation

Francis Crick initially proposed that morpho-gen gradients can form by simple diffusion(Crick 1970). To examine the role of Dpp dif-fusion in the wing disc, a Dpp–green fluores-cent protein (GFP) fusion was created allowingthe morphogen gradient to be directly visual-ized (Entchev et al. 2000; Teleman and Cohen2000). Surprisingly, fluorescent recovery afterphotobleaching (FRAP) experiments usingthis Dpp–GFP suggested a diffusion coefficientthat was too slow to account for production ofthe observed gradient in the wing disc (Kichevaand Gonzalez-Gaitan 2008). This result led tothe development of two alternative models, oneinvolving multiple rounds of receptor-mediatedligand uptake and resecretion, termed transcy-tosis (Kicheva and Gonzalez-Gaitan 2008), anda second based on directed transport of Dppalong extended actin-based filipodia, termed cy-tonemes (Ramirez-Weber and Kornberg 1999;Roy and Kornberg 2011).

Although the final resolution of the truemechanism may not be fully settled, single mol-ecule imaging of tagged Dpp in the wing disc hasallowed for measurements of the diffusion co-efficient, revealing two distinct populations ofDpp that diffuse at different rates (Zhou et al.2012). A significant portion of Dpp diffuses ex-tremely slowly, as predicted for ligand bound tocell surface proteins such as receptors, and an-other pool of Dpp diffuses much more rapidly,consistent with free diffusion that is hindered

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only by the viscosity of the extracellular fluidand the tortuosity of intercellular paths (Zhouet al. 2012). Theoretical calculations suggest thatthis highly mobile pool of Dpp is sufficient toform the observed gradient in the disc. Further-more, wild-type cells can sense Dpp even whencompletely surrounded by cells lacking Tkv.This observation argues against the need forreceptor-mediated transcytosis (Schwank et al.2011). To date, no strong support for cytoneme-mediated dispersal of Dpp throughout the wingdisc has been reported, although it appears thatcytonemes convey Dpp from the wing disc toother interacting tissues such as the trachea (Royand Kornberg 2011; Roy et al. 2014).

Modifiers of the Dpp gradient

Because the FRAP studies and other microscopytechniques highlighted above suggest that mostDpp is bound to the cell surface, it is likely thatany molecule regulating ligand–receptor inter-actions will dramatically affect either gradientformation or the signaling output. Indeed, ge-netic approaches identified the products of thegenes division abnormally delayed (dally) anddally-like protein (dlp), which encode two mem-bers of the HSPG family, as proteins that poten-tially affect Dpp gradient formation and/or sig-nal output (Fujise et al. 2003; Belenkaya et al.2004; Akiyama et al. 2008). However, theoreticalcalculations now suggest that HSPGs do notrestrict the fast free diffusion of Dpp (Zhouet al. 2012). Instead, they exert their primaryeffect on signaling output and not gradient for-mation, likely by facilitating Dpp–receptor in-teractions (Kuo et al. 2010).

Another less understood aspect of morpho-gen gradients is their ability to scale accordingto size of the tissue, (Wartlick et al. 2011a). Anovel secreted factor, Pentagone (Pent), hasbeen shown to be important for Dpp gradientscaling as the wing disc grows during the larvalstages (Vuilleumier et al. 2010; Hamaratogluet al. 2011). Pent contains follistatin-like, thyro-globulin type I and EF hand calcium-bindingdomains and interacts with HSPGs to alter theslope of the Dpp signaling output (Vuilleumieret al. 2010).

Because pent expression is negatively regu-lated by Dpp signaling, cells along the flankssense less Dpp as the disc grows in size, andbegin to produce Pent, which then throughfeedback appears to facilitate Dpp gradient ex-pansion (Vuilleumier et al. 2010; Ben-Zvi et al.2011). The ability of Pent to facilitate internal-ization of Dally and Dlp has been proposed tomodify the ability of cells to trap and transduceBMP by fine-tuning the levels of the BMP re-ception system at the plasma membrane (Nor-man et al. 2016).

The Role of Gbb in Wing Growth andPatterning

Although the previous sections emphasize thecritical role of Dpp as a morphogen that speci-fies wing growth and patterning, it is importantto recognize that, just like in the embryo, a sec-ond BMP ligand (Gbb) is necessary for properdisc growth and wing patterning. Gbb is widelyexpressed in the wing disc, contrary to Dppwhose expression is restricted to cells alongthe A/P compartment boundary (Khalsa et al.1998). Gbb loss results in similar wing growthand patterning defects, as does Dpp loss, al-though generally not as severe (Ray and Whar-ton 2001). As in the embryo, both heterodimersand homodimers of these BMP ligands likelyexist, but their respective roles in activatingthe observed spatial and temporal BMP signalresponses are not clear (Ray and Wharton 2001;Bangi and Wharton 2006). As in the embryo,genetic evidence suggests that BMP signal trans-duction in the wing disc uses heteromeric re-ceptor complexes containing combinations ofthe type I receptors Tkv or Sax together withtwo molecules of the type II receptor Punt. Al-though Tkv appears to mediate signals fromboth Dpp and Gbb, Sax shows the novel abilityto both promote and antagonize signaling, andthis ability is proposed to be dependent on itsreceptor partner (Bangi and Wharton 2006).When partnered with Tkv, Sax is thought tofacilitate BMP signaling, but, as a homodimer,it fails to signal effectively and instead sequestersGbb. This model proposes a balance betweenantagonizing and promoting BMP signaling

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across the wing pouch that helps shape the BMPactivity gradient.

Transcriptional Responses to BMP Signalingin the Wing Disc

The morphogen model predicts that targetgenes of BMP signaling should be differentiallyexpressed within the morphogen gradient field.Indeed, spalt (sal), optomoter blind (omb), andbrinker (brk) were identified early on as likelyBMP-responsive genes (Fig. 3A) that are differ-entially expressed along the A/P axis (Lecuitet al. 1996; Nellen et al. 1996; Campbell andTomlinson 1999; Jazwinska et al. 1999; Minamiet al. 1999). Both omb and sal encode transcrip-tion factors that are required for proper pattern-ing and cell survival in the central region of thedisc, whereas brk encodes a repressor that isexpressed in the lateral regions of the disc. brkexpression is negatively regulated in the centerof the disc by silencer elements (SE) that bind aheteromeric complex of transcription factorscomposed of pMad and Medea, together withanother transcription factor, Schnurri (Kim

et al. 1997; Affolter and Basler 2007). Surpris-ingly, the induction of omb and sal by Dpp ac-tually occurs by repressing brk, which is itself arepressor of omb and sal. The result is that highlevels of Dpp signaling directly silence brk in thecenter of the disc so that its expression pattern isan inverse complement to that of the Dpp gra-dient, low in the middle and high at the edges(Fig. 3A). The graded expression of brk in themore central regions of the disc in turn gives riseto the nested expression patterns of omb and sal(Fig. 3A).

Although expression of omb and sal are in-directly controlled by Dpp signaling, direct tar-gets have also been identified, including dad,which codes for an inhibitory Smad (Tsunei-zumi et al. 1997). Dad affects the robustness ofthe Dpp signaling gradient (Ogiso et al. 2011)by blocking Tkv-meditated phosphorylation ofMad and oligomerization of pMad (Inoue et al.1998). Within the central region of the disc, dadexpression is controlled by an activating ele-ment (AE) in its regulatory sequences that di-rectly responds to Dpp signals. The AE DNAsequence is similar to that of the SE and recruits

sal

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Figure 3. Dpp signaling during Drosophila wing development. (A) During larval development, dpp (blue, top) isexpressed along the A/P border and its gene product spreads to both compartments (blue arrows). High Dppsignaling in the middle of the disc results in activation of Mad (blue, below), which silences brk (red). The inversegradients of pMad and Brk form the nested expression pattern of sal and omb. pMad is slightly lower in cellsabutting the A/P axis due to local tkv down-regulation (Funakoshi et al. 2001). (B) Posterior crossvein (PCV)forms between the L4 and L5 veins (top), which begins during pupal development. Dpp–Gbb heterodimersbound by Sog (purple) and crossvein (dark green) allow for facilitated diffusion of ligands into the presumptivePCV space. Tolloid-related (Tlr) cleaves Sog and allows for signaling to occur. The initial broad signaling inducesexpression of CV-2, which further sharpens the signaling, resulting in PCV formation.

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a trimeric pMad–Medea complex, but differs atcertain nucleotide positions, such that the AEdoes not recruit Schnurri (Weiss et al. 2010). Asa result, it is able to promote rather than represstranscription. The lateral boundary of dad acti-vation is controlled by Brk repression. The com-bined activities of AE and SE elements, togetherwith Brk-binding sites, appears to explain thebulk of BMP response gene expression patternsso far identified in the wing disc.

Role of Dpp in Regulating Tissue Growth

In addition to controlling patterning within thewing disc, Dpp also regulates tissue growth.Loss of dpp leads to very small discs (Spenceret al. 1982; Zecca et al. 1995) while gain of Dppsignaling throughout the disc produces over-growth (Capdevila and Guerrero 1994; Burkeand Basler 1996; Martin-Castellanos and Edgar2002). Even expressing extra dpp within its nor-mal source using dpp-Gal4 causes a mild en-largement of the larval wing disc with someextra vein defects (Entchev et al. 2000; Telemanand Cohen 2000). When expressed locally inclones, it can also lead to the formation of anentirely new secondary winglet attached to anormal wing (Zecca et al. 1995).

How BMP signaling can regulate bothgrowth and patterning in the wing disc hasbeen an enigma for quite some time. The di-lemma arises because cell proliferation withinthe wing disc is uniform (Milan et al. 1996)and patterning is clearly coupled to gradientformation and interpretation. Explaining howuniform proliferation can be produced by agraded pattern of Dpp has proven difficult. Sev-eral hypotheses have been put forth includingthe idea that proliferation depends on the slopeof the Dpp gradient (Day and Lawrence 2000),the rate of change in Dpp signal reception (Wart-lick et al. 2011b), and the “growth equalizationmodel” (Restrepo et al. 2014). At present, themechanism for producing uniform growth isunclear because all the models have some con-flicts with certain observations. The major con-flict arises from analysis of brk mutants and ex-pression. That Brk plays a central role inregulating wing disc growth was initially re-

vealed by analysis of loss-of-function clones,which in the flanks of the disc also leads to wing-let formation as does the gain of BMP signaling(Campbell and Tomlinson 1999). Subsequently,it was found that increased expression of brk inthe entire disc leads to a severe reduction in discsize (Martin et al. 2004; Schwank et al. 2008)similar to loss of dpp. The real surprise, however,was that wing discs not only grow, but actuallyovergrow if they lose both Brk and Dpp (Camp-bell and Tomlinson 1999; Schwank et al. 2008,2012). This observation suggests that neither theslope model nor the temporal dynamics modelis correct. One alternative is that Dpp and Brk donot directly regulate proliferation of wing disccells, but instead modulate an underlying non-uniform proliferation pattern that is the result ofother growth promoting signals such as insu-lin–TOR and/or the Hippo–Fat, and otherTGF-b signaling pathways (Restrepo et al. 2014).

Two additional studies using novel indepen-dent reagents, suggest that the Dpp gradient isnot required for uniform growth (Hamaratogluet al. 2011; Akiyama and Gibson 2015). In thefirst study, dpp is deleted from the cells inthe wing disc that normally express Dpp, usinga UAS.GAL4/FRT system (Akiyama and Gib-son 2015). Surprisingly, the disc cells proliferateuniformly, resulting in a normal size tissue, eventhough brk expression is up-regulated in thewing pouch. This result argues against theDpp-gradient and the growth equalizationmodel and instead supports the temporal dy-namics model perhaps due to other non-A/Pboundary disc cells that still produce Dpp. Inthe second method, Dpp diffusion is restrictedby a nanobody trapping method and once againthere is uniform disc growth, supporting thegrowth equalization model (Harmansa et al.2015). Going forward it will be important toreconcile these observations. Furthermore, ifand how Gbb signaling contributes to discgrowth in the absence of a Dpp gradient willbe interesting to explore.

BMP Signaling in Pupal Wing Development

Dpp and Gbb also play important roles in veinspecification and positioning, which begins in

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late larval stages and continues throughout pu-pal development (de Celis 2003; O’Connor et al.2006). During the late larval stages, induction ofthe BMP targets, sal and omb, dictate wheresome of the longitudinal veins (LVs) will form(de Celis 1997, 2003). Soon after pupariation,dpp expression is lost at the A/P boundary andbecomes localized to cells that form the LVs, andhigh levels of pMad are detected in these pre-vein cells (Conley et al. 2000). Intriguingly, theposterior crossvein (PCV), that forms slightlylater at �20 h after puparium formation, alsorequires BMP signaling, but Dpp is not ex-pressed in primordial PCV cells. Nevertheless,these cells receive high levels of BMP signal asevidenced by strong pMad staining (Ralstonand Blair 2005). The mechanism appears to besimilar to that used in the embryo where theDpp or a Dpp–Gbb heterodimer is shuttledaway from the primordial LVs and concentratedat the site of the future PCV (Matsuda andShimmi 2012). Just as in the embryo, this shut-tling process requires several extracellular BMPmodulators, including Sog, CV-2, CV-1 (a Tsghomolog), and Tlr (a Tld homolog) (Fig. 3B)(O’Connor et al. 2006). One novel componentnot used for shuttling in the embryo is cross-veinless-D, which encodes a vittelogenin-likelipoprotein that appears to regulate PCV forma-tion by modulating Dpp movement as part of alipid–Dpp–lipoprotein complex (Chen et al.2012).

Mad Is a Focal Point For Integration of Cross-Pathway Signals

Dpp signals are integrated with numerous othergrowth and patterning signals during imaginaldisc development. In many cases, this interac-tion occurs through Mad, and two importantpoints of integration within the wing disc in-volve the Hippo and Wingless (Wg) signalingpathways (reviewed in Baena-Lopez et al. 2012).The Hippo pathway can promote disc growthvia regulation of the nuclear localization of thetranscription factor Yorkie (Yki; Yap in verte-brates), which binds to promoters of certaincyclins and the microRNA encoded by bantam(ban) (reviewed in Oh and Irvine 2010). The

microRNA ban is particularly interesting be-cause it also responds to Dpp signaling (Martinet al. 2004) and a direct link between the controlof ban levels by Dpp and Hippo has been estab-lished (Oh and Irvine 2011). On its own, Yorkieis not able to bind DNA but can do so when in acomplex with other transcription factors. Madcan form a complex with Yki (Oh and Irvine2011), and this complex is mediated through aPPXY motif located in the linker region betweenthe Mad homology 1 (MH1) DNA-binding do-main of Mad and the carboxy-terminal MH2domain, a more typical site of regulatory inter-action between Mad and other proteins. TheMad–Yki complex was also shown to bind toa novel enhancer element in the ban promoterto augment ban transcription, thus providingan integration point for these two growth pro-moting signals (Oh and Irvine 2010).

Mad is also the focal point of interactionwith the canonical Wg pathway (Eivers et al.2009, 2011). Both in vivo and in vitro studiesshowed that unphosphorylated Mad forms acomplex with Armadillo (homolog of verte-brate b-catenin) to regulate wg target genes inboth the embryo and wing disc. Upon Dpp sig-naling, Mad is titrated away from this complex,resulting in Wg–reporters being significantlydown-regulated (Eivers et al. 2011). Once again,the linker region between the MH1 and MH2domains of Mad plays an important role in me-diating regulatory interactions between the twopathways. The linker region possesses severalserines and tyrosines, which are phosphorylatedby various kinases to control Mad stability(Kretzschmar et al. 1997; Pera et al. 2003; Alar-con et al. 2009). In particular, glycogen synthasekinase-3 (GSK3)-mediated phosphorylation ofthe linker is important for initiating Mad deg-radation via Smurf1, and it was proposed thatboth the Wg and Dpp functions of Mad areterminated by GSK3-mediated phosphoryla-tion of the linker (Eivers et al. 2011). An addi-tional complexity in the interplay between thesetwo pathways has also been uncovered where inone region of the wing blade, phosphorylationof the Mad linker by GSK3 does not lead todegradation. Instead, the linker-phosphorylat-ed Mad controls certain cell divisions that lead

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to the formation of selected sensory bristles onthe wing (Quijano et al. 2011).

A third signal intersection converging onMad takes place between the activin and theBMP signaling branches in the wing disc. Al-though these two branches appear to have di-verged from one another long ago, the activin-specific type I receptor Babo retains an ability toactivate Mad by carboxy-terminal phosphory-lation (Peterson et al. 2012). Whether this car-boxy-terminal Mad phosphorylation activity isused during normal development is still notclear, but results from using a dSmad2 null mu-tant further supports the idea that cross-signal-ing by the activin pathway impinges on Madactivity (Sander et al. 2010; Peterson andO’Connor 2013). Interestingly, when dSmad2protein is absent, Babo sends an ectopic signalthat leads to pronounced overgrowth of thewing disc. The excessive growth is caused bydown-regulation of brk, and this down-regula-tion requires binding of a Mad–Medea–Schnurri complex to the brk SEs. However, therewas no detectable increase of carboxy-terminalMad phosphorylation, leading to the intriguingunresolved issue of how Mad is hyperactivatedby Babo in the absence of dSmad2. Taken to-gether, these studies indicate that Mad is clearlyat the nexus of integrating numerous signalingevents that control wing growth and patterning.

BMP Signaling in Other Imaginal Discs

So far, our discussion has focused on the role ofBMP signaling in wing disc growth and pattern-ing because it is the best studied of the imaginaltissues. Not surprisingly, however, BMP signalsalso play important roles in the development ofall imaginal discs. In fact, the name decapenta-plegic indicates that all 15 (decapenta) discs aredefective ( plegic) (Spencer et al. 1982). Here, webriefly examine the role of BMP signaling in theleg, eye and genital imaginal discs. More de-tailed reviews cover the development of the eye(Curtiss and Mlodzik 2000), leg (Estella et al.2012), and genital discs (Keisman and Baker2001; Sanchez et al. 2001; Estrada et al. 2003).

In the leg disc, Dpp synergizes with Wg togive proximal/distal (PD) positional informa-

tion to the tissue with distal being the center(Lecuit and Cohen 1997; Estella and Mann2008). Along the A/P boundary of the leg discdpp is expressed in the dorsal compartment andwg in the ventral. This creates a zone in thecenter of the disc where both ligands are presentat high concentrations, which is required to in-duce the expression of the distal marker distalless (dll). Cells surrounding the dll expressiondomain sense moderate levels of Dpp and Wgand turn on dachshund (dac), a medial legmarker. Cells in the periphery give rise to prox-imal domains of the leg and sense low levelsof the ligands leading to expression of homo-thorax and teashirt. Patterning of dll and dacare thought to result from brk repression, amechanism that poses striking similarity withthe wing disc (Estella and Mann 2008). Thus,although the details of patterning in the leg discare different from those in the wing, the basicfunction of Dpp as a morphogen that functions,in part, through the repression of brk, is never-theless the same in both tissues.

In the eye imaginal disc, the responses toDpp are notably different from those in otherdiscs. In this tissue, dpp is expressed only in themorphogenetic furrow, which migrates posteri-or to anterior during late larval stages (Baker2001). The Dpp signaling response varies de-pending on the position of the cells relative tothe furrow. Cells anterior of the furrow are stim-ulated to proliferate, whereas cells in and abut-ting the furrow undergo cell-cycle arrest (Baker2001). Cell-cycle arrest is necessary for propereye differentiation because mutant clones defec-tive in Dpp signaling are delayed in entering G1

arrest and do not fully differentiate (Horsfieldet al. 1998; Firth et al. 2010). Although themechanism for the dual nature of Dpp signalingto induce both cell proliferation and G1 arrest isunresolved, it is likely that multiple signalingpathways, including those activated by Wg,Hedgehog (Hh), Notch, and EGF, finely tunethis process (Amore and Casares 2010).

Dpp signaling also plays an important rolein genital disc development, which gives rise tothe adult internal and external genitalia andanalia. Several distinguishing characteristics ofthis disc set it apart from the others. It is the

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only disc that is located medially, formed by thefusion of cells originating from three embryonicsegments, it is an unpaired disc (i.e., not one foreach side of the larva) and it is sexually dimor-phic (Sanchez and Guerrero 2001; Estrada et al.2003). Nevertheless, the genital disc has somesimilarity to other discs, such as the leg, in thesense that hh expression in the posterior com-partment induces expression of dpp and wg inthe anterior compartment, which in turn in-duce dll expression that is required for develop-ment of the disc in both males and females(Gorfinkiel et al. 1999; Sanchez and Guerrero2001; Sanchez et al. 2001).

TGF-b FAMILY SIGNALING IN THENERVOUS SYSTEM

Activins and BMPs at the NeuromuscularJunction

The Drosophila NMJ, a collection of synapsesmade on body wall muscles by motor neurons,has long served as an excellent model for study-ing synapse formation and function. The NMJgrows substantially during larval life to accom-modate the rapidly growing body wall muscles,and several trans-synaptic signaling pathwayshave been shown to play important roles in co-ordinating the pre- and postsynaptic develop-ment at the larval NMJ (Dani and Broadie2012). Both branches of TGF-b family signalinghave important roles at the NMJ (Fig. 4), andBMP signaling has been particularly well stud-ied. The primary ligand involved is Gbb, whichis expressed in the muscles and the central ner-vous system (CNS) beginning at the embryonicstage (McCabe et al. 2003). Mutations in gbbcause a loss of pMad accumulation in motorneuron nuclei as well as a reduced NMJ size,aberrant presynaptic ultrastructure, and defec-tive neurotransmission (McCabe et al. 2003).The defective synaptic growth of gbb null mu-tants is only fully rescued when gbb expression isrestored in muscles and not in the CNS. Theseobservations suggest that the Gbb signal actsin a retrograde fashion to control presynapticgrowth in the motor neurons (McCabe et al.2003).

Similar phenotypes in pMad level, NMJsize, neurotransmitter release, and synaptic ul-trastructure were also observed in mutations ofthe type II receptor wit (Aberle et al. 2002; Mar-ques et al. 2002). The expression of wit is re-stricted to motor neurons in the neuromuscularsystem and restoration of wit in the motor neu-rons rescues synaptic size, supporting the ideathat Wit acts as the primary type II receptor forGbb in motor neurons (Aberle et al. 2002; Mar-ques et al. 2002). Likewise, null mutations in allother BMP signal transduction components,including tkv, sax, mad, and medea, phenocopythe gbb and wit null mutations indicating thatcanonical BMP signaling is the main regulatorof synaptic growth at the NMJ (Rawson et al.2003; McCabe et al. 2004).

The identities of target genes in motor neu-rons that regulate synaptic growth are largelyunknown. However, recent studies using molec-ular genetic tools that allow for temporal regu-lation of Gbb signaling at the NMJ identifiedsome molecular mechanisms through whichGbb affects synapse structure and function.These studies showed that synaptic structureand function use genetically separate pathwayswith an early transient embryonic signal neededfor synaptic growth, whereas continuous Gbbsignaling is required for maturation of neuro-transmitter release properties (Berke et al.2013). One BMP target gene required for regu-lating synaptic growth is trio, which encodes aguanine nucleotide exchange factor (GEF) thatacts on Rac GTPases. (Ball et al. 2010). Becauseprevious studies show that GTPases controlaxon growth and guidance through alterationsin the actin cytoskeleton (Dickson 2001; Fanet al. 2003), it was postulated that Trio couldpromote synaptic growth also by modulationof the actin cytoskeleton via Rac (Ball et al.2010). However, a direct relationship betweenRac activation and changes in actin filaments isyet to be shown. In parallel to stimulating Trioexpression, Wit signaling also leads to stimula-tion of Lim domain kinase 1 (Limk1) activitythrough a non-canonical pathway. Because Limkinase is implicated in rapid bouton budding inresponse to elevated synaptic activity and syn-aptic stabilization (Eaton and Davis 2005; Pic-

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Tkv

/Sax

Wit

Mad

MedTrio

D

Figure 4. Bone morphogenetic protein (BMP) and activin signaling at the Drosophila neuromuscular junction(NMJ). Motor neuron-released Actb induces Babo-mediated activation of dSmad2, facilitating association withMedea in the muscle. The pdSmad2-Medea complex then activates the transcription of glurIIB and an unknownfactor(s) that controls posttranscriptional processing or mRNA stability of glurIIA. On the other hand, muscle-released Gbb binds Wit and the Tkv-Sax complex in the motor neuron, and stimulates Mad phosphorylation. Theresultant pMad–Medea complex activates the transcription of trio and twit whose products promote synapticgrowth and control spontaneous vesicle release, respectively. The expression of Gbb in the muscle is fine-tuned byglia-released Mav. The dotted lines in this model depict speculations that should be verified in future studies.

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cioli and Littleton 2014), this may be yet anoth-er means by which the Gbb signal influencessynaptic growth.

In addition to promoting NMJ growthand presynaptic structural maturation, BMPsignaling also plays a pivotal role in regulatingsynaptic transmission at the NMJ. Strong BMPpathway mutants display reductions in the am-plitude of evoked junctional potential (EJP) andthe frequency of miniature EJPs (mEJP) (Aberleet al. 2002; Marques et al. 2002; McCabe et al.2003; Rawson et al. 2003). Unlike synapticgrowth that is predominantly regulated by ret-rograde BMP signaling, the defective synaptictransmission phenotypes of gbb mutants areonly completely rescued when gbb expressionis restored by a pan-neuronal driver (Aberle etal. 2002; McCabe et al. 2003), indicating thatpresynaptic Gbb is required for proper neuro-transmission. The smaller magnitude of EJPshas also implied that homeostatic regulationof synaptic efficacy is compromised in theBMP pathway mutants. It was later shown thatBMP signaling is continuously required in themotor neurons to confer competence for therapid induction of synaptic homeostasis (Gooldand Davis 2007). In terms of the effects on neu-rotransmission, the only BMP-responsive targetgene identified to date is twit, which encodes aLy6 neurotoxin-like molecule. Restoring twitexpression in wit mutant motor neurons par-tially rescues neurotransmitter release defects,although the molecular mechanism remainsobscure (Kim and Marques 2012).

Because BMP signaling has profound effectson NMJ structure and function, it is not sur-prising that its activity is tightly controlledto prevent uncoordinated growth of the synaps-es. Multiple molecular mechanisms have beenimplicated in regulating BMP signaling atthe NMJ. In muscles, Gbb release is inhibitedby Drosophila Cdc42-interacting protein 4(dCIP4) acting downstream of Cdc42 andactivating the Wsp (Drosophila orthologue ofmammalian Wiskott–Aldrich syndrome pro-tein, Wasp) pathway (Nahm et al. 2010a). TheCdc42-Wsp pathway is, in turn, inhibited byDrosophila Rich (dRich), a Cdc42-selectiveGAP (Nahm et al. 2010b). Therefore, a complex

regulatory loop appears to be used to controlthe release of Gbb. In the presynaptic motorneurons, hyperactivation of BMP signaling isprohibited by Highwire, a ubiquitin ligase thatpresumably targets Medea for protein degrada-tion (McCabe et al. 2004). The activated recep-tors are also subject to negative regulationby internalization. Mutations in nwk, spin, andspict whose products act in, or interact with,endocytic machinery result in overgrowth ofthe synapses (Sweeney and Davis 2002; Wanget al. 2007; O’Connor-Giles et al. 2008). Fur-thermore, genetic interactions of these geneswith BMP receptors have been documented(Sweeney and Davis 2002; Wang et al. 2007;O’Connor-Giles et al. 2008). Finally, retrogradesignaling of Gbb also occurs in the central mo-tor circuit, which is necessary to strengthen thesynaptic activity. Therefore, it appears that theretrograde signaling is a general theme by whichGbb regulates synaptic development and func-tion (Baines 2004). On the other hand, releaseof Gbb from the presynaptic side is promoted byCrimpy, a homolog of vertebrate Cysteine-richtransmembrane BMP regulator 1 (CRIM1),which binds Gbb and delivers it to a densecore vesicle (DCV) (James and Broihier 2011).Interestingly, the ectodomain of Crimpy is alsoreleased together with Gbb from the DCV onexocytosis. This has been proposed to help dis-criminate the presynaptic and postsynapticpools of Gbb, thus setting the directionality ofBMP signaling at the NMJ.

The retrograde Gbb signaling at the NMJresults in accumulation of pMad in the nucleiof motor neurons. In addition to the nuclearpMad, another pool of pMad has been identi-fied. This pool of pMad is localized to the pre-synaptic side of the synapses at the NMJ and isregulated differently from the nuclear pMad(Sulkowski et al. 2014, 2016). Specifically, thelevel of synaptic pMad is independent of Gbb,but is positively regulated by the activity of post-synaptic iGluRs containing glutamate receptorIIA (type A receptor). The synaptic pMad inturn stabilize the A-type glutamate receptorssuggesting a positive feedback loop betweenthe presynaptic and postsynaptic molecules.This has been proposed to be a mechanism to

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coordinate the maturation of synapses to theactivity.

In contrast to BMP signaling, whose role insynaptic formation and function is well docu-mented, relatively little is known about the roleof the activin pathway in the regulation of NMJdevelopment and activity, even though all threeactivin ligands are expressed in at least one celltype of the neuromuscular system (Brummelet al. 1999; Lo and Frasch 1999; Zheng et al.2003; Serpe and O’Connor 2006; Gesualdi andHaerry 2007). Studies of babo, dSmad2, andactb mutants indicate that Actb is a motor neu-ron-derived anterograde signal that is necessaryfor achieving normal levels of GluRIIA andGluRIIB densities in muscle at the postsynapticside of the NMJ (Fig. 4), as well as proper restingmembrane potential of the muscle (Kim andO’Connor 2014). Actb appears to control thesynaptic GluRIIA density through a posttran-scriptional mechanism while it regulates theGluRIIB density via dSmad2-dependent tran-scriptional activation.

Lastly, glia-secreted Mav, a more distantlyrelated TGF-b family ligand whose signalingbranch is unclear, has been implicated in regu-lating Gbb release from the muscle (Fuentes-Medel et al. 2012). This may provide a way tofinely tune presynaptic axon growth and bud-ding with muscle growth. Together, the findingssuggest that the two branches of TGF-b familysignaling play distinct roles in regulating syn-apse growth, maturation and function at theNMJ (Fig. 4).

Activin Signaling Stimulates NeuroblastProliferation

In addition to its role at the NMJ, activin sig-naling also has other roles in the nervous systemincluding regulation of neuroblast prolifera-tion, control of neurite outgrowth and targetselection, as well as neuronal remodeling duringmetamorphosis. These activities are accom-plished by deployment of the activin-type li-gands, that is, Actb, Daw, and Myo, from differ-ent source cells in the nervous system acting ondifferent target cells. In most cases, this signal-ing is canonical through Babo and dSmad2, but

there is one example in which noncanonicalsignaling appears to be used.

One major function of activin signaling inthe nervous system is to stimulate production ofneurons, which are generated from neuroblastsin the brain. Loss-of-function mutations inbabo and dSmad2 produce small brains due tofewer neuroblasts and reduced proliferation ofneuroblasts and their daughter cells, whereasreexpression of Babo specifically in neuroblastsrescues brain size (Zhu et al. 2008). The ligandsActb and Daw have been implicated in this pro-cess. actb is expressed in numerous neuronswithin the central brain and ventral ganglia,whereas daw is expressed in subsets of glial cells.Myo may also be involved, since it, too, is ex-pressed in many glial cells (Lo and Frasch 1999).Mechanistically, activin signaling appears tocontrol proliferation at the S to M phase transi-tion of the cell cycle (Zhu et al. 2008).

Activin Signaling and Axon Guidance andTarget Selection

In addition to generating neurons, activin sig-naling also regulates several aspects of axon tar-geting in different contexts. In the embryonicnervous system, activin signaling plays a role inguiding motor neuron axons to their targetmuscles. In daw mutant embryos, motor neu-rons leaving the CNS have path-finding defectsin both intersegmental nerve and segmentalnerve roots (Parker et al. 2006; Serpe andO’Connor 2006). These defects are also seenin babo and dSmad2 mutant embryos (Parkeret al. 2006; Serpe and O’Connor 2006) indicat-ing that canonical signaling is responsible. Be-cause expressing dominant-negative forms ofeither Babo or Punt in the motor neurons alsocause path-finding defects, the target of Dawsignaling appears to be the motor neuron itselfand not the muscle (Parker et al. 2006). In ad-dition, because Daw expression in either themuscle or glia rescues path-finding defectsand excess Daw expression is not detrimental,Daw appears to provide a permissive cue formotor neuron axon guidance (Parker et al.2006). Interestingly, mutants in tlr also pheno-copy the path-finding defects of daw mutants

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(Serpe and O’Connor 2006). Tlr was found tocleave the prodomain of Daw, as well as Actband Myo, and, in the case of Daw, cleavage en-hances signaling in cell culture assays. Theseobservations suggest that, as described abovefor Scw, prodomain processing of activin-likeligands at sites distinct from the normal furinmaturation sites may be important for full sig-naling activity, perhaps by stimulating completedissociation of the prodomain from the car-boxy-terminal ligand domain (Serpe andO’Connor 2006). Similar activating cleavagesin prodomains of several vertebrate ligandshave also been reported (Wolfman et al. 2003).

During the larval stage, photoreceptor ax-ons must be targeted properly for light and col-or information to be processed correctly, andActb plays a role in regulating proper photore-ceptor axon termination in the medulla. Clonalloss of babo or dSmad2 in R7 photoreceptorscauses their axons to target to the correct layer,but move laterally and enter the wrong column(Ting et al. 2007). Actb is expressed in R7 pho-toreceptors, and Actb RNAi in the photorecep-tors also causes axon mis-targeting (Ting et al.2007). These studies indicate that Actb expres-sion in R7 photoreceptors restricts their target-ing to a single column in the medulla throughan autocrine signaling mechanism (Ting et al.2007).

Activin also signals in a juxtacrine mannerto negatively regulate dendritic branching andtermination of medulla interneurons, which re-ceive input from photoreceptors. In this case,Actb derived from R7 or R8 photoreceptors sig-nals to Dm8 or Tm20 medulla neurons, respec-tively, to regulate their dendritic field size (Tinget al. 2014). Loss-of-function babo and dSmad2mutant clones in medulla interneurons produceexpanded dendrites, whereas neurons with in-creased reception of activin signals have reduceddendrites. In loss-of-function mutants the ex-panded dendrites form ectopic membrane con-nections with presynaptic sites on multiplephotoreceptors, potentially disrupting visualprocessing (Ting et al. 2014).

Activin signals also function to regulate MBneuron b-lobe axon growth. MB neurons areresponsible for olfactory learning and memory.

Expression of activated Babo causes axon trun-cation, whereas loss of babo results in over-growth (Ng 2008). This is independent ofdSmad2 and requires the actin cytoskeleton reg-ulators Limk1, Rho, and Rac. The identity of theresponsible ligand has not been determined.This example is one of only two cases in Dro-sophila in which non-Smad signaling is respon-sible for mediating a response.

Activin Signaling and Neuronal Remodeling

Apart from roles in axon guidance and targetselection, activin signaling is also required dur-ing metamorphosis for MB remodeling. TheMB undergoes stereotypic remodeling duringthe pupal phase to eliminate larval-specific con-nections and to form new connections requiredfor proper adult behaviors. The larval MB hastwo major branches of g neurons: medial anddorsal. Both branches are pruned during meta-morphosis, and only the medial lobe remains inthe adult MB. Mutations in babo and dSmad2prevent pruning of dorsal and medial g neuronsof the MB, and these defects are rescued by ex-pressing the proper wild-type gene in the MBneurons (Zheng et al. 2003). One major targetof activin signaling in the remodeling MB neu-rons is the gene encoding the ecdysone receptorEcR-B1. Ectopic expression of EcR-B1 in theMB neurons partially rescues the pruning defect(Zheng et al. 2003). Although it was originallysuggested that Actb was the responsible ligandbased on RNAi phenotypes, further studies us-ing loss-of-function mutants show that glial-derived Myo provides the signal (Awasaki et al.2011). In this example, Myo appears to signalprimarily through the BaboA isoform (Awasakiet al. 2011) and can use either Punt or Wit as atype II receptor because only when both areeliminated is a MB remodeling defect observed(Zheng et al. 2003).

This signaling event is also noteworthy be-cause efficient signaling requires a novel core-ceptor in addition to the normal type I and typeII receptors. Genetic screens for additional fac-tors that regulate MB remodeling identifiedplum, a gene that codes for an immunoglobulinsuperfamily protein. Mutations in plum lead to

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loss of EcR-B1 expression and defective MB re-modeling. Because these phenotypes can be res-cued by expressing activated Babo in MB neu-rons, but not by overexpressing Plum in babomutant MB clones, Plum appears to act up-stream of receptor activation (Yu et al. 2013).Limited structure-function studies show thatthe extracellular domain of Plum is requiredfor rescue, but the cytoplasmic domain is dis-pensable (Yu et al. 2013). At present, the bestguess is that Plum acts to either stabilize recep-tor complexes and/or to facilitate Myo bindingto the receptor complex, although no biochem-ical interactions between any of these compo-nents have been reported.

ROLE OF TGF-b FAMILY SIGNALING INPHYSIOLOGICAL AND METABOLICHOMEOSTASIS

Roles of TGF-b family signaling in develop-mental processes are well established and exten-sively studied. Nevertheless, TGF-b family sig-naling components continue to be expressedthroughout the lifespan of an animal and invarious adult tissues, signifying potential rolesof the TGF-b family signaling pathways in keyphysiological or homeostatic processes. Investi-gating the role of TGF-b family signaling inphysiology is an active field of research, andthe involvement of the TGF-b family in the reg-ulation of physiological homeostasis in Droso-phila larvae and adults has been shown in anumber of studies. In this section, we highlightour understanding of how TGF-b family signal-ing in Drosophila regulates the following fourkey homeostatic and physiological processes:(1) metabolic homeostasis, (2) hormonal sig-naling, (3) innate immunity, and (4) tissue ho-meostasis.

TGF-b Family Signaling in MetabolicHomeostasis

TGF-b family signaling components are ex-pressed in metabolically active tissues in bothvertebrates and invertebrates, and their roles inregulating metabolism are gaining attention(Andersson et al. 2008; Bertolino et al. 2008;

Zamani and Brown 2011; Ghosh and O’Connor2014). In Drosophila, both the TGF-b familyligand Dawdle (Daw) and the BMP ligandGlass-bottom boat (Gbb) have been implicatedin metabolic regulation.

The activin-like ligand Daw regulates insu-lin/insulin-like growth factor signaling (IIS) infeeding third instar larvae (Ghosh and O’Con-nor 2014). Consistent with loss of IIS, dawmutant larvae show a significant increase incirculating sugar (glucose þ trehalose) con-centration, total triacylglycerol and glycogencontent, and a significant reduction in Akt ac-tivation in the peripheral tissues (Ghosh andO’Connor 2014). Although these mutant ani-mals did not show any change in expression ofdilps in the insulin producing cells (IPCs), theywere defective in the release of Dilps from theIPCs (Ghosh and O’Connor 2014).

Independent of its role in regulating IIS,canonical Smad signaling downstream fromDaw also regulates the internal pH balance,most likely by altering the output of acidicTCA cycle intermediates primarily in the fatbody (FB) (Ghosh and O’Connor 2014). FB-specific RNA sequencing of daw mutants andcontrol flies revealed increased expression ofmultiple nuclear-encoded mitochondrial genes,including enzymes of the TCA cycle, electrontransport chain, b-oxidation and ketogenesisin the daw mutants (Ghosh and O’Connor2014). Additionally, metabolomics analysis re-vealed increased accumulation of multiple TCAcycle intermediates in these mutants. These re-sults point toward a transcriptional role of Dawin mitochondrial biogenesis, energy metabo-lism, TCA cycle activity and pH balance. Theinvolvement of Daw in expression of nuclearencoded mitochondrial genes may be reminis-cent of the proposed role of activin A in mito-chondrial biogenesis in vertebrates (Li et al.2009; Zamani and Brown 2011).

Daw may regulate metabolic processes in asystemic dose-dependent manner. Expressingdaw in any larval tissue could completely rescuelarval lethality associated with mutations indaw, and had a graded effect in rescuing themetabolic phenotypes of daw mutants depend-ing on the size of the tissue. Analyses of phos-

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pho-dSmad2 (pdSmad2) levels in the FBshowed that Smad2 activation in the larval FBis primarily mediated by Daw and that the FB ispoised to respond to a wide range of Daw sig-naling. Consistent with the endocrine nature ofDaw signaling, expressing daw in a variety oflarval tissues could rescue pdSmad2 levels in adaw mutant FB (Ghosh and O’Connor 2014).These results indicate that Daw acts like a met-abolic hormone capable of dose-dependentlyregulating cellular metabolism. Intriguingly,unlike most hormones, daw is expressed in mul-tiple larval tissues, and it will be interesting tolearn how each of these tissues uses this meta-bolic ligand for systemic communication. Onepossibility is that daw expression in each tissueis regulated by different environmental stimuliand that Daw then acts on other tissues to bringabout systemic changes to cope with the stimuli.

The involvement of the Drosophila TGF-bfamily ligand Daw in sugar homeostasis is fur-ther shown by work showing that nutrient-sen-sitive expression of Daw is required for inducingglucose repression (GR) in the Drosophila intes-tine (Chng et al. 2014). GR is a phenomenonwhereby feeding of excessive dietary glucoseleads to repression of amylase in the gut, pre-sumably in an effort to protect the animalagainst deleterious effects of a high sugar diet(Hickey and Benkel 1982; Benkel and Hickey1986; Musselman et al. 2011; Na et al. 2013).Smad signaling activated by Babo and dSmad2mediates GR by negatively regulating the ex-pression of digestive enzymes (amylases, hydro-lases, and lipases) in the gut in response to highsugar diet (Chng et al. 2014). This effect is me-diated by the Babo isoform C in the gut, whichis specific to Daw signaling (Jensen et al. 2009;Chng et al. 2014). Importantly, in response tohigh sugar diet, Daw is released from the adultfat body and signals to the gut to repress expres-sion of digestive enzymes including amylase,further confirming the endocrine nature ofDaw signaling. It is interesting to note that ex-pression of oxido-reductase genes is also signif-icantly suppressed in the gut when flies are kepton a high sugar diet. Because a high sugar dietincreases Daw signaling in the gut, this findingis consistent with a role for Daw signaling in

repressing the expression of mitochondrialgenes, including oxido-reductases, similar towhat is seen in whole animal daw mutants(Ghosh and O’Connor 2014).

Additional evidence suggests that Daw isone of the primary effectors of a conserved sug-ar-sensing pathway mediated by the ChREBP/MondoA-Mlx complex (Mattila et al. 2015).The MondoA-Mlx transcription factors are ac-tivated by sugars and are essential for mediatingmultiple downstream processes essential forconversion of sugars to lipids and usage of sug-ars. The daw promoter contains a carbohydrateresponse element that is occupied by Mlx inboth S2 cells and in Drosophila larvae indicatingthat daw expression is directly regulated byMondo-A/Mlx. Additionally, Daw is requiredfor and regulates the expression of a secondkey target of Mondo-A/Mlx called sugarbabe(sug) (Mattila et al. 2015). These findings putDaw firmly in a major sugar sensing and sugarmetabolism pathway in Drosophila. Whether ac-tivin signaling in vertebrates is also a down-stream target and effector of a Mondo-A/Mlxcomplex remains to be determined.

Daw has also been implicated in lifespanextension in Drosophila. A screen for transcrip-tional targets of dFOXO identified daw as a po-tential target, and muscle-specific knockdownof daw leads to increased lifespan (Bai et al.2013). Loss of daw in the muscle affects lifespanpossibly by increasing macro-autophagy thathelps remove age-related accumulation of pro-tein aggregates in the muscle, thereby improv-ing muscle function (Bai et al. 2013). Interest-ingly, daw-RNAi in the muscle can reduce Dilp2levels in circulation, whereas daw-RNAi in theFB leads to an increase in Dilp2 in the hemo-lymph (Bai et al. 2013). Although these obser-vations support a role of Daw in regulating IISin the adult fly as well, it remains to be explainedhow tissue-specific knockdown of a ligand,which clearly works as an endocrinal systemicsignal in the larva, can lead to opposing pheno-types in the adult. The relative contributions ofthe muscle and FB to circulating Daw levels andpotential compensatory changes in daw expres-sion when it is knocked down in any single tis-sue could explain some of these findings.

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The BMP ligand Gbb has also been pro-posed to regulate metabolic processes, initiallybased on a striking morphological defect exhib-ited by gbb mutants in which the FB takes on aglassy appearance instead of its normal opaque-ness (hence, the name Glass bottom boat).Characterization of these mutant larvae re-vealed that the FB shows nutrient storage andmorphological characteristics resembling a stateof starvation (Ballard et al. 2010). Additionally,gbb mutants show alterations in the expressionof multiple starvation response genes. Surpris-ingly, monitoring lipid uptake using a fluores-cently labeled lipid derivative (Bo-C12) revealsthat the gbb mutants show an increased rate ofnutrient uptake, indicating defects in the abilityto store nutrients. Although Gbb produced bythe FB regulates FB morphology and nutrientstorage in a cell-autonomous manner, Gbb sig-naling in the FB may only be partially responsi-ble for the increased nutrient uptake phenotypeobserved in gbb mutants (Ballard et al. 2010).The mechanistic details of how Gbb affects us-age of absorbed nutrients remains unexplained.

TGF-b Family Signaling Regulates HormonalControl of Drosophila Development

Like other insects, Drosophila uses two primaryhormonal signals, juvenile hormone (JH) andecdysone (E), to coordinately regulate transi-tions in larval development. TGF-b family sig-naling appears to work upstream of these hor-monal signals by controlling the production ofboth these hormones, presumably in responseto developmental or environmental cues (Gib-bens et al. 2011; Huang et al. 2011). Both gainand loss-of-function studies show that activinsignaling is required in the prothoracic gland(PG), the site of E production and release, togenerate the large pulse of E necessary for en-tering metamorphosis (Gibbens et al. 2011). Inthe absence of canonical activin signaling in thePG, the larvae arrest at the third instar stage anddo not undergo metamorphosis. This pheno-type results from a requirement for activin sig-naling in the PG to make it competent torespond to two other hormonal signals, pro-thoracicotropic hormone (PTTH) and insulin.

In the absence of activin signaling, expression ofTorso, the PTTH receptor, and InR, the insulinreceptor and several other downstream compo-nents in the insulin signaling pathway, are alldown-regulated in the PG. Because each of thesetwo signaling pathways is required for high-levelexpression of several key E biosynthetic en-zymes, their simultaneous loss eliminates pro-duction of the E pulse that is required for initi-ating metamorphosis. A remaining question isthe identity of the TGF-b ligand that signals inthe PG because loss of any one ligand (actb,daw, or myo) does not show larval developmen-tal arrest phenotypes. This observation suggestsredundancy among the ligands for providingcompetence to the PG for reception of PTTHand insulin signals.

In contrast to the involvement of activinsignaling in the control of E production, JHsynthesis is under the control of the BMP ligandDpp (Huang et al. 2011). JH is critical for de-termining the nature of developmental transi-tions in insects. In the presence of JH, E inducesa larval– larval molt, whereas, in the absence ofJH, E induces a larval–pupal or pupal–adultmolt. Using an elegant genetic screen, Tkv andMad were identified as positive regulators of JHsignaling in the larva (Huang et al. 2011). JH issynthesized in the corpus allata (CA), and glu-tamatergic signals emanating from the braininduce dpp expression in the CA. Dpp, thencell-autonomously induces production of theJH-synthesizing enzyme JH acid methytrans-ferase to stimulate JH production in the devel-oping larvae.

TGF-b Family Signaling in Drosophila InnateImmunity

In vertebrates, TGF-b signaling plays diverseroles in the regulation of immune responses(Li et al. 2006). This is also true in Drosophilain which both the BMP and activin pathwaysregulate different aspects of the innate immuneresponse (Clark et al. 2011). Expression of bothdaw and dpp responds to immune challenge,although the nature and context of the responsediffer between the two ligands. Expression ofdaw goes up in response to an infection. In this

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case, Daw appears to play an important role insuppressing infection-induced activation of theimmune response, in an effort to protect theanimal against immune-induced pathology. Incontrast, dpp expression increases and persists inresponse to sterile wounding, and Dpp has beenproposed to suppress unnecessary expression ofantimicrobial peptides (AMP). The presence ofa nearby Mad-Medea-Schnurri SE in manyAMP genes suggests that Dpp may directly reg-ulate AMP gene expression (Clark et al. 2011).

Role of TGF-b Family Signaling in TissueHomeostasis

Adult tissues showing a rapid turnover of livingcells require a constant supply of differentiatedcells to replenish old ones to maintain tissuehomeostasis. Adult stem cells accomplish thiscrucial task of tissue homeostasis. Each adultstem cell undergoes a stereotypical asymmetriccell division giving rise to one progenitor cellthat commits to differentiation and one stemcell that ensures maintenance of a healthy pop-ulation of precursor cells throughout the life ofan animal. This behavior of stem cells and stemcell identity itself are maintained by specializedmicroenvironments or “niches” generated bysupport cells. Studies using Drosophila haveshown crucial involvement of TGF-b family sig-naling components in stem cell niche mainte-nance and stem cell self-renewal.

In the Drosophila female germline, two orthree germline stem cells (GSCs) are located inclose contact with the niche cells (terminal fila-ment cells and cap cells) at the tip of each ovar-iole (Fig. 5A) (Lin 2002; Yamashita et al. 2005).TGF-b family signaling plays a crucial role dur-ing both the development of this niche at latelarval stages and during maintenance of theadult GSCs. In the late larval stages, the activinreceptor Babo is required for proliferation ofsomatic precursor cells that give rise to the ter-minal filament (TF) cells. Additionally, duringthe final differentiation of these precursor cellsactivin signaling interacts with ecdysone signal-ing to regulate expression of the target gene Br-Z1, and thereby regulates differentiation of theTF cells (Lengil et al. 2015). In the adult female

germline, GSCs undergo asymmetric cell divi-sion that give rise to one GSC and a cystoblast(CB). Each CB undergoes four rounds of syn-chronous divisions to produce 16 interconnec-ted cystocytes (CC) that ultimately differentiateinto ovarian follicles (Lin 2002). The DrosophilaBMP ligand Dpp plays a prominent role inmaintenance of GSCs in this tissue. Dpp is pro-duced and released by the TF and cap cells, re-sulting in a shallow gradient that elicits a strongBMP response in the GSCs but not in the distallylocated CBs (Xie and Spradling 1998, 2000; Kaiand Spradling 2003). BMP signaling in the GSCfunctions cell-autonomously through Mad andMedea to maintain GSC identity and inhibit dif-ferentiation (Xie and Spradling 1998). ExcessiveBMP signaling induced by ectopic overexpres-sion of dpp produces enlarged germaria filledwith cells that resemble GSCs, while null muta-tions in sax encode the BMP type I receptor andshorten the half-life of GSCs (Xie and Spradling1998; Kai and Spradling 2003). The effect ofBMP signaling on GSC maintenance is primar-ily mediated by the BMP target gene bag-of-mar-bles (bam) (Chen and McKearin 2003; Song et al.2004). Expression of bam is directly regulated bybinding of the Mad–Medea complex at repres-sor elements in the promoter region of bam(Chen and McKearin 2003; Song et al. 2004).Consequently, bam expression is high in differ-entiating, early germ cells and has been shown tobe necessary and sufficient for differentiation offemale GSCs.

The Drosophila BMP ligands, Dpp and Gbbhave also been shown to repress bam levels in themale germline. The function of this regulationdiffers between the male germline and the fe-male germline. In the female germline, bam ex-pression initiates differentiation of the stem cellprogeny. However, in the male germline, expres-sion of bam determines the number of tran-sient-amplifying divisions that spermatogoniaundergo before differentiating into spermato-cytes (Fig. 5B) (Shivdasani and Ingham 2003;Schulz 2004). BMP signaling in the male germ-line may also play a permissive role in the sur-vival and proliferation of the GSCs but does notdetermine GSC fate (Shivdasani and Ingham2003).

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TGF-b Family Signaling in Drosophila

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Apart from the germline tissue, BMP signal-ing also plays a role in tissue homeostasis in theadult midgut. Based on morphological andfunctional characteristics, the Drosophila adultmidgut can be broadly divided into three re-gions, the anterior midgut (AM), the middlemidgut (MM) and the posterior midgut (PM)(Fig. 6). Each of these compartments housesintestinal stem cells (ISCs), which are responsi-ble for the renewal of the various cell types inthe midgut (Micchelli and Perrimon 2006; Ohl-stein and Spradling 2006). The ISCs in the Dro-sophila midgut divide to produce a daughter cellcalled the enteroblast (EB), which, dependingon the level of Notch signaling, differentiatesinto either an enterocyte (EC) or an enteroen-docrine cell.

In the adult midgut, Dpp is expressed andsecreted by a group of ECs that are located atthe boundary between the PM and MM (Fig.6). Anterior to this junction, and containedwithin the MM, is an acidic region called thecopper cell region (CCR). The CCR houses spe-cialized “copper” cells, which secrete acids thatmaintain the lumen of the CCR at low pH. Dppsignaling plays an essential role in the specifica-tion of the coppercells from EBs (Guo et al. 2013;Li et al. 2013a). Disrupting the Dpp gradient inthe MM by overexpressing dpp in all ECs causesan increase in the presence of copper cells in theCCR and a progressive expansion of the CCR inthe AM (Li et al. 2013a). Conversely, silencingmad or tkv in the CCR leads to a complete loss ofthe low luminal pH associated with CCR, and aloss of Labial-expressing cells in the CCR, indi-cating a loss of functional copper cells (Guo et al.2013).

BMP signaling may also play an importantrole in regulating the choice between ISC and EBcell fate in the intestine. Loss of BMP signaling inprecursor cells has been shown to cause rapidloss of ISCs accompanied by an increase in thenumber of EB (Tian and Jiang 2014). Addition-ally, the strength of BMP signaling is asymmetricin ISC and EB cell pairs, with the basally locatedISCs showing much stronger BMP signalingthan apically located EBs, indicating a role forBMP signaling in maintaining ISC identity.Consistent with these observations, loss of

BMP in precursor cells leads to formation ofmore EB–EB pairs, and gain of BMP in the pre-cursor cells results in formation of large ISC-likecell clusters and favors symmetric ISC–ISC di-vision of ISCs (Tian and Jiang 2014).

Apart from the physiological role in speci-fying copper cells and maintaining ISC identity,BMP signaling has also been proposed to regu-late ISC proliferation in the AM and PM regions(Guo et al. 2013; Li et al. 2013b; Tian and Jiang2014). The source of Dpp or Gbb that mediatesthis role and the nature of this regulation remaincontroversial based on seemingly contradictoryresults. Work from two groups suggests thatDpp signaling inhibits ISC proliferation in themidgut (Guo et al. 2013; Li et al. 2013a). Oneshows that Dpp secreted from the trachea indi-rectly affects ISC proliferation by protecting theloss of ECs (Fig. 6B). The second group suggest-ed that Dpp released from visceral muscle inresponse to intestinal injury works directlyon the ISCs to inhibit ISC proliferation andthereby helps contain injury-induced prolifera-tion of ISCs (Fig. 6A) (Guo et al. 2013). In con-trast, a third study proposed that both Gbb andDpp released from the ECs indirectly regulateISC proliferation by acting on the ECs andinhibiting ISC proliferation signals (Fig. 6C)(Tian and Jiang 2014). Another showed thatDpp can have a dual role in ISC proliferation,and that in response to gut damage, Dpp re-leased from the hemocytes activates ISC prolif-eration by signaling through Sax and Smox.Subsequently, to prevent overproliferation, theISCs switch on tkv expression and Dpp signal-ing through Tkv and Mad inhibits ISC prolifer-ation (Ayyaz et al. 2015). Although more workwill be necessary to fully understand the sourceof BMP ligands and the mechanism by whichthey regulate ISC proliferation and mainte-nance, these results clearly highlight a signifi-cant role of BMP signaling in maintaining Dro-sophila midgut homeostasis.

SUMMARY AND OUTLOOK

Studies of TGF-b family signaling in Drosophilahave uncovered a wide variety of roles for bothactivins and BMPs in the regulation of diverse

A. Upadhyay et al.

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TGF-b Family Signaling in Drosophila

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processes, including developmental patterning,neural development and function, innate im-munity, as well as metabolic and tissue homeo-stasis. However, numerous questions remain.Several noncanonical signal transducers werefound to interact with TGF-b family signaling,but most of the factors have not been identified.Accessory factors to core TGF-b family signal-ing components, such as the putative Myo co-receptor Plum, need to be further investigatedto determine the nature of their interactionswith the canonical signaling machinery. Otherinteresting areas of study include unravelinghow many ligands can signal in different direc-tions at structures like the synapse, and, impor-tantly, how their downstream effects can becompartmentalized. Determining to what de-gree different ligands act systemically throughthe circulatory system also requires attention,as does the issue of differential processing with-in ligand prodomains and how that might affectligand activity. The mechanistic basis of howTGF-b family signaling regulates metabolic ho-meostasis still remains to be explained, and ad-ditional roles of the pathway in regulating otheraspects of physiology need to be explored.Moreover, identification of nutritional and en-vironmental cues that work upstream to TGF-bfamily signals to regulate their roles in metabo-lism and homeostasis is also an important un-resolved question. Undoubtedly, addressingthese issues will keep Drosophila researchersbusy for some time, and the results shouldalso help inform studies of TGF-b family signal-ing in other systems.

ACKNOWLEDGMENTS

The authors thank MaryJane O’Connor and Ai-dan Peterson for critical reading of the manu-script and for helpful suggestions. This work issupported by Grant GM R01 GM95746 and R35GM118029 to M.B.O.

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TGF-b Family Signaling in Drosophila

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published online January 27, 2017Cold Spring Harb Perspect Biol  Ambuj Upadhyay, Lindsay Moss-Taylor, Myung-Jun Kim, Arpan C. Ghosh and Michael B. O'Connor 

Drosophila Family Signaling in βTGF-

Subject Collection The Biology of the TGF-β Family

Development Family Signaling in Early VertebrateβTGF-

Joseph Zinski, Benjamin Tajer and Mary C. MullinsDifferentiation

Family Signaling in MesenchymalβTGF-

Peterson, et al.Ingo Grafe, Stefanie Alexander, Jonathan R.

ApproachesBased Therapeutic−Bone Morphogenetic Protein

Jonathan W. Lowery and Vicki Rosen

1 Signaling and Tissue FibrosisβTGF-

ChapmanKevin K. Kim, Dean Sheppard and Harold A.

and Branching Morphogenesis Family Signaling in Ductal DifferentiationβTGF-

MoustakasKaoru Kahata, Varun Maturi and Aristidis

Homeostasis and DiseaseBone Morphogenetic Proteins in Vascular

et al.Marie-José Goumans, An Zwijsen, Peter ten Dijke,

Function Signaling in Control of CardiovascularβTGF-

Marie-José Goumans and Peter ten Dijke TransitionMesenchymal−Differentiation and Epithelial

Family Signaling in Epithelial βTGF-

MoustakasKaoru Kahata, Mahsa Shahidi Dadras and Aristidis

and Cancer Progression Family Signaling in Tumor SuppressionβTGF-

Joan Seoane and Roger R. GomisSkeletal Diseases

Family Signaling in Connective Tissue andβTGF-

Dietz, et al.Elena Gallo MacFarlane, Julia Haupt, Harry C.

Signaling for Therapeutic GainβTargeting TGF-Rosemary J. Akhurst

Family in the Reproductive TractβThe TGF-

A. PangasDiana Monsivais, Martin M. Matzuk and Stephanie

Family Signaling MoleculesβDisease by TGF-Regulation of Hematopoiesis and Hematological

Kazuhito Naka and Atsushi Hirao

Drosophila Family Signaling in βTGF-

Kim, et al.Ambuj Upadhyay, Lindsay Moss-Taylor, Myung-Jun

Differentiation, Development, and Function Family Signaling in Neural and NeuronalβTGF-

Emily A. Meyers and John A. KesslerOther Signaling Pathways

/Smad andβSignaling Cross Talk between TGF-

Kunxin Luo

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

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