Abstract
Correct scaling of body and organ size is crucial for proper development and survival of all organisms. Perturbations in circulating hormones, including insulins and steroids, are largely responsible for changing body size in response to both genetic and environmental factors. Such perturbations typically produce adults whose organs and appendages scale proportionately with final size. The identity of additional factors that might contribute to scaling of organs and appendages with body size is unknown. Here we report that loss-of-function mutations in Drosophila Activinβ (Actβ), a member of the TGF-β superfamily, lead to production of small larvae/pupae and undersized rare adult escapers. Morphometric measurements of escaper adult appendage size (wings, legs), as well as heads, thoraxes, and abdomens, reveal a disproportional reduction in abdominal size compared to other tissues. Similar size measurements of selected Actβ mutant larval tissues demonstrate that somatic muscle size is disproportionately smaller when compared to fat body, salivary glands, prothoracic glands, imaginal discs and brain. We also show that Actβ control of body size is dependent on canonical signaling through the transcription-factor dSmad2 and that it modulates the growth rate, but not feeding behavior, during the third instar period. Tissue and cell-specific knockdown and overexpression studies reveal that motoneuron derived Actβ is essential for regulating proper body size and tissue scaling. These studies suggest that, unlike in vertebrates where Myostatin, and certain other Activin-like factors act as systemic negative regulators of muscle mass, in Drosophila Actβ is a positive regulator of muscle mass that is directly delivered to muscles by motoneurons. We discuss the importance of these findings in coordinating proportional scaling of insect muscle mass to appendage size.
Introduction
Some members of the animal kingdom, including most species of fish, amphibians, lizards, turtles, and salamanders, undergo indeterminate growth and increase their biomass throughout their lifespan. In contrast, birds, mammals and many insect species exhibit determinate growth whereby ideal body length and weight is fixed upon reaching sexual maturity. This process produces a more limited range of sizes that are characteristic for the species (HARIHARAN et al. 2015). In these animals, growth rate can vary during development and is influenced by both intrinsic and extrinsic factors. For example, in humans, at the conclusion of the high pubertal growth period, the long bone growth plates are ossified thereby preventing additional increase in overall skeletal size (KRONENBERG 2003; SHIM 2015). Similar to mammals, holometabolous insects also exhibit determinate growth. In Drosophila, a larva increases its mass 200-fold (70% of which occurs in the last larval instar) before terminating growth at pupariation (CHURCH AND ROBERTSON 1966). During the non-feeding pupal stage, the adult structures differentiate from larval imaginal tissue and there is no net increase in body mass. Thus, the final body size is set by the rate of larval growth and the timing of its termination.
In recent years, numerous studies have centered on elucidating the molecular mechanisms that regulate hormonal activity during larval development in holometabolous insects to better understand how growth rate and duration are controlled (reviewed in (REWITZ et al. 2013; BOULAN et al. 2015). In Drosophila, growth is largely regulated by the Insulin/IGF Signaling (IIS) and Target of Rapamycin (TOR) pathways, which are themselves regulated by different nutritional inputs. IIS is regulated by systemic sugar concentrations and TOR by circulating amino acid levels. Mutations that attenuate either pathway lead to slower growth rates resulting in diminutive animals with smaller and fewer cells ((CHEN et al. 1996; BOHNI et al. 1999; OLDHAM et al. 2000; RULIFSON et al. 2002)). Conversely, activation of either pathway can lead to larger organs and cells if there are adequate nutrients (LEEVERS et al. 1996; GOBERDHAN et al. 1999; STOCKER et al. 2003). Interestingly, systemic manipulation of IIS/TOR pathways typically leads to smaller or larger animals, with proportional effects on organ and appendage size (allometric growth) (SHINGLETON et al. 2007; SHINGLETON AND FRANKINO 2013).
While IIS/TOR are central regulators of growth rate in holometabolous insects, the major regulator of growth duration is the steroid hormone 20-hydroxyecdysone (20E) (reviewed in (YAMANAKA et al. 2013a). During the final larval stage, a pulse of 20E extinguishes feeding, terminates growth and initiates pupariation. The timing of the 20E pupariation pulse is triggered, in part, by the neuropeptide prothoracicotropic hormone (PTTH) which in Drosophila is produced by the two pairs of neurons in each brain hemisphere that innervate the prothoracic gland (PG) (MCBRAYER et al. 2007; SHIMELL et al. 2018). PTTH binds to its receptor Torso and stimulates synthesis and secretion of ecdysone from the PG (REWITZ et al. 2009; YAMANAKA et al. 2013a). PTTH production/release responds to a variety of environmental signals including nutritional status, light, and tissue damage as well as internal signals such as juvenile hormone (JH) to further tune the timing of pupariation (YAMANAKA et al. 2013b; DE LOOF et al. 2015; SHIMELL et al. 2018).
In addition to IIS/TOR signaling and steroid hormones, other signaling pathways have also been identified that affect final body mass and proportion scaling in both vertebrates and invertebrates. In particular, the TGFβ signaling pathway has known roles in controlling cell, tissue, and body size. TGFβ super-family ligands signal by binding to a heterotetrameric complex of type I and type II serine-threonine receptor kinases. Ligand binding triggers type II receptors to phosphorylate type I receptors, thereby activating its kinase (HELDIN AND MOUSTAKAS 2016). In canonical signaling, the activated type I receptor phosphorylates its major substrates, the R-Smads (reviewed in (HATA AND CHEN 2016). Once phosphorylated, R-Smads oligomerize with a co-Smads and translocate to the nucleus where together with other cofactors they regulate gene transcription (review in (HILL 2016). The ligand super-family is broadly divided into two major sub-divisions based on phylogenetic and signaling analysis (KAHLEM AND NEWFELD 2009). These include the TGFβ/Activins, which in vertebrates signal through R-Smads 2/3, while the BMP/GDF-type factors signal through R-Smads 1/5/8 (MACIAS et al. 2015).
TGFβ family members contribute to tissue and body size growth by a variety of mechanisms. For instance, in mammalian mammary cells, TGFβ cell-autonomously regulates cell size via mTOR during epithelial-mesenchymal transition (EMT) (LAMOUILLE AND DERYNCK 2007). In addition, BMPs have been shown to control cell proliferation at the long bone growth plate and have been identified by genome-wide association studies as regulating human height (HIRSCHHORN AND LETTRE 2009; WOOD et al. 2014). Another particularly stunning example is Myostatin, a circulating Activin-type ligand, whose loss causes skeletal and muscle hypertrophy in vertebrates (MCPHERRON et al. 1997; MCPHERRON AND LEE 1997). TGFβ-type factors also affect the body size of invertebrates. For example, in C. elegans, a BMP-type ligand DBL-1, is secreted from neurons and signals via small (sma), a worm Smad, in the hypodermis to regulate expression of cuticle genes (TUCK 2014; MADAAN et al. 2018).
To further explore how TGFβ ligands influence body size, we investigated the role of Drosophila Actβ in regulating these traits using both loss and gain-of-function studies. In Drosophila, genetic studies as well as phylogenetic analysis suggests that Actβ signals via Baboon (Babo) and Punt, type I and type II receptors respectively, to phosphorylate dSmad2 (reviewed in (UPADHYAY et al. 2017). We find that canonical Actβ signaling through dSmad2 regulates adult viability, body size, and tissue scaling. Actβ mutants produce small larvae and pupae along with rare adult escapers. Compared to controls, these rare mutant adults exhibit small abdomens while other structures such as the head, thorax, leg, and wing are of relatively normal size. In larvae, muscle size is most profoundly affected while imaginal discs and the larval brain are of normal size. Furthermore, Actβ mutants have a slower overall growth rate, but show no defects in food intake. Using tissue specific gain and loss-of-function, we demonstrate that motoneuron derived Actβ is required for proper muscle growth and adult viability. Conversely, hyperactivation of Activin signaling in muscles by overexpression of activated Babo produces a much larger animal with bigger muscles, but smaller imaginal discs. These observations demonstrate that muscle size can be perturbed without having proportional effects on the size of the imaginal tissues. Therefore, we suggest that either muscles and appendages do not typically coordinate their growth or that such coordination requires Actβ signaling.
Results
Actβ is required for adult viability, normal body size, and correct tissue scaling
Drosophila Actβ has been shown to be involved in a diverse group of developmental processes, including neuroblast proliferation, photoreceptor tiling, regulation of Akh signaling, inter-organ regulation of mitochondrial and hemocyte function (TING et al. 2007; ZHU et al 2008; MAKHIJANI et al. 2017; SONG et al. 2017a; SONG et al. 2017b). However, in none of these studies was the lethal stage or the gross morphological phenotype carefully documented. To examine this issue, we initially characterized mutant phenotypes using the previously reported putative Actβed80 null allele (nonsense mutation) (ZHU et al. 2008). However, since the Actβ locus is on the fourth chromosome, additional recessive background mutations on the Actβ ed80 chromosome cannot be removed by recombination and therefore could complicate the phenotypic analysis of homozygous Actβed80 mutants. To resolve this issue, we generated several independent deletion alleles (Actβ4E, Actβ10E and Actβ 4dd) in the w1118 background using the CRISPR/Cas9 system (REN et al. 2013; SEBO et al. 2014). All phenotypes initially described using Acted80 homozygotes were confirmed using different combinations of transheterozygous alleles to rule out 4th chromosome background effects.
All examined Actβ mutant alleles are predominantly late pupal (pharate) stage lethal (Fig. 1A and B). Many of the pharates show limited movement inside the pupal case, but most never eclose. Manual cracking of the operculum allowed a small percentage (∼1%) to escape and produce viable adults that exhibit severe locomotive defects, and held out immobile wings rendering them flightless (Movie 1,2). Despite these behavioral/physical defects, females could mate and produce offspring from wildtype males. Actβ mutant males were unable to produce progeny with either mutant females or wildtype females. Whether this is a behavioral issue (i.e. unable to initiate courtship behavior) or a fertility defect was not determined.
In addition to pharate lethality, Actβ mutants exhibit a small body size at all stages of development. Actβed80 homozygous pupae (mixed male and female populations) are 21% smaller by volume relative to w1118 or heterozygous pupae (Fig. 1C). Similar to the Actβed80 homozygous phenotype, all trans-heterozygous combinations (Actβed80/4E, Actβ10E/ed80, Actβ10E/4E) are also significantly smaller (21%, 22%, and 26%, respectively) compared to w1118 control (Fig 1D) indicating that the small pupal size is not caused by secondary mutations on the mutant chromosome. Taken together, these data indicate that Actβ is required to produce normal pupal volume and adult viability.
Appendage size is proportionally scaled with body mass in Drosophila (Mirth and Shingleton, 2012). To examine if the adult body components of Actβ mutants are proportionally reduced, we collected 1 day old escaper males and females and measured various traits. We found that the Actβed80 homozygous male weights are reduced on average 28% compared to the control (Fig. 2A, female 20% not shown). We next measured the abdomen, thorax, and prothoracic leg lengths, along with head projection area and wing surface area of Actβ mutant males and controls. Interestingly, the size of some adult structures of Actβ mutants are more severely affected than others (Fig. 2B-L). The abdomen length in Actβ mutants is reduced by a much greater proportion, −24% (Fig. 2D, F) than any other measured component: head projection area, −8% (Fig. 2B, C), thorax length, −4% (Fig. 2D, E), prothoracic leg length, −2% (Fig. 2G, J), wing area −4% (Fig. 2 H, K). Using the wing trichome density as a proxy, we found no difference in cell size between Actβ mutants and the w1118 control (Fig. 2K, I, L), indicating that the minor reduction in wing size is likely caused by a subtle defect in cell proliferation at some time during development.
Actβ disproportionately affects larval muscle and certain polyploid tissue sizes
To understand the size discrepancy of adult structures in Actβed80 mutants, we examined directly the size of various larval tissues including brain, wing and leg discs, and body-wall muscles, and indirectly the sizes of several polypoid tissues including the fat-body, proventriculus, salivary and prothoracic gland cells using the size of the nucleus as a proxy for cell size.
The most pronounced defect of Actβed80 mutant larvae is exhibited by the body wall muscles which in males are reduced by 37% (Fig. 3 A-C), and muscle nuclear size by 53% (Fig. 3D-F). The muscle size reduction is not caused by an earlier myoblast fusion defect since mutant muscles contain the same number of nuclei as wildtype (Fig. S1). In contrast, neither the brain volume (Fig. 3J, K) nor the 2-D projected surface area of the wing, leg, and haltere disc (Fig. 3K-P) were significantly affected. Interestingly, we note that the nucleus size of several other polyploid tissues including the fat body and the prothoracic glands are also significantly reduced, but to a lesser degree than the muscle nuclei (22% for fatbody, Fig. 3FD-I, 37% prothoracic gland vs 53% muscle Fig S2 G-I). Curiously, the nuclear sizes of the cells within the proventriculus remained unchanged (Fig. S2 A-C) and the average salivary gland cell nuclear size actually increased. (Fig. S2 D-F). We conclude that the small pupal volume and reduced escaper weights are primarily due to the disproportionate reduction in muscle size rather than alterations in mitotic tissue growth such as the brain and imaginal discs.
Actβ mutants feed normally but grow slowly
Body size is largely determined by two factors, the duration of growth and the growth rate, or some combination of the two parameters. In addition, a slower growth rate may reflect reduced food intake, diminished absorption of nutrients, or an alteration in metabolic flux. We examined several of these parameters to determine if they were altered in Actβ mutants. First, we measured the larval growth rate during the L3 period, when most of the larval growth occurs. At the start of the L3 stage, there is no difference in mass of the mutants versus the controls; however, over the course of 36 hours, a slower rate of mass accumulation becomes apparent such that, at the time when larvae begin to wander, the Actβed80 mutants weigh 18% less than w1118 controls (Fig. 4A). This difference in growth rate likely accounts for a large portion of the reduced body size phenotype. To examine whether the diminished growth rate might reflect reduced food intake, we measured feeding rates of foraging early L3 larvae by the mouth-hook contraction assay (WU et al. 2003; WU et al. 2005). Surprisingly, we found no difference in the head contraction rates of the Actβ mutants (Fig. 4B) suggesting that the slow growth rate of these mutants is not likely caused by reduced food intake, but instead may reflect an alteration in nutrient absorbance or dysfunctional metabolic flux.
Next, to determine whether the small body size might also involve a reduced growth period, we measured the time to pupariation as well as the critical weight (CW), which is a nutritional checkpoint that ensures larvae have enough nutrient stores to produce viable adults (NIJHOUT AND CALLIER 2015). In both the control and Actβed80 mutant, starvation after just 2 hours into the L3 stage blocks pupariation (Fig. 4C). In the Actβed80 mutant, starvation beginning 4 hours after L2/L3 ecdysis results in delayed pupariation, while starvation 12 hours after L3 ecdysis results in no developmental delay, indicating attainment of CW. The w1118 control achieves CW 8 hours after L2/L3 ecdysis (Fig. 4D, E). Given the four hour difference between the time the Actβed80 mutant and the w1118 control reach CW, and considering that Actβ mutants grow slower, we calculated that the weight at the time when CW is reached for Actβ mutants is similar to that of controls (0.88 mg for w1118 vs 0.84 mg for Actβed80, Fig. 4F). Therefore, we conclude that Actβed80 does not affect the CW checkpoint.
Although the CW represents the lower threshold of mass necessary for pupariation without delay, body size can be altered by either a shorter or longer terminal growth period which occurs after CW has been reached (NIJHOUT AND CALLIER 2015). Therefore, we also measured the total time to pupariation. We find that, although Actβed80 homozygous mutants grow slowly, they pupariate on average 4 hours earlier when compared to w1118 at 25°C. (Fig. 4G). However, the Actβed80/+ heterozygotes also pupariate earlier than w1118 (Fig 4G) suggesting either a haplo-insufficient effect of Actβed80 or that this phenotype is attributable to the genetic background differences between Actβed80 and w1118. To examine this issue, we repeated the experiment with two CRISPR alleles. In this case, we find no difference in pupariation timing during the third instar stage between trans-heterozygous Actβ4dd/Actβ10E mutants or Actβ4dd/unc13 heterozygotes compared to w1118 controls (Fig. 4H). However, we also observe that certain allelic combinations such as w1118/unc13 wild-type controls and Actβ4dd/w1118 heterozygotes also pupariate 2 hours earlier than w1118 controls (Fig. 4H). Therefore, we conclude that the slight timing differences reflect either differences in genetic background, slight differences in the duration of first and second instar growth, or the limits of timing resolution in this experiment and that Actβ loss does not substantially affect developmental timing.
Overexpression of Actβ in its normal pattern produces larger and slower-growing larvae
Since loss of Actβ results in small developmentally arrested pupae, we asked whether overexpression of Actβ in its endogenous pattern would have the opposite effect on pupal size, viability, and whether it might also affect developmental timing. For this purpose, we overexpressed Actβ using an Actβ-Gal4 promoter enhancer line (ZHU et al. 2008; SONG et al. 2017a). Relative to either the UAS-Actβ-3B2 or the Actβ-Gal4 controls, overexpression of Actβ (Actβ > Actβ-3B2) results in a significant increase in pupal volume and a delay in developmental timing (Fig. 5A,B). Strikingly, pupariation is delayed over 20 hours compared to either w1118 or Actβ mutants however, there is no change in viability (Fig. 5B,C). Together, these data show that Actβ regulates body size and perhaps developmental timing in a dose dependent manner.
Tissue and cell-type specific rescue experiments identify several potential sources of Actβ for controlling body size
To investigate how Actβ affects body size, developmental timing and viability, we first sought to determine if one or more cell types serves as the source(s) of the ligand that controls different aspects of the mutant phenotype. Several features of endogenous Actβ transcription have been previously described including expression in motoneurons, mushroom body neurons, peripheral neurons including multi-dendritic and chordotonal neurons, developing photoreceptors in the eye disc, and in midgut enterocytes (GESUALDI AND HAERRY 2007; TING et al. 2007; ZHU et al. 2008; KIM AND O’CONNOR 2014; TING et al. 2014; MAKHIJANI et al. 2017; SONG et al. 2017a). We examined the Actβ expression pattern in the larvae by crossing an Actβ-Gal4 to UAS-cd8GFP or UAS-GFP. We also confirmed expression in particular cell types using RNA in situ hybridization (Fig. S3). As previously described, Actβ is almost exclusively expressed in the central and peripheral nervous systems (Fig. 6E). More detailed examination reveals that in the central brain lobes, Actβ-Gal4>UAS-GFP is expressed strongly in mushroom body neurons and in a 14-cell cluster in the anterior medial region of each brain lobe (Fig. 6A-A’’). A subset of 7 cells within this 14 cell cluster also stain with α-Dilp5 (Fig. 6A’), which marks the ∼7 insulin producing cells (IPCs) (BROGIOLO et al. 2001). In the ventral nerve cord, Actβ >Gal4 is expressed strongly in the motoneurons, marked by α-p-Mad (Fig. 6G-G’’) (MARQUES et al. 2002). We also see strong staining in all α-Dimm-marked neuroendocrine cells (Fig. 6H-H”) (PARK et al. 2008).
Because of the possible developmental timing defects, we were particularly interested in whether Actβ is expressed in the neurons that innervate the ring gland (RG), the major endocrine organ of larvae, or in any of the RG cells themselves. We found strong expression in the corpus cardiacum (CC) cells that produce the hormone Akh which is involved in regulating sugar metabolism (Fig. 6 C-C’’ Fig S3D) (Lee and Park, 2004). While we observed no expression in the cells of the prothoracic gland (PG), which produces the steroid hormone ecdysone (YAMANAKA et al. 2013a), or in the corpus allatum (CA) which produces juvenile hormone (RIDDIFORD et al. 2010), we did see signal in axons tracts that innervate each of these tissues (Fig 6F-F’). The PG neurons produce prothoracicotropic hormone (PTTH) and innervate the PG portion of the ring gland to regulate ecdysone production (SIEGMUND AND KORGE 2001; MCBRAYER et al. 2007). Co-staining of Actβ>cd8GFP brains with α-PTTH reveals strong expression in the PG neurons (Fig. 6B-B”). While we have no specific antibody that marks the CA neurons, the GFP positive innervations that we observe on the CA are highly suggestive that the CA neurons express Actβ (Fig. 6F-F’). Actβ is also found in various other unidentified neurons within the central brain and ventral nerve cord. Outside the nervous system, we observe Actβ expression only in a limited number of enterocytes in the midgut (Fig. 6 D-D”) and non neuronal tissue expression includes the enteroendocrine cells (Fig. 6D-D”, Fig S3H) as previously reported (SONG et al. 2017a), and the ovariole follicle and border cells (Fig. 6 I and J, Fig. S3G) and some tracheal cells. (Fig. 6K, Fig. S3F). Our observation that the rare escaper females are fertile suggests that Activin signaling in the follicle cells is either not required for full fertility or that its expression might be redundant with another activin like ligand such as Dawdle or Myoglianin.
Motoneuron-derived Actβ regulates body size and viability
To determine which Actβ-expressing cell types influence size and viability, we attempted rescue experiments using different tissue specific Gal4 drivers to overexpress the Actβ transgene in the Actβed80 mutant background. Actβed80 mutants with one copy of either the UAS-Actβ or the various Gal4 transgenes served as negative controls. Since overexpression of Actβ-GAL4 driving UAS-Actβ is sufficient to increase body size (Fig. 5A), here we asked whether it is able to rescue the small body size (pupal volume) and pupal lethality of Actβ mutants. Indeed, Actβ>Actβ in the mutant background is not only sufficient to rescue body size, but actually produces larger animals (47% bigger Fig. 7A) similar to what we see upon overexpression in a wild type background (Fig.5A). Overexpression of Actβ in its normal pattern also resulted in strong rescue of lethality (Fig. 7B, 42.7% viability vs 1-4% viability of mutant controls; the test cross viability rate for w1118 is 73.8%, Fig. 7B).
To narrow down the relevant source of ligand that regulates each phenotype, we used increasingly restrictive (tissue specific) Gal4 lines to overexpress Actβ and then measured body size and viability. Overexpression of Actβ using the pan-neuronal driver, nrv2-Gal4, rescues both body size and adult viability (Fig. 7C and D). Surprisingly, overexpression of Actβ from the motoneurons, OK371>GAL4, alone rescues both body size and viability (Fig. 7E and F). Interestingly, Actβ overexpression in Dimm+ neuroendocrine cells (C929>Gal4), also rescues both body size and viability (Fig. 7G and H). Just like overexpression of Actβ from its endogenous sources, we also found that overexpression of Actβ in either motoneurons or neuroendocrine cells in wildtype animals also produces large adults (Fig. S4). Lastly, overexpression of Actβ in only the IPCs (dilp2>Gal4), which makes up a much smaller subset of all neuroendocrine cells, does not rescue either phenotype (Fig. 7I and J).
The finding that expression in only the motoneurons rescues body size suggests that Actβ may be supplied directly to the muscles via the neuromuscular junctions. However, we also find that overexpression in neuroendocrine cells is sufficient to rescue body size, which suggests that Actβ may be able to function as a systemic endocrine signal and need not be directly delivered to the muscle via the neuromuscular junction synapse. Therefore, we asked if expression of Actβ from non-neuronal, but highly secretory tissues, was able to rescue various aspects of the null phenotype. Interestingly, expression of Actβ using the ppl-Gal4 (fat body and muscle) driver increases pupal volume beyond wild-type levels and partially rescues adult viability (Fig. 7K and L). Overexpression in only the body wall muscles (MHC-Gal4) also increases body size beyond wild type levels, but does not rescue adult viability (Fig. 7M-N). However, we note that overexpression of Actβ using either MHC-Gal4 or ppl-Gal4 in a wild type background results in most animals dying as large oversized and curved pupae (Fig. S5). These phenotypes are likely due to hyperactivation of TGFβ signaling in the muscles because we observe a similar phenotype when a constitutively activated version of Babo is overexpressed in the muscles (Fig. S5). Taken together, these results suggest that, although Actβ signaling in muscles is required for proper body size, too much signaling in muscles can be deleterious. We were not able to specifically test the ability of enteroendocrine-derived Actβ to rescue mutant phenotypes, because overexpression of Actβ using the midgut enteroendocrine cell driver (EE-Gal4) (SONG et al. 2017a) is lethal in both wild type and Actβ mutant backgrounds, likely due to overexpression in many cells besides enteroendocrine cells, including fat body, CNS and PNS (data not shown). In summary, we conclude that since overexpression of Actβ from motoneurons or neuroendocrine cells rescues both body size and viability and can increase body size when overexpressed from these sources in wild type animals, they are likely the most important endogenous sources of ligand for viability and body size control.
Motoneuron derived Actβ signals through the canonical Babo/dSmad2 pathway to control muscle and body size
The rescue experiments described above suggest that either motoneurons or Dimm+ neuroendocrine cells, or both can produce enough Actβ to regulate body size. Since data from overexpression alone does not reflect the in vivo importance of various endogenous ligand sources, we sought a complementary set of loss-of-function data using tissue specific RNAi knockdown. First, we tested all publicly available (TRiP, VDRC, and NIG) Actβ RNAi lines to phenocopy the Actβ mutant. Using the ubiquitous driver da-GAL4 to overexpress dicer2 along with the various Actβ RNAi lines, we find that only the TRiP stock (BDSC#29597) can phenocopy the small, dead pharates similar to Actβ null alleles (data not shown). Most other lines produce viable flies of normal size suggesting that they are not very effective in knocking down endogenous Actβ. Both NIG lines (1162R-1 and 1162R-2) produce a more severe phenotype (early larval lethality) compared to the null suggesting it may have off-target effects.
Using the TRiP 29597 RNAi line, we tested whether knockdown of Actβ in either all neurons, motoneurons or neuroendocrine cells alone phenocopies any aspect of null alleles. We find that knockdown in Dimm+ neuroendocrine cells (C929-Gal4) produces viable normal-sized flies (data not shown). In contrast, knockdown in all neurons (Elav>Gal4 Fig. 8A) or motoneurons (OK371-Gal4, Fig. 8A,B) completely phenocopies Actβ nulls giving rise to small, dead pharates with rare escapers that hold out their wings and have a slow gait (Movies 3,4). The OK371 driver is not expressed in the Dimm+ neuroendocrine cells (Fig. S6) leading us to conclude that the motoneurons are the major source of endogenous Actβ that regulates body size and viability.
We next determined if motoneuron-derived Actβ signals to the muscle via the canonical Smad2 pathway. Canonical TGFβ signaling is mediated by the Activin receptor, Babo, and the signal transducer, dSmad2. In muscles, overexpressed Babo is localized to the postsynaptic neuromuscular junction, perhaps sensitizing the muscles to receive motoneuron derived Actβ (KIM AND O’CONNOR 2014). Indeed, RNAi knockdown of Babo or dSmad2 in the body wall muscle results in smaller pupal volume (Fig. 8C and D). Furthermore, on Western blots, we detect lower levels of phosphorylated dSmad2 in Actβ mutant carcass extracts (containing somatic muscle, cuticle and associated cells) compared to the w1118 control (Fig. 8E). Conversely, overexpression of Actβ in motoneurons both increases pupal/adult size (Fig. S4) and p-Smad2 levels in the carcass (Fig. 8D-E), which is similar to overexpression of activated dSmad2 in the muscle, producing large larval muscles (Fig. 8F-H) or overexpression of activated Babo or dSmad2 results in adult flies with extended abdomens (Fig. S7A-C). Interestingly, the MHC>dSmad2(SDVD) animals with larger body size have slightly smaller wings, not larger wings as expected if organs are actively scaled to maintain size proportion between muscles and appendages (Fig. S7D).
Discussion
Identifying and characterizing how inter-organ signals regulate physiologic and metabolic homeostasis during development and adulthood is of central importance. Various types of inter-organ signals are also likely to be necessary for coordinating growth between organs during development to achieve proper body proportions (DROUJININE AND PERRIMON 2016). In this report, we demonstrate that Actβ is a key brain-derived factor that regulates somatic muscle size in Drosophila by signaling through the canonical Smad-dependent pathway. Furthermore, we find that disruption of Actβ signaling alters larval and adult organ allometry suggesting that Actβ might be a component of an inter-organ signaling pathway that helps coordinate muscle growth with appendage growth.
Localized versus systemic effects of Actβ
The question of whether Actβ acts locally or systemically via the hemolymph to target tissues is an important issue raised by our study and previous work (SONG et al. 2017a; SONG et al. 2017b). On the one hand, we find that Actβ is strongly expressed in most if not all neuroendocrine cells. We also find that overexpression of Actβ from these cells results in the rescue of mutant phenotypes and overgrowth of wild type animals indicating that direct tissue contact is not necessary for Actβ signaling to control muscle size. However, we also find that depleting Actβ expression in just the motoneurons phenocopies Actβ mutants while depletion in neuroendocrine cells does not do so, at least with the 929>Gal4 driver. Therefore, we conclude that, while high systemic concentrations of Actβ produced by overexpression is capable of regulating muscle growth, the endogenous systemic level supplied by the combination of the neuroendocrine and the enteroendocrine cells is not sufficient to do so.
Whether local or systemic Actβ signaling is important in other contexts is less clear. Interestingly, Actβ has also been implicated in regulating hemocyte proliferation and adhesion within hematopoietic pockets localized on the larval body surrounding a number of peripheral neurons that express Actβ including da and chordotonal peripheral neurons (MAKHIJANI et al. 2017). In contrast, enteroendocrine derived Actβ is able to affect AKH receptor levels in the fatbody to regulate glycemic index on a high sugar diet (SONG et al. 2017a). In addition, it has been reported that upon mitochondrial perturbation, muscle-derived Actβ signals to the fat body to regulate triglyceride levels (SONG et al. 2017b). In either case these observations raise the question: what dictates the requirement of a local versus a systemic signal for Actβ function? In the muscle motoneuron synapses and hematopoietic pocket paradigms, there may be physical barriers that help concentrate ligand from a local source to levels sufficient to produce a response. In the case of muscles, motoneuron synapses are embedded within the muscle fiber (PROKOP 2006; PROKOP AND MEINERTZHAGEN 2006) and therefore delivery of Actβ directly to the NMJ via the synapse likely provides a highly effective signal, especially since its receptor Babo is also highly concentrated at the postsynaptic NMJ (KIM AND O’CONNOR 2014). This possibility might also account for the discrepancy between our findings (Fig. S3) that muscles do not express Actβ under normal conditions, while mitochondrial perturbations in muscle appears to release Actβ for signaling to the fat body (SONG et al. 2017b). Perhaps disturbing mitochondrial function in muscle disrupt synaptic structure such that Actβ is liberated from defective NMJ synapses. Likewise, the hematopoietic pockets might provide a similar restricted niche-like signaling environment that is able to modulate hemocyte proliferation and adhesion. These types of physical constraints may limit the ability of endogenous circulating Actβ to produce sufficient levels of signaling at these locations, except under overexpressed conditions.
Another factor influencing the cellular response to Actβ levels is the composition of the surface receptors. The babo locus produces three receptor isoforms that only differ in the extracellular ligand binding domain, and each likely has a different affinity for the three Activin-like ligands (JENSEN et al. 2009; UPADHYAY et al. 2017). Therefore, the complement of receptor isoforms on a cell’s surface is apt to determine the sensitivity of the cell or tissue to Actβ signals.
Mechanisms of Actβ control of tissue size
The molecular mechanism(s) by which Actβ affects tissue growth are unclear. The most well characterized factors that regulate insect body size are all systemic signals such as juvenile hormone, ecdysone and the IIS/TOR pathways (REWITZ et al. 2013; MIRTH AND SHINGLETON 2014; BOULAN et al. 2015; KOYAMA AND MIRTH 2018). For example, Ptth mutants delay ecdysone accumulation allowing larvae to grow for an additional 24 hours ultimately leading to larger flies (SHIMELL et al. 2018). In this report, we demonstrate that Actβ, although it is expressed in the PTTH-producing neurons, does not appear to affect ecdysone signaling since Actβ loss affects neither critical weight nor developmental timing.
Interestingly, we also see Actβ positive innervation of the CA organ which produces juvenile hormone (JH), and lowering JH levels in Drosophila leads to production of smaller flies by slowing the overall growth rate (RIDDIFORD et al. 2010.; MIRTH et al. 2014) Since we also observe a slower growth rate in Actβ mutants it is possible that Actβ might work to slow growth via reduction of JH signaling. However, given the strong expression of Actβ in all Dimm+ neuroendocrine cells which secrete numerous peptides that regulate many aspects of behavior and metabolism including insulin, many other mechanisms must be considered for slowing growth. While food intake does not seem to be altered in Actβ mutants, it is possible that nutrient absorption or metabolic flux is disrupted. The latter possibility is particularly attractive since we see strong expression of Actβ in the CC organ, which produces the Drosophila glucagon-like hormone (Akh), and in the insulin producing cells (IPCs) in the brain. As previously noted, Actβ has been implicated in regulating AkhR levels in the fat body (SONG et al. 2017a) and perhaps it may also influence Akh synthesis or release. Furthermore, Dawdle, another Drosophila Activin-like ligand that also signals through dSmad2 has been previously shown to regulate metabolism and carbohydrate utilization (CHNG et al. 2014; GHOSH AND O’CONNOR 2014). Therefore, Actβ signaling through dSamd2 may also regulate global carbohydrate synthesis or aspects of metabolism to adjust the larval growth rate.
Regardless of how overall growth defects occur, it is important to remember that not all larval tissues respond equally to Actβ. The brain and imaginal discs, for example, are of normal size while fat body and muscle are significantly smaller. In addition, the size and viability defects can be largely rescued by expression of Actβ solely in motoneurons. Therefore, it seems unlikely that a primary defect in systemic levels of insulin or Akh would account for the tissue specific responses. Rather, it is likely that alterations in muscle metabolism and perhaps factors secreted by muscles could account for the small muscle/body size.
Larval versus Adult requirements for Actβ
The requirement of Actβ for adult eclosion raises several issues. The first is whether the low eclosion rate is primarily a muscle defect or a neuronal problem since both must be coordinated to produce the complex set of motor behaviors required for eclosion. Interestingly, ablation of Dimm+, Eclosion hormone (EH) producing neuroendocrine cells, (PARK et al. 2008), results in a defective eclosion motor program, which involves a series of coordinated head, thorax, and abdominal muscle contractions that ejects the animal through the operculum and out of the pupal case (MCNABB et al. 1997). It may be that the small adult muscles lack the power to properly execute the eclosion motor program. In addition, the small muscle phenotype may also partially explain why the Actβ mutant adult escapers walk slowly and cannot move their wings. However, this must be reconciled with the observation that Actβ mutant larvae exhibit no obvious defect in locomotion, even though they have a similar proportional reduction in overall body and muscle size.
Improper synaptic development or NMJ function could also potentially account for adult locomotion defects. However, we have previously shown that, at least in larvae, the NMJ size and bouton number are not affected in babo and dSmad2 mutant larvae when normalized to the smaller muscle size (KIM AND O’CONNOR 2014). Nevertheless, we did uncover a number of electrophysiological alterations including a decrease in the number and frequency of miniature excitatory potentials and a depolarized muscle membrane resting potential, both of which were primarily attributed to defective Actβ signaling in muscles (KIM AND O’CONNOR 2014). Despite these defects, the large action potentials in babo and dSmad2 mutants are relatively normal and, as described, there are no obvious larval locomotion defects (KIM AND O’CONNOR 2014). Since adult muscles are formed de novo during metamorphosis, it is possible that during this time more extreme defects in muscle or neuron physiology develop in Actβ mutants, perhaps leading to a more strongly depolarized muscle, for example, that would interfere with proper muscle function.
The motoneuron source of Actβ also raises questions concerning whether Actβ production/release is muscle/neuron activity dependent. We find that overexpression of Actβ in motoneurons can produce bigger muscles, but whether increased muscle activity also accompanies higher Actβ expression/secretion triggering increased muscle growth is an interesting issue to address. We note, however, that adult muscles, which develop during the immobile pupal stage, are also likely smaller than wild type in Actβ mutants suggesting that significant muscle activity is not likely required for Actβ release.
Body-appendage scaling
One of the more novel features of the Actβ null phenotype is the disproportionate effect it has on muscle size compared to other tissues. One might expect that evolutionary pressures fine tune mechanisms to coordinate muscle size with the size of the appendage that it moves. This is perhaps especially true in winged insects where flight muscle and wing size should be coordinated to produce efficient flight. Such coordination between wing and body size in response to environmental perturbations has been best studied in Manduca sexta (NIJHOUT AND GRUNERT 2010; NIJHOUT AND CALLIER 2015). In this insect, nutritional restriction can result in as much as a 50% reduction in body size, with the wing scaling proportionally and containing half as many cells (NIJHOUT AND GRUNERT 2010). This scaling mechanism utilizes a shift in the amplitude and kinetics of steroid hormone production during the last instar stage. Since this mechanism involves systemic factors that adjust the growth rate of the whole body, presumably affecting muscles and discs simultaneously, it does not really address whether specifically perturbing muscle growth can directly or indirectly affect growth of the wing or other appendages.
In Drosophila, alteration in the growth properties of one imaginal disc perturbs growth of other wild type discs in a coordinated manner so that adults emerge with properly proportioned structures (SIMPSON AND SCHNEIDERMAN 1975; SIMPSON et al. 1980; STIEPER et al. 2008). Once again, the inter-organ signaling mechanism involves alteration in the levels of systemic hormones (PARKER AND SHINGLETON 2011; MIRTH AND SHINGLETON 2012; GOKHALE et al. 2016). In these reports, is not clear whether muscle size was also altered to produce isometric scaling between it and the imaginal discs. However, it is interesting to note that growing Drosophila at low temperatures produces hyperallometric scaling where the wing size is disproportionally larger relative to body size (SHINGLETON et al. 2009). Since we observe similar phenotypes in Actβ mutants grown at normal temperatures, it is intriguing to speculate that Actβ signaling might mediate hyperallometric scaling between wing and body in response to temperature.
The only other report that we are aware of where Drosophila larval muscle size was specifically manipulated, and the effect on growth of other tissues examined, involved genetic alteration of insulin signaling (DEMONTIS AND PERRIMON 2009). Similar to our analysis of Actβ, the level of insulin signaling in muscle is directly correlated with muscle, appendage, and overall animal size. Nevertheless, our findings for Actβ show several notable differences. First, insulin gain-of-function signaling in muscle lead to larger bodies and larger wings (DEMONTIS AND PERRIMON 2009), while we find that increased Actβ signaling in muscles results in larger bodies, but slightly smaller wings. In the insulin loss-of-function case, both muscles and wings were smaller, the latter due to a reduction in cell size not cell number. However, in the case of Actβ mutants, we see only a 4% decrease in wing size with no change in cell size. In both cases, the effect on muscle size appears to be much more dramatic than the effect on appendage size. Therefore, if a scaling mechanism exists, then either insulin or Actβ loss disrupts it, or it is not isometric as is found for the nutrient-dependent body-wing scaling response in M. sexta. The general similarity in phenotypes produced by insulin or Actβ signaling suggests that Actβ may exert its effect on muscle and body size, in part, through the insulin signaling pathway, a possibility that we are currently exploring.
TGFβ control of body size in other animals
TGFβ regulation of body or muscle size has been reported in both C. elegans and mammals. In C. elegans, BMP-type factors are also secreted from a specific set of neurons and appear to act systemically to regulate the size of the hypodermis through a canonical Smad dependent pathway (TUCK 2014). In fact, the term Smad is a compound word derived from the C. elegans gene sma meaning small, and the Drosophila gene mad (mothers against dpp) which were the founding members of the Smad family of TGFβ signal transducers (DERYNCK et al. 1996). Recently, many transcriptional targets for the Sma factors in C. elgans have been identified, among which are several collagens that are major structural components of the hypodermal body-wall (MADAAN et al. 2018). Hence, we speculate that one set of targets for both Actβ and insulin signaling in the Drosophila muscle could be structural proteins that build muscle. It is also possible that target gene expression is indirectly regulated by DNA copy number. In Drosophila, larval and adult muscle are polyploid tissues where DNA content is controlled by endocycling. Both Actβ (this report) and insulin signaling (DEMONTIS AND PERRIMON 2009) appear to regulate nuclear size in many polypoid tissues, possibly indicating that control of the endocycle maybe the primary mechanism regulating tissue size. However, at least in Actβ mutants not all polyploid tissues show regulation in the same direction, i.e. muscle, fatbody and the prothoracic gland all show smaller nuclei while the salivary gland has larger nuclei. Whether systemic Actβ signaling is directly regulating size of polyploid tissues or acts indirectly through a muscle-derived myokine needs to be determined.
In mammals, the best characterized example of a TGFβ-type factor that regulates body and muscle size is provided by Myostatin (Mstn). Loss of Mstn was discovered to cause the muscle overgrowth phenotype of Belgian blue cattle and subsequent work in many other species including humans confirmed that Mstn is a negative regulator of muscle mass (MCPHERRON et al. 1997; MCPHERRON AND LEE 1997). Mstn mutant muscles have both an increase in myofiber number (TRENDELENBURG et al. 2009; MATSAKAS et al. 2010) as well as myofiber size (MCPHERRON AND LEE 1997; ELASHRY et al. 2009). The molecular basis for the phenotype appears to be an alteration in protein homeostasis, where proteasome and autophagic degradative capacity is reduced relative to protein synthesis (LEE et al. 2011; LOKIREDDY et al. 2012). Mstn signals through Smads2/3 and is therefore considered to be within the TGFβ/Activin subgroup in the TGFβ superfamily. Additional studies of Activin ligands themselves suggest that that they also act as negative regulators of muscle mass similar to Mstn (ZHOU et al. 2010). Furthermore, studies on the role of BMP signals in muscle size control suggest that they function as dominant positive regulators of muscle mass by promoting protein synthesis instead of breakdown (SARTORI et al. 2013; WINBANKS et al. 2013).
Our present work shows that in Drosophila, Actβ is a positive regulator of muscle mass, by affecting myofiber size not number. It is worth noting that Drosophila has a close Myostatin homolog that is called myoglianin (myo) and recent studies suggest that loss of myo in muscle produces larger fibers similar to the vertebrate homolog (AUGUSTIN et al. 2017). How Actβ and Myo interact will be interesting to examine as will the role for BMPs in Drosophila muscle size determination. Lastly, the question of whether Mstn loss in vertebrates affects scaling of other tissues is largely unexplored although it does appear that bone density is increased in myostatin mutant animals (ELKASRAWY AND HAMRICK 2010). Additional studies of how local versus systemic roles of TGFβ ligands might affect growth and scaling between tissues and organs in vertebrates should be enlightening.
Materials and Methods
Fly lines
For overexpression experiments single copies of Gal4 and UAS transgenes were used. Actβ-Gal4 and UAS-Actβ (3B2) were previously described (ZHU et al. 2008). C929-Gal4, dilp2-Gal4, Elav-Gal4, Mef2-Gal4, MHC-Gal4, Nrv2-Gal4, OK371-Gal4, ppl-Gal4, UAS-dicer2, UAS-cd8::GFP, and UAS-Actβ RNAi Ok6>Gal4 were all from the Bloomington stock center. UAS-babo RNAi, UAS-dSmad2 RNAi, were from O’Connor lab stocks. (details of construction available upon request). UAS-dSmad2SDVD (constitutively activated dSmad2) was previously described (GESUALDI AND HAERRY 2007).
The Actβed80 allele is an EMS-induced substitution leading to a premature stop codon and presumed to be a null mutation (ZHU et al. 2008). The chromosome carrying the Actβed80 allele (fourth) also contains a variegating w+ transgene (P{hsp26-pt-T}39C-12, FlyBaseID= FBti0016154) inserted between Hcf and PMCA (John Locke, personal communication). This w+transgene causes red speckles with dominant inheritance in an otherwise w- background.
Actβ4E and Actβ10E and Actβ4dd were all generated using the CRISPR/Cas9 system. Two guide RNAs were cloned into the BbsI site of pU6-BbsI-chiRNA plasmid (obtained from Addgene) and injected by Best Gene into w1118; PBac{y[+mDint2]=vas-Cas9}VK00027 on chromosome 3 (Bloomington Stock Center #51324). The following guides were used to target the genomic locus, guide 1: 5’-GGGTTGTGGAAATGACTTCC-3’, guide 2: 5’-GCGATTGCACGGGCTCTTTT-3’. G0 male flies were backcrossed to a balancer stock (CiD/unc13-GFP) to isolate w1118;;;Actβ?/unc-13-GFP stocks. To identify new Actβ alleles, DNA from homozygous (non-GFP) larvae was used to PCR amplify the genomic region flanking the CRISPR target sites using the following primers (FWD: 5-CTGCTGCAACAGCCTTGGCTCCC-3; REV: 5-GGGGCGCAACACGGTCGCATTCC-3). Line 4E and 4dd are independent ∼3 kb deletions that remove that remove exons 2 and 3. Line 10E is a ∼1.3 kb deletion that removes exon 4 and 5. Exact deletion junction sites are available upon request.
Rearing Conditions
Eggs were collected over a 2-3 hour time period on apple juice plates inoculated with yeast paste and aged until hatching into first instar larvae. Larvae of the desired type were then transferred to vials containing standard cornmeal food (Bloomington recipe) and incubated at 25°C in a 12 hour light/dark cycle until scoring. Animals were transferred to vials at a low density (30 or 40 per vial) to prevent crowding affects.
Size measurements of larval tissues and nuclei
To measure size of larval organs, tissues were prepared using standard protocols for immunohistochemistry (see below). To measure size of larval body wall muscles, larval fillets of late wandering L3 larvae were prepared, and the surface area of muscle #6 of the A2 segment was measured in FIJI by outlining the muscle segment using the free-hand selection tool. Larval brains were stained with DAPI and rhodamine-phalloidin and placed onto a glass microscope slide between two #2 coverslips that acted as a bridge to prevent deforming the shape of the brain lobes. Confocal Z-stacks of the entire lobe were captured, and manual 3D segmentation using ITK-SNAP (PMID: 16545965) was used to measure lobe volume. Imaginal discs were stained with DAPI, imaged using confocal microscopy, and then maximum intensity projections were generated and processed in FIJI using the threshold and measure functions to obtain a 2D area of each disc. For fat body, proventriculus, muscle salivary and the prothoracic glands, tissue was stained with DAPI and rhodamine-phalloidin and then Z stacks obtained. Nuclear size was measured using FIJI (SCHINDELIN et al. 2012) at the sections where nuclei were largest.
Pupal Volume determination
Pupal volume was calculated from the length and width of individual pupae assuming a prolate spheroid shape [V = (4/3) π (width/2)2 (length/2)] (DEMONTIS AND PERRIMON 2009). Pupal length was measured from the anterior tip midway between spiracles to the base of the posterior spiracles. Pupal width was measured at the mid-length of the pupae.
Measurement of Adult appendage sizes
Adult specimens were fixed in 95% Ethanol. Structures were dissected and mounted in Canadian Balsam (Sigma, C1795) and Wintergreen oil (Sigma, M2047) solution (50:50). To measure size (length or area) of adult body parts images were processed in FIJI using either the free-hand or polygon tool (illustrated by red lines in Fig. 2).
Developmental Timing and Growth Assay
To measure developmental timing, flies were transferred to a constant light environment for at least 2 days prior to egg lay and all subsequent assays were carried out under constant light conditions to avoid circadian rhythms. Eggs were collected on apple juice plates with yeast paste for two hours. The next day, early L1 larvae were transferred to standard cornmeal food with yeast paste. For Actβed80/Actβed80 mutants the time to midpoint of pupariation was measured. For the CRISPR alleles, an additional synchronization step was employed. After ∼48 hours of growth, 20-30 synchronized L2-L3 ecdysing larvae were transferred to cornmeal food without yeast paste to measure time to pupariation. Pupariation was scored every 2 hours by monitoring for anterior spiracles eversion and larval movement. The half point is the time it takes for half of the population to pupariate, which is calculated using a simple linear regression.
To measure growth rate, L3 larvae were cultured for appropriate times after L2-L3 ecdysis, washed in water and weighed individually on a Mettler Toledo XP26 microbalance. For adult mass, groups of 8-10 animals were weighed on the microbalance.
Statistics
Data were analyzed using either Graph Pad – Prism or R-studio. A single test variable was compared to a single control using a Welch Two Sample t-test. Multiple test variables were compared to controls using a one way ANOVA followed by Tukey’s multiple comparison test. For rescue experiments with two controls and one test cross, the test cross must be significantly different in the same direction (e.g. larger) to be considered a significant result. Where the test cross was reported to be x units different than the controls, the different was in reference to the control with smaller variation. P values designation: ns= not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Immunohistochemistry
Wandering third instar larvae were rinsed, dissected, fixed in 3.7% formaldehyde in PBS for 25 minutes, and then washed three times in PBS-(0.1%) TritonX-100. Samples were incubated with primary antibody overnight at 4°C followed by secondary antibodies for 2 hours at 25°C. Tissues were mounted in 80% Glycerol. The following stains and antibodies were used: Rhodamine-phalloidin (Molecular Probes R415), α-Dachshund (DSHB, mAbdac2-3), α-PTTH (guinea pig) a gift from P. Leopold, α-p-Mad (Eptitomics), α-DIMM a gift from P. Taggert.
Microscopy
Confocal images were generated using a Zeiss Axiovert microscope with a CARV attachment or Zeiss LSM710. Pupae, adult heads, and body were imaged live with Zeiss Stemi stereo microscope using a 1X objective. Adult wings and legs were imaged using Nikon Optiphot light microscope with a 4X objective. Trichomes were imaged using a 40X objective.
Western Blots
L3 larvae were dissected and all organs were removed from the carcass samples. Carcass samples were lysed with reducing gel loading buffer. Bands were resolved on 4-12% gradient gels (Invitrogen), and transferred to PVDF membrane (Biorad). Membrane blocking and antibody incubation were performed using standard protocols for ECL detection. α-pSmad2 (CST, 138D4) and α-tubulin (Sigma, T9026) were used at 1/1000 dilutions. Bands were visualized using Pierce ECL Western Blotting Substrate (#32209).
Data Availability Statement
Strains and plasmids are available upon request. Supplemental files available at FigShare. Movie S1 illustrates the defective shock response of Actβ escaper females compared to heterozygous controls. Movie S2 shows a close up view of Actβ mutant females exhibiting poor locomotion and a held out wing phenotype compared to heterozygous controls. Movie S3 demonstrates a defective shock response of adults in which Actβ was knocked down in motoneurons using RNAi (Ok371>Gal4, UAS Actβ RNAi). Movie S4 shows a close up view of Ok371>Gal4, UAS Actβ RNAi Actβ knockdown adults exhibiting poor locomotion and a held out wing phenotype similar to that exhibited by Actβ null escaper flies (Movie 1).
Competing interest
The authors declare no competing or financial interests
Author Contribution
Conceptualization: M.B.O., L.M.T., experimentation and data analysis: L.M.T., A.U., X.P., M.J.K., M.B.O., writing: M.B.O., A.U., L.M.T., funding acquisition: L.M.T., M.B.O.
Funding
This work was supported by the National Institute of Health grant 1R35GM118029 to M.B.O. and the American Heart Association Predoctoral Fellowship grant 15PRE25700041 to L.M.T.
Acknowledgements
We would like to thank: Aidan Peterson, MaryJane O’Connor, and Heidi Bretscher for comments on the manuscript; David Zhitomirsky for making guide RNA constructs to generate CRISPR/Cas9 alleles, P. Leopold for the anti-PTTH, P. Taggert for the anti-Dimm antibody, M. Titus for providing the microscope for live imaging of feeding larvae, and the Bloomington Drosophila Stock Center for providing numerous fly lines.