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Amalgam plays a dual role in controlling the number of leg muscle progenitors and regulating their interactions with the developing Drosophila tendon [1]

['Blandine Moucaud', 'Gred Institute', 'Umr Cnrs', 'Inserm', 'University Of Clermont-Auvergne', 'Clermont-Ferrand', 'Elodie Prince', 'Elia Ragot', 'Yoan Renaud', 'Krzysztof Jagla']

Date: 2024-10

Formation of functional organs requires cell–cell communication between different cell lineages and failure in this communication can result in severe developmental defects. Hundreds of possible interacting pairs of proteins are known, but identifying the interacting partners that ensure a specific interaction between 2 given cell types remains challenging. Here, we use the Drosophila leg model and our cell type-specific transcriptomic data sets to uncover the molecular mediators of cell–cell communication between tendon and muscle precursors. Through the analysis of gene expression signatures of appendicular muscle and tendon precursor cells, we identify 2 candidates for early interactions between these 2 cell populations: Amalgam (Ama) encoding a secreted protein and Neurotactin (Nrt) known to encode a membrane-bound protein. Developmental expression and function analyses reveal that: (i) Ama is expressed in the leg myoblasts, whereas Nrt is expressed in adjacent tendon precursors; and (ii) in Ama and Nrt mutants, myoblast-tendon cell–cell association is lost, leading to tendon developmental defects. Furthermore, we demonstrate that Ama acts downstream of the FGFR pathway to maintain the myoblast population by promoting cell survival and proliferation in an Nrt-independent manner. Together, our data pinpoint Ama and Nrt as molecular actors ensuring early reciprocal communication between leg muscle and tendon precursors, a prerequisite for the coordinated development of the appendicular musculoskeletal system.

Here, we generate transcriptomic data sets from myogenic precursors and analyze them together with our previously published data sets on isolated tendon precursors [ 25 ] and identify Neurotactin (Nrt) and its binding partner, Amalgam (Ama), as candidates selectively expressed in a specific tendon cluster and in leg myoblasts, respectively. We show that knockdown of Ama results in a dramatic decrease of the leg disc myoblast population independently of Nrt. However, later during metamorphosis, Ama and Nrt are both required for keeping tendon and myogenic precursor cells closely associated, thus ensuring correct development of appendicular muscles. These results highlight an unexpected double role of Ama during leg muscle formation in both maintaining the pool of leg muscle precursor cells and coordinating tendon-muscle precursor growth.

Nrt is a transmembrane protein of the serine esterase-like family and belongs to the family of neuronal cell adhesion molecules [ 13 – 17 ]. Ama is a secreted protein of the immunoglobulin superfamily [ 18 ] that plays a role as a cell adhesion molecule involved in axon guidance [ 15 , 16 ] by acting through its ligand, Nrt. In the central nervous system, Ama/Nrt also regulate neuroblast specification [ 19 ], and Ama affects glial cell proliferation/survival and migration through the receptor tyrosine kinase (RTK) signaling by modulating the expression of Sprouty, a negative regulator of the RTK pathway [ 20 – 22 ]. More recently, 2 independent single-cell transcriptomic studies indicated that Ama and Nrt are expressed in a subset of flight muscle progenitors. In the wing disc, depletion of Ama results in a reduced pool of wing disc-associated myoblasts [ 23 ], and Nrt is critical for proper direct flight muscle development [ 24 ].

Thus, convergent observations in vertebrates and invertebrates suggest the existence of a common genetic circuitry that controls muscle and CT progenitor interactions, and coordination during appendicular development. The molecular signals that ensure the specificity of interactions between subpopulations of myoblasts and their respective tendon precursors remain largely unknown. This prompted us to use the Drosophila model to search for candidates affecting early interactions between muscle and tendon precursors.

Drosophila leg musculature develops from the appendicular muscle precursors that lie on the surface of the leg disc in close vicinity of the leg tendon precursors that derive from the disc epithelium. At the onset of metamorphosis, tendon precursors undergo a collective cell migration and form long internal structures inside the developing leg, whereas myoblasts aggregate and engage in a coordinated migration following the invaginating tendon precursors. Consequently, the perturbation of tendon formation during the early steps of leg development affects the spatial localization of the associated myoblasts [ 7 ]. Interestingly, Drosophila leg myoblasts express ladybird (lb), the vertebrate ortholog of Lbx1, which is also expressed in vertebrate myoblasts and required for their migration into the limb buds [ 8 – 10 ]. Moreover, appendicular tendon specification and invagination/migration rely on Stripe (Sr), Dar1 and Odd-skipped (Odd), 3 transcription factors whose vertebrate orthologs are markers of limb tendon and MCT cells [ 11 , 12 ]. Strikingly, in the limb of chick embryo, a subset of MCT cells expressing Odd skipped-related 1 (Osr1), the ortholog of Odd, were observed in close association with migrating Lbx1+ myogenic progenitors [ 11 ]. In Osr1 mutant, the pool and distribution of myogenic cell is altered, suggesting that muscle progenitors require Osr1+ MCT cells to survive/proliferate and/or to reach the correct position [ 11 ].

The limb musculoskeletal system is a remarkable model to study the integration of multiple cell types. It is composed of muscle fibers, motor neurons, blood vessels, and connective tissue (CT) cells. CTs include tendons connecting muscles to bones and irregular muscle CT (MCT) cells surrounding and connecting muscle fibers. During vertebrate development, CT progenitors from the lateral plate mesoderm and myogenic cells from the myotome undergo a coordinated migration into the limb bud and differentiate coordinately to build the appendicular musculoskeletal organ. Flies do not possess an internal skeleton. Instead, their muscles are connected to the exoskeleton through specialized muscle attachment cells known as apodemes. These structures are regarded as the functional counterparts of tendons found in vertebrates. Tendon and muscle progenitors of the Drosophila larval muscle system develop independently from each other [ 2 ]. However, in the Drosophila leg, the behavior of tendon and muscle progenitors are intimately linked [ 3 , 4 ]. This suggests that, like in vertebrates, there are reciprocal interactions between muscle and CT progenitors shaping the appendage musculature [ 5 – 7 ].

Development of organs in a multicellular organism requires the orchestration of cell–cell interactions that control the assembly of various cell types. Altered cellular communication or inappropriate responses to intercellular signals can lead to major developmental defects [ 1 ]. Research on multicellular organism development generally focuses on one specific tissue. Classically, these studies highlight how the activation of signaling pathways, in a cell-autonomous or non cell-autonomous manner, influences the fate or behavior of a particular cell type. However, bi-directional communication between different cell types, and how these cells are integrated into a functional physiological unit, have been less investigated.

Results and discussion

Transcriptomics data identify Ama and Nrt as a candidate pair for cell–cell interaction We previously reported transcriptional signatures of leg tendon precursor cells [25] at the developmental time point of 0 h after pupae formation (APF) when subpopulations of myoblasts localize around clusters of tendon precursors. Here, we analyzed the transcriptome of leg disc myoblasts at the same time of development to identify potential interacting pairs. After fluorescence-activated cell sorting and RNA sequencing of the myoblasts (see Material and methods and GSE245192), we performed differential gene expression analysis and identified a set of 2,236 differentially expressed (DE) genes in myoblasts compared to all other cells in the leg disc (Fig 1A and S1 Data). Among enriched genes (FC > 1.5), we consistently found genes specifically expressed in leg myoblast or involved in leg muscle development, such as twist, htl, zfh1, him, mef2, vg, cut [3,10,26–30]. Gene ontology (GO) analysis of enriched genes showed an overrepresentation of GO terms related to muscle development (Fig 1B). We then searched for corresponding interacting partners expressed in muscle and tendon precursors. Considering an RPKM>5, we identified up to 290 pairs of cell surface or secreted proteins known to interact with each other. To identify pairs of interactors more likely to participate in specific interactions between tendon cells and myoblasts, we narrowed down this list to genes exhibiting a more specific expression (FC > 1.5) in tendon cells and myoblasts compared to other disc cells (Fig 1C). Among the most enriched genes, we found Nrt encoding a transmembrane protein (RPKM = 234, FC = 2.5) specifically in tendon cells and Ama encoding its binding partner. Ama was highly enriched both in myoblasts (RPKM = 385, FC = 3) and in tendon cells (RPKM = 221, FC = 1.7). These data prompted us to analyze in detail the expression pattern of Ama and Nrt during leg disc development. PPT PowerPoint slide

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TIFF original image Download: Fig 1. DE genes and interactome data in leg disc myoblasts. (A) Volcano plot showing DE genes, including 533 enriched genes (red) and 1,703 down-regulated genes (blue). Those not significantly changed (fold change < 1.5; P > 0.05) are in gray (S1 Data). Examples of genes known to be specifically expressed in myoblasts are annotated, as are Nrt and Ama. For graphical representation, outliers (-log10 (p-value) >100 and log FC >20) were excluded. Generated using Volcano Plot tool (Galaxy V.0.0.5). (B) GO analysis of enriched genes in leg myoblasts. Graph shows selected GO terms for biological process, GO term accession numbers, fold enrichment, and p-value. Data were obtained by GO overrepresentation test using PANTHER. (C) List of identified pairs of interacting partners expressed in myoblasts and tendon cells (Flybase protein interactions browser). Among the 290 pairs of interacting genes expressed in myoblasts and tendon cells (RPKM>5), 9 of them have corresponding interacting genes with a Fold Change >1.5. DE, differentially expressed; GO, gene ontology. https://doi.org/10.1371/journal.pbio.3002842.g001

Ama controls myoblast number in an Nrt-independent way Ama was previously shown to regulate the pool of glial cells and flight muscle progenitors [22,23]. Interestingly, Ama is expressed in leg disc myoblasts from the early larval stage onwards (S1A–S1C Fig). Ama down-regulation in myoblasts in R32D05-Gal4>UAS-GFP leg discs (Fig 3) leads to a strong reduction in GFP fluorescence, suggesting that the number of myoblasts could have been reduced. Because the number of total leg disc myoblasts and their proliferative rate have never been precisely determined, we first counted them at different stages of development using a Twist antibody. The number of myoblasts grows from a pool of around 10 per disc primordia at the end of embryonic stage to 36 (+/−10) at mid-L2 stage and to 653(+/−82) by the end of L3 stage. To thoroughly assess the effect of Ama knockdown on the pool of myoblasts, we used a Twist antibody to compare the number of muscle progenitors in L3 leg discs from R32D05-Gal4>UAS-AmaRNAi, R32D05-Gal4>UAS-mcherryRNAi, and w1118 strains. Myoblast-specific RNAi-knockdown of Ama leads to a severe reduction in muscle precursors with almost no Twist+ myoblasts (<10/disc) when compared to both controls (Fig 4A, 4B, and 4E and S2 Data). No significant differences could be observed between R32D05-Gal4>UAS-mcherryRNAi and w1118 controls, with 643 and 653 myoblasts on average, respectively, by the end of L3. Thus, Ama depletion results in a severe reduction of Twist+ myoblasts associated with the leg disc. As a decrease in the number of cells may be due to a defect in cell proliferation and/or to an increase in cell death, we tested these 2 variables by immunostaining L3 leg discs with antibodies against either the mitotic marker phospho-histone H3 (pH3) or the apoptotic marker caspase-activated Dcp1. Because these 2 antibodies were raised in rabbit, as was the Twist antibody, we could not use this latter to visualize the myoblasts. So, we performed the anti-Dcp1 and anti-pH3 immunostainings on R32D05-Gal4>UAS-AmaRNAi and R32D05-Gal4>UAS-mcherryRNAi leg discs expressing UAS-GFP to visualize the myoblasts (Fig 4F–4J). Then, we quantified the number of GFP cells that were pH3+ or Dcp1+. In control leg discs, 1.8% of cells were pH3+/GFP+. After Ama RNAi induction, this ratio decreased at 0.7% (Fig 4F–4H and S2 Data), and 2.5% of GFP+ cells were also Dcp1+ in controls, indicating the cells were apoptotic. This ratio increased to 13.8% when Ama was knocked down (Fig 4H–4J). The low number of total myoblasts in L3 leg discs after Ama knockdown (<10) can make these outcomes difficult to interpret. Thus, to increase the number of myoblasts at the time of counting (L3), we delayed the expression of the UAS-AmaRNAi transgene using Gal80ts, a thermosensitive form of the Gal4-inhibitor Gal80 [34]. In this way, we induced UAS-AmaRNAi expression from L2 stage, and we could count an average of 100 GFP+ cells in L3 stage. The percentage of mitotic cells was still significantly lower compared to controls, and the percentage of apoptotic cells was higher (Fig 4H), indicating that Ama depletion affects both viability and the proliferation rate of leg disc myoblasts. Since Ama expression was also observed in tendon cell (Figs 2 and S2), we tested whether AmaKD could similarly impact the viability and the proliferation rate of these cells. However, anti-Ph3 and anti-Dcp1 immunostainings showed no difference between Sr-Gal4>UAS-AmaRNAi and control leg discs (S3A–S3C Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Ama controls the pool of myoblast number independently of Nrt. (A–D) L3 leg discs immunostained for Twi (magenta). (A, B) Expression of UAS-AmaRNAi in myoblasts using R32D05-Gal4 driver leads to strong depletion of myoblasts when compared to R32D05-Gal4>UAS-mCherryRNAi control. (C, D) Comparison between leg discs of w1118 control and Nrt1/Nrt2 transheterozygous show no difference in myoblast number. (E) Dot-plot graph showing the mean number of Twi-positive myoblasts per disc, in R32D05-Gal4>UAS-mCherryRNAi (control) leg disc versus R32D05-Gal4>UAS-AmaRNAi (Ama KD) leg disc, and between w1118control leg discs versus Nrt1/Nrt+ and Nrt2/Nrt+ heterozygous leg discs and Nrt1/Nrt2 transheterozygous leg discs. (F, G) R32D05-Gal4>UAS-GFP (green) leg discs immunostained for pH3 (magenta); a small portion of myoblasts are proliferating in control (F), while only few cells remained after UAS-AmaRNAi expression in the myoblasts (G); none of them are pH3-positive. (I, J) R32D05-Gal4>UAS-GFP (green) leg discs immunostained for dcp1 (magenta); several apoptotic cells can be found among the GFP-positive cells remaining after UAS-AmaRNAi expression (arrows in J). (H) Graphs showing the percentage of mitotic myoblasts (on left) and the percentage of apoptotic myoblasts (on right). Compared to UAS-mCherryRNAi (control), the percentage of mitotic myoblasts is significantly reduced when UAS-AmaRNAi is expressed in the myoblasts from early larval stages (Ama KD) and when it is expressed from the beginning of L2 stage using Gal80ts (Ama KD from L2). Compared to UAS-mCherryRNAi (control), the percentage of apoptotic myoblasts is significantly higher when UAS-AmaRNAi is expressed in the myoblasts from early larval stages (Ama KD) and when it is expressed from the beginning of L2 stage using Gal80ts (Ama KD from L2). (K) Dot-plot graph showing the mean number of Twi-positive myoblasts per disc, in R32D05-Gal4 leg discs crossed with different UAS-transgenic lines affecting the FGFR pathway. Statistical analysis reveals an increase in the total of myoblasts when overexpressing an activated ERK (rl OE), a constitutive active FGFR (FGFR OE) or when down-regulating sty expression (Sty KD), compared to control RNAi. Inversely, overexpressing sty (Sty OE) or a dominant negative FGFR (FGFR DN) reduce the number of myoblasts. Note that overexpressing Ama (Ama OE) is not sufficient to induce an increase of myoblast number. (L) Dot-plot graph showing the mean number of Twi-positive myoblasts per disc in rescue experiments using R32D05-Gal4 driver. Overexpressing UAS-Ama together with UAS-Sty (Ama OE; Sty OE) show no significant difference in myoblast number when compared to UAS-LacZ, UAS-Sty overexpression (Sty OE). Co-expressing UAS-StyRNAi with UAS-AmaRNAi (Sty KD; Ama KD) is not sufficient to rescue the loss of myoblasts after UAS-AmaRNAi; UAS-LacZ (Ama KD) expression. Co-expressing UAS-Ama with UAS-HtlDN (FGFR DN; Ama OE) partially rescues the decrease of myoblast number of UAS-HtlDN; UAS-mCherryRNAi (FGFR DN) expression. Co-expression of UAS-Ama and UAS-StyRNAi (Sty KD; Ama OE) together enhances the phenotype of UAS-StyRNAi (Sty KD). In all graphs, error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, ns: not significant, (Mann–Whitney test). n = number of discs (S2 Data). Scale bars = 50 μm. https://doi.org/10.1371/journal.pbio.3002842.g004 A previous study suggested that Ama-depleted flight muscle precursors were unable to proliferate but the authors could not assay a defect in proliferation rate [23]. Moreover, Nrt was expressed in flight muscle precursors and its depletion resulted in a flight muscle defect, suggesting that Ama may interact with Nrt to regulate the pool of flight muscle myoblasts [23,24]. To test this possibility in leg myoblasts, we counted the number of Twist+ cells in leg discs of Nrt1/Nrt2 transheterozygous mutants. We observed no differences compared to w1118 or with heterozygous Nrt1/Nrt+ and Nrt2/Nrt+ leg discs (Fig 4C–4E). This result is consistent with the absence of Nrt expression in myoblasts at L3 stage (Fig 2). Moreover, myoblast depletion can already be observed in Ama-RNAi expressing leg discs of earlier stage L2 (S3A–S3F Fig), a stage when Nrt could not be detected in leg disc even in tendon cells, which are specified at the beginning of L3 stage (S3G–S3I Fig). We conclude that Ama controls the number of leg myoblasts in an Nrt-independent way during larval stages.

Ama is a potential downstream effector of FGF pathway in the regulation of myoblast number A previous study proposed that Ama could modulate the RTK pathway activity during Drosophila brain development by regulating Sprouty (Sty) expression, a negative regulator of this pathway [22]. In this study, when Ama was specifically attenuated in glial cells, single-cell RNA-sequencing showed a strong down-regulation of cell cycle genes such as PCNA and Cyclins, as well as high expression of the pro-apoptotic gene hid, supporting a role of Ama in regulating cell survival and proliferation. Interestingly, we had previously shown that the RTK pathway, FGFR, is involved in regulating the number of leg disc myoblasts [10]. To test whether Ama could regulate myoblast number through the modulation of the RTK pathway, we conducted a set of gain- and loss-of-function experiments using the R32D05-Gal4 line. First, we counted the total number of Twist+ myoblasts in leg disc expressing an active form of the downstream effector of the RTK pathway, ERK, also named Rolled (UAS-rlsem), a dominant-negative form (UAS-HtlDN) or a constitutively active form (UAS-HtlCA) of the FGF receptor Heartless (Htl) (Fig 4K and S2 Data). Compared to control (mean = 643 myoblasts/disc, n = 37), we found that the number of myoblasts was significantly increased in rlsem (mean = 1032, n = 14) and in HtlCA (mean = 1,706, n = 27) expressing leg discs, and reduced after HtlDN induction in myoblasts (mean = 410, n = 19). This result confirmed that RTK pathways, and notably the FGFR pathway, are involved in controlling the number of total myoblasts. Overexpression of Sty (UAS-Sty) resulted in a significant decrease of myoblast number (mean = 502, n = 19), whereas its reduction (UAS-StyRNAi) leads to more myoblasts (mean = 839, n = 18), in accordance with its role of inhibiting FGFR pathway [20]. As Ama negatively regulates Sty expression in glial cells, we asked whether Ama could counteract Sty-induced changes in myoblast numbers in the developing leg. We found that Ama overexpression (Ama-HA) alone cannot rescue the reduced number of myoblasts caused by Sty overexpression (UAS-lacZ; UAS-Sty) (Fig 4L and S2 Data). Similarly, no rescue was observed when co-expressing AmaRNAi with StyRNAi compared to UAS-lacZ; UAS-AmaRNAi (Fig 4L). These results indicate that the hypothesis that Ama stimulates the FGFR pathway by negatively regulating Sty expression is not sufficient to explain the role of Ama in controlling leg myoblast number (see Material and methods for myoblast counting details). Nevertheless, we continued to investigate possible connections between Ama and the FGFR pathway. Previous work has shown that the expression of the constitutively active form of ERK can rescue the phenotype of Ama depletion in glial cells [22]. Therefore, we tried to rescue the UAS-AmaRNAi phenotype by co-expressing the UAS-rlsem. However, rlsem overexpression was insufficient to rescue myoblast depletion (Fig 4L). Since we could not show an upstream effect of Ama on the FGFR pathway, we asked if Ama could act downstream. Strikingly, leg discs co-expressing UAS-AmaHA and UAS-HtlDN showed a significantly higher number of myoblasts compared to the ones expressing UAS-HtlDN; UAS-mcherryRNAi. This suggests that Ama could, in fact, be a target of the FGFR pathway. Lastly, when Ama was overexpressed in a context where Sty expression was decreased (UAS-StyRNAi; UAS-AmaHA), the increase in the number of myoblasts was even higher than when only Sty expression was reduced (Fig 4L). However, the mere overexpression of Ama (UAS-AmaHA) without modifying Sty expression had no impact on the number of myoblasts (Fig 4K). Thus, our results indicate that Ama does not promote myoblast proliferation by alleviating Sprouty’s inhibitory effect on the RTK pathway, in contrast to the previously described effect on glial cells [22]. However, the fact that Ama overexpression and Sty knockdown show an additive effect on myoblasts number and that Ama overexpression could improve loss of myoblasts in the HtlDN context supports a model in which Ama acts downstream of FGF/RTK pathway. Another, non-mutually exclusive, possibility is that Ama enhances myoblast responsiveness to proliferation-inducing FGF signals. This view is supported by 2 studies. First, Ama knockdown was found to reduce the suppressive effect on retinal degeneration of the E3 ligase SORDD1 [35]. Second, a two-hybrid screen found Ama interaction with Syndecan [36], a proteoglycan acting as a co-receptor that modulates ligand availability for the FGF receptor [37].

Ama and its receptor Nrt are required for myoblast-tendon adhesion Given the restricted Nrt expression in one unique tendon precursor, the tilt, and the known role of Nrt as an adhesion molecule [13–15], we hypothesized that Ama and Nrt interact to ensure adhesion between myoblasts and the tilt. To test this hypothesis, we used an alternative myoblast specific driver, R15B03-Gal4 (S1A–S1F Fig), to induce Ama attenuation (UAS-AmaRNAi) after the extensive larval phases of proliferation. We combined the R15B03-Gal4 driver with R79D08-lexA that induced lexAop-GFP transgene in tendon cells (S4G–S4I Fig), allowing us to observe the developing tendons while inducing UAS-AmaRNAi in myoblasts from late L3 stage. We then analyzed the consequences of late myoblast-specific Ama knockdown on myoblast distribution at 5 h APF, a time when myoblasts are properly aligned along the elongating tilt tendon (Soler and colleagues, Fig 5A and 5A’). Late myoblast-specific Ama knockdown results in an apparent misdistribution of the myoblasts around the tilt tendon (Fig 5B and 5B’ compared to Fig 5A and 5A’). To quantify the myoblast position variability relative to the tilt, we measured the shortest distance between each myoblast and the surface of the tilt and calculated the myoblast-to-tendon mean distance (dMT) for each disc (for detailed protocol, see Material and methods and S5 Fig). In controls, myoblasts are located at an overall average dMT of 4 μm from the tilt across all discs, whereas in AmaRNAi-expressing leg discs, this value increased slightly but significantly to 5.4 μm. This result indicates that Ama knockdown, leads to myoblasts mis-distribution around the tilt, suggesting that the reduction of Ama level could affect myoblast-tendon adhesion (Fig 5D and S4 Data). To better assess whether Ama knockdown increases the probability of the myoblasts being located farther away from the tilt than in control, we calculated the percentage of discs for which the dMT was greater than 4 μm (average dMT across all control discs), and 68% of the discs expressing R15B03-Gal4>AmaRNAi had a dMT greater than 4 μm, compared to 45% of the control discs (Fig 5E and S4 Data). Thus, reducing level of Ama expressed by the myoblasts significantly increases the proportion of discs in which myoblasts are not properly associated with the tilt. In parallel, we generated a strain carrying the Nrt2 allele and the R79D08-lexA>lexAop-GFP transgenes that we crossed with the line carrying the Nrt1 allele. F1 were then analyzed the same way as Ama knockdowns for myoblast positioning relative to the tilt. R79D08-lexA>lexAop-GFP; Nrt2/Nrt1 transheterozygous mutants exhibit an average dMT of 6.2 μm, significantly elevated compared to the average dMT of R79D08-lexA>lexAop-GFP of 4.3 μm and R79D08-lexA>lexAop-GFP; Nrt2/Nrt+ controls (4.8 μm) (Fig 5D). As expected, the percentage of discs with a dMT greater than the average dMT of control discs (4.3 μm) was much higher in transheterozygous mutants (84%) compared to controls (48% and 45%, respectively, Fig 5E). Thus, in both Ama and Nrt knockdown contexts, myoblasts lose their ability to aggregate and to align along their corresponding tendon. These results support the hypothesis that Ama/Nrt heterophilic interactions play a role in myoblasts for tendon precursor adhesion. Given that Ama is a secreted protein [18] and Nrt is only present on the surface of the tendon, it is likely that Ama interacts with another membrane-associated protein at the surface of the myoblasts. Alternatively, the presence of a short hydrophobic domain at its COOH-terminal end suggests that Ama could directly tether to the membrane via a glycosyl-phosphotidylinositol (GPI) anchor [18] similar to other cell adhesion molecules [38]. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Myoblast positioning and tendon morphogenesis require Ama and Nrt. (A–C) Confocal optical sections of 5 h APF leg disc immunostained for Twi from (A) R79D08-lexA>lexAop-mCD8::GFP; R15B04-Gal4>UAS-mCherryRNAi, from (B) R79D08-lexA>lexAop-mCD8::GFP; R15B04-Gal4>UAS-AmaRNAi and from (C) R79D08-lexA>lexAop-mCD8::GFP; Nrt1/Nrt2 transheterozygous. (A’–C’) Higher magnifications of the dorsal femur regions from (A), (B), and (C), respectively. (A, A’) In control leg discs expressing UAS-mCherryRNAi using R15B03-Gal4 late myoblast driver, the tilt (green) has elongated within the dorsal femur cavity to form a long internal structure along which the myoblasts (magenta) are aligned. (B, B’) When UAS-AmaRNAi is expressed in the myoblasts, they lose their adhesion with the tilt; the tilt itself appears wider and shorter compared to control. (C, C’) The same observations can be made in Nrt1/Nrt2 transheterozygous leg discs, with a misdistribution of the myoblasts along the tilt and an elongation default of the tilt. (D) Dot-plot graph showing the mean distance between myoblasts and the tilt surface; each dot corresponds to the mean distance between the tilt and all the myoblasts for one disc. The average of mean distance for R79D08-lexA>lexAop-mCD8::GFP; R15B04-Gal4>UAS-AmaRNAi (Ama KD) leg discs is statistically higher compared to R79D08-lexA>lexAop-mCD8::GFP; R15B04-Gal4>UAS-mCherryRNAi (control RNAi) leg disc (p < 0.0016). This average is also higher when comparing R79D08-lexA>lexAop-mCD8::GFP; Nrt1/Nrt2 transheterozygous leg discs with both R79D08-lexA>lexAop-mCD8::GFP (+/+) (p < 0.0008) and R79D08-lexA>lexAop-mCD8::GFP; Nrt2/Nrt+ (p < 0.0201) control leg discs. (E) Graphs showing the percentage of discs for which the mean distance between the myoblasts and the tilt is higher than the average mean distance of the controls UAS-mCherryRNAi and +/+, respectively. (F) Dot-plot graph showing the volume of the tilt, each dot corresponds to one disc. No statistically significant difference is observed between the control lines and the lines expressing AmaRNAi, as well as the mutant lines for Nrt. (G) Dot-plot graph showing the lengths of the tilt, each dot corresponds to one disc. UAS-AmaRNAi (Ama KD) myoblast late expression leads to shortening of the tilt length compared to UAS-mCherryRNAi control (p < 0,0042). This shortening is also evident in transheterozygous mutants Nrt1/Nrt2 compared to wild-type Nrt homozygous (p < 0,025) and Nrt2/ Nrt+ (p < 0,011). #Tendon-specific AmaKD in R79D08-lexA>lexAop-mCD8::GFP; Sr-Gal4>UAS-AmaRNAi discs does not significantly affect either the volume or length of tendons. In all graphs, error bars represent SD; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, ns: not significant, Mann–Whitney test (D) and (G), Fisher’s exact test (E). n = number of discs; (S4 Data). APF, after pupae formation. https://doi.org/10.1371/journal.pbio.3002842.g005 Thus, Ama could mediate tendon-myoblast adhesion by interacting with Nrt on the tendon side and myoblast-myoblast adhesion on the other side, directly or through another unidentified transmembrane receptor. Ama is orthologous to several Ig-like neuronal cell adhesion molecules in vertebrates, including the IgLON 4 and 5 GPI-anchored membrane proteins that regulate myoblast adhesion during myogenesis and muscle regeneration by providing an essential microenvironment that ensures muscle stem cell survival [39,40]. Thus, the conservation of Ama’s function could highlight a pivotal role in sustaining muscle tissue homeostasis and development across evolution.

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