InteractiveFly: GeneBrief

Myosin 31DF: Biological Overview | References

Gene name - Myosin 31DF

Synonyms - myosin-IA

Cytological map position - 31F1-31F4

Function - cytoskeletal motor protein

Keywords - cytoskeleton, left-right asymmetry, hindgut, testes

Symbol - Myo31DF

FlyBase ID: FBgn0086347

Genetic map position - 2L:10,491,682..10,506,780 [-]

Classification - Myosin motor domain, type I myosins and Myosin tail

Cellular location - cytoplasmic

NCBI links: EntrezGene

Myo31DF orthologs: Biolitmine
Recent literature
Amcheslavsky, A., Lindblad, J. L. and Bergmann, A. (2020). Transiently "Undead" Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut. Cell Rep 33(8): 108408. PubMed ID: 33238125
This study reveals surprising similarities between homeostatic cell turnover in adult Drosophila midguts and "undead" apoptosis-induced compensatory proliferation (AiP) in imaginal discs. During undead AiP, immortalized cells signal for AiP, allowing its analysis. Critical for undead AiP is the Myo1D-dependent localization of the initiator caspase Dronc to the plasma membrane. This study shows that Myo1D functions in mature enterocytes (ECs) to control mitotic activity of intestinal stem cells (ISCs). In Myo1D mutant midguts, many signaling events involved in AiP (ROS generation, hemocyte recruitment, and JNK signaling) are affected. Importantly, similar to AiP, Myo1D is required for membrane localization of Dronc in ECs. It is proposed that ECs destined to die transiently enter an undead-like state through Myo1D-dependent membrane localization of Dronc, which enables them to generate signals for ISC activity and their replacement. The concept of transiently "undead" cells may be relevant for other stem cell models in flies and mammals.
Farrell, L., Puig-Barbe, A., Haque, M. I., Amcheslavsky, A., Yu, M., Bergmann, A. and Fan, Y. (2022). Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation. PLoS Genet 18(12): e1010533. PubMed ID: 36469525
Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. This study identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP.

The internal organs of animals often have left-right asymmetry. Although the formation of the anterior-posterior and dorsal-ventral axes in Drosophila is well understood, left-right asymmetry has not been extensively studied. This study demonsrates that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA [also referred to as MyoID in mammals; Gillespie, 2001; Morgan, 1995), and is the first actin-based motor protein to be implicated in left-right patterning. Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, it was found that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F (myosin-IB), another myosin I, reverses the handedness of the embryonic gut, and its knockdown also causes a left-right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left-right patterning. It is suggested that the actin cytoskeleton and myosin I proteins may be crucial for generating left-right asymmetry in invertebrates (Hozumi, 2006). An accompanying paper (Spéder, 2006, see below) demonstrates that Myo31DF regulates dextral looping of genitalia.

Mechanisms that create characteristic left-right asymmetry have been studied extensively in vertebrates. However, although the organs of many invertebrate species also have left-right asymmetry, the mechanisms by which this asymmetry arises are largely unknown. In Drosophila, several organs have left-right asymmetry, including the embryonic gut, the adult brain and the genitalia (Hozumi, 2006).

To identify genes involved in left-right asymmetry of the Drosophila embryonic gut, a genetic screen was performed using a collection of P-element lines. The embryonic gut is composed of three major parts, the foregut, midgut and hindgut, all of which have characteristic left-right asymmetry. 75.7% of homozygous Myo31DFsouther embryos show synchronous inversion of the midgut and hindgut. In these embryos, the hindgut and midgut are the mirror-image of those in wild-type embryos, rather than showing randomized patterning. In contrast, foregut handedness was normal in all cases examined, indicating that this phenotype was heterotaxic. Myo31DFsouther is a background mutation of the Gene Search Drosophila line GS14508. Deficiency mapping was used to map the cytological location of Myo31DFsouther to between 30D and 31F. Complementation tests were performed between Myo31DFsouther and lines bearing mutations that map to this region. Myo31DFsouther failed to complement Myo31DFK1 (Spéder, 2006), an allele of Myo31DF encoding Drosophila MyoID (Hozumi, 2006).

The transposable element gypsy was inserted into the 5'-untranslated region of the Myo31DF gene in Myo31DFsouther. Myo31DFL152 was one of five ethylmethanesulfonate (EMS)-induced Myo31DF alleles isolated in a large-scale EMS mutant screen. Myo31DFL152 has a base substitution that introduces a premature stop codon at amino acid 331, resulting in a putative truncated product. Myo31DF overexpression from UAS-Myo31DF driven by byn-Gal4 in the hindgut and posterior midgut and their primordial counterparts rescued the left-right defects of Myo31DFsouther embryos, indicating that Myo31DF was responsible for the heterotaxia. In Myo31DFsouther embryos, NP2432-driven expression of Myo31DF in the hindgut epithelium, but not in other parts of the embryonic gut, such as the midgut and mesoderm, was sufficient to rescue this heterotaxia, suggesting that Myo31DF is required in the hindgut epithelium. Furthermore, the frequency of the handedness defect was similar in Myo31DFL152 homozygous and Myo31DFL152/Df(2L)J2 embryos. Thus, Myo31DFL152 is probably a null mutant of Myo31DF. All homozygous Myo31DF mutants isolated in this study were viable and fertile, with normal hindgut tissue specification, suggesting that Myo31DF function is largely restricted to left-right patterning. No maternal phenotype or Myo31DF gene contribution was detected. Notably, the foregut became a mirror-image of its wild-type counterpart when Myo31DF was overexpressed in the entire embryo, but other parts of the gut were normal. Therefore, it is suggested that Myo31DF is not involved in the left-right asymmetrical development of the foregut in wild-type embryos, but can reverse foregut handedness (Hozumi, 2006).

The adult hindgut and testes, which are regenerated during metamorphosis, were examined. These also showed inversed handedness in the Myo31DF homozygote. In most Myo31DFL152 adults, the loop of the hindgut and spiral of the testes were reversed, although not always synchronously. The function of the Myo31DF gene was knocked-down using RNA interference (RNAi) in vivo. The expression of double-stranded Myo31DF RNA driven by byn-Gal4 caused inversion of the adult, but not the embryonic, hindgut. Thus, the left-right pattern involving Myo31DF is not transmitted during metamorphosis (Hozumi, 2006).

In situ hybridization revealed Myo31DF expression in the amnioproctodeal invagination at stage 6. At stages 12-14, Myo31DF messenger RNA was strongly detected in the primordial midgut and hindgut, and in the proventriculus, midgut and hindgut. A sense-strand probe of Myo31DF gave no detectable signal. Immunostaining of wild-type embryos with an anti-Myo31DF antibody (anti-Myo31DF-1P) also labelled the midgut and hindgut. These signals were absent in Myo31DFsouther and Myo31DFL152 homozygotes, indicating that the staining was specific. Myo31DF mRNA and protein were detected in a symmetrical pattern before the development of left-right asymmetry. Myo31DF protein is expressed in the adult gut (Morgan, 1995). No Myo31DF expression was detected in the foregut, which may account for the absence of any laterality defect in the foregut of Myo31DF mutants (Hozumi, 2006).

Myo31DF protein binds to actin in an ATP-dependent manner (Morgan, 1995). The co-localization of Myo31DF was examined and the actin cytoskeleton in cultured Drosophila S2 cells. A green fluorescent protein (GFP)-tagged Myo31DF (Myo31DF-GFP) had wild-type function, given that its overexpression rescued Myo31DFsouther. Myo31DF-GFP co-localized with actin, mostly at cell protrusions. In epithelial cells of the hindgut, endogenous Myo31DF was detected as punctate staining, partly overlapping with cortical actin (Hozumi, 2006).

byn-Gal4-driven misexpression of GFP-tagged moesin, an actin-binding protein, in wild-type embryos caused a reduction in actin filaments in the apical region of the hindgut epithelium, where Myo31DF function is required. Notably, the midgut and hindgut always had the same handedness, but the handedness was random (not inversed), in wild-type and Myo31DF homozygous embryos. Embryo handedness was also affected by NP2432-driven GFP-moesin expressed in the hindgut epithelium only. However, GFP-moesin expression in the midgut only did not affect handedness (Hozumi, 2006).

To investigate further the functional link between Myo31DF and the actin cytoskeleton, the phenocritical period for inducing left-right defects was determined using Myo31DF RNAi and GFP-moesin misexpression, following the TARGET method. Both Myo31DF knockdown and GFP-moesin expression in the hindgut 0-24 h after pupation caused similar defects in adult gut handedness, suggesting that Myo31DF and the appropriate organization of actin filaments are required at the same time. GFP-moesin also affected handedness in the Myo31DF embryo, suggesting that the default handedness, which may be manifested in the Myo31DF homozygote, also depends on the actin cytoskeleton. The involvement of three Rho GTPase family proteins, Rho, Rac and Cdc42, which regulate the organization of the actin cytoskeleton, was examined. Expression of dominant-negative forms of these proteins, especially Rho, in the hindgut induced synchronous left-right defects in the embryonic midgut and hindgut. Together, these results suggest that Myo31DF depends on the actin cytoskeleton to generate left-right asymmetry (Hozumi, 2006).

Cell division and cell death do not occur during left-right asymmetric development of the hindgut. It is therefore speculated that the rearrangement of hindgut epithelial cells may be part of this process. To test this possibility, a time-lapse analysis was performed. The position of each cell was visualized by labelling the nucleus with GFP. Cell rearrangement, which coincided with the left-right bias associated with left-handed rotation of the hindgut, was suggested by the significant intercalation of some cells (Hozumi, 2006).

Another myosin I protein, Myo61F (Drosophila MyoIB, also referred to as MyoIC in mammals), has been reported in Drosophila. Myo61F protein is detected in the embryo and adult gut. To test whether Myo61F is also involved in left-right asymmetry, Myo61F was overexpressed using UAS-Myo61F or GS9889 driven by byn-Gal4. Unexpectedly, Myo61F overexpression resulted in inversion of the midgut and hindgut in both cases. In contrast, Myo31DF overexpression did not affect the handedness of these organs. These results suggest that Myo31DF and Myo61F have antagonistic functions in creating the left-right asymmetry of these organs. The involvement of Myo61F in left-right asymmetry is also supported by the finding that its knockdown by RNAi results in the left-right defect in the embryonic midgut (Hozumi, 2006).

Homozygous Myo31DF embryos show reversed handedness of embryonic and adult visceral organs, which may represent the default state of left-right asymmetry in Drosophila. This situation is similar to the function of the sinistral gene in the freshwater snail, Limnea (although the sinistral gene is required maternally) (Wood, 1997; Shibazaki, 2004). Normal handedness is still seen in 25% of Myo31DF homozygotes. It is speculate that some other myosin gene(s) has a redundant function in left-right patterning. Inversion of the anteroposterior axis does not affect laterality, suggesting that left-right pattering occurs zygotically; this is consistent with the zygotic function of Myo31DF. These results also suggest that an actin-based mechanism, which can align itself to either an anteroposterior-dorsoventral reference or the pre-existing sinistral handedness, exists to direct the rotation of the hindgut epithelium. Since myosin I proteins are involved in vesicular transport (Huber, 2000), it is proposed that Myo31DF and Myo61F, which on the basis of their structures are believed to move to the plus ends of actin filaments, carry left-right determinants with opposite activities. Thus, both left-right determinants would be concentrated in the plus ends of actin filaments that have a hypothetical planar polarity. In the Myo31DF mutant, only the opposing determinant is concentrated here, which reverses the handedness. According to this model, disruption in actin organization would result in left-right randomization, as was indeed observed experimentally (Hozumi, 2006).

Type ID unconventional myosin controls left-right asymmetry in Drosophila

Breaking left-right symmetry in Bilateria embryos is a major event in body plan organization that leads to polarized adult morphology, directional organ looping, and heart and brain function. However, the molecular nature of the determinant(s) responsible for the invariant orientation of the left-right axis (situs choice) remains largely unknown. Mutations producing a complete reversal of left-right asymmetry (situs inversus) are instrumental for identifying mechanisms controlling handedness, yet only one such mutation has been found in mice (inversin; Morgan, 1998) and snails (Ueshima, 2003; Freeman, 1982). The conserved type ID unconventional myosin 31DF gene (Myo31DF) has been identified as a unique situs inversus locus in Drosophila. Myo31DF mutations reverse the dextral looping of genitalia, a prominent left-right marker in adult flies. Genetic mosaic analysis pinpoints the A8 segment of the genital disc as a left-right organizer and reveals an anterior-posterior compartmentalization of Myo31DF function that directs dextral development and represses a sinistral default state. As expected of a determinant, Myo31DF has a trigger-like function and is expressed symmetrically in the organizer, and its symmetrical overexpression does not impair left-right asymmetry. Thus Myo31DF is a dextral gene with actin-based motor activity controlling situs choice. Like mouse inversin (Nurnberger, 2002), Myo31DF interacts and colocalizes with β-catenin, suggesting that situs inversus genes can direct left-right development through the adherens junction (Spéder, 2006).

In wild-type males, the genital plate, to which the spermiduct is attached, undergoes a 360° clockwise (dextral) rotation when viewed from the posterior pole. This directional looping is reminiscent of other coiling processes such as mammalian heart tube looping and snail spiral development. As in other species, one direction is dominant among the Drosophilidae, the dextral rotation, with no sinistral species reported to date. This study identified an insertional mutation, KG02246, which shows a striking inverted phenotype when combined with deficiencies covering the 31DF genomic region. In KG02246/Df(2L)Exel7048 or KG02246/Df(2L)J3 males, genitalia rotation is variable, with ~60% of individuals showing sinistral rotation. Imprecise excision of KG02246 generated two genomic deletions, KG022461 and KG022462, both of which presented a stronger, 100% inverted (sinistral) genitalia rotation phenotype. Thus, KG02246 alleles identify the first situs inversus mutations in Drosophila and provide genetic evidence for the existence of a left-right axis in this organism (Spéder, 2006).

Drosophila Myo31DF is a conserved myosin belonging to the Myo1D family. It is known to interact with F-actin (Morgan, 1995) and it colocalizes with actin-rich structures in different tissues. After mouse inversin, Drosophila Myo31DF represents the second situs inversus gene to be molecularly identified. The mouse inversin gene (Invs) encodes a protein with ankyrin repeats and two IQ domains (Morgan, 2002; Eley, 2004) that bind calmodulin, a Ca2+ -dependent regulatory protein (Bahler, 2002). Like inversin, Myo31DF contains two IQ domains essential for its function. Indeed, a Myo31DF form lacking IQ domains (Myo31DFDeltaIQ) is unable to rescue Myo31DF mutations (Spéder, 2006).

To determine the function of Myo31DF in genitalia rotation, its expression was examined in the genital disc, the precursor of adult genitalia. The genital disc is composed of segments A8, A9 and A10, each with an anterior and a posterior. Immunostaining of wild-type flies with two polyclonal antibodies directed against overlapping regions of the Myo31DF tail domain, anti-Myo31DF-1P and anti-Myo31DF-3P, or using a Myo31DF-Gal4 enhancer-trap line (NP1548) revealed symmetrical expression of Myo31DF in a double chevron-like pattern restricted to the ventral domain of the male genital disc. This expression, starting in third instar larvae and remaining unchanged during this stage, was absent in Myo31DFK2 mutant discs. Double staining with an A8-specific marker in the genital disc (tsh-Gal4 > myr-RFP) indicated that Myo31DF is expressed in the A8 segment, with one chevron in the posterior compartment and the other in the anterior compartment. Consistently, expression of two copies of an inhibitory RNA gene (2 x Myo31DFRNAi) specifically in the A8 segment led to loss of Myo31DF expression and to inverted phenotypes that mimicked Myo31DF mutations. Together, these data identify the A8 segment as a left-right organizer that is required for situs choice in Drosophila (Spéder, 2006).

To investigate the relationship between the anterior-posterior and left-right axes, the putative domain(s) of Myo31DF function was mapped by selectively silencing the gene in different compartments, using specific Gal4 lines driving 2 x Myo31DFRNAi. The combinatorial removal of Myo31DF function in the anterior and/or posterior domains led to distinct phenotypes, indicating a dual function for the Myo31DF protein in A8. First, blocking Myo31DF function posteriorly in A8 (using hh-Gal4 or en-Gal4) resulted in a striking non-rotated genitalia phenotype. This finding was confirmed in a complementary experiment using dpp-Gal4 to rescue Myo31DFK1 in the anterior compartment. The absence of situs choice observed in these experiments indicates that posterior Myo31DF has an instructive role in dextral looping. Second, blocking Myo31DF function solely in the anterior compartment (in dpp-Gal4 > 2 x Myo31DFRNAi or in Myo31DFK1/Myo31DFK1; hh-Gal4 > Myo31DF rescued males) led to partial dextral rotation, suggesting a permissive role for Myo31DF in this compartment. Comparing this outcome to the effect of concomitant removal of both anterior and posterior functions (the only context that led to complete reversal) indicates that the function of Myo31DF in the anterior compartment is to repress sinistral looping. These experiments demonstrate that Myo31DF is essential both anteriorly and posteriorly. A model illustrating the dual function of Myo31DF in regulating dextral development within the A8 segment is presented. In this model, A8 contains information to specify both sinistral and dextral rotation, with sinistral information being anterior and dextral information posterior. Dextral information is dominant over sinistral information, and Myo31DF function is required both posteriorly, to induce dextral development, and anteriorly, to repress sinistral development. Only in the absence of Myo31DF can sinistral development occur as a default state (Spéder, 2006).

As expected of a left-right determinant with a function that precedes asymmetry, Myo31DF is expressed symmetrically in A8. In addition, as with mouse inversin (Watanabe, 2003), symmetrical overexpression does not lead to left-right defects (AbdB-Gal4, tsh-Gal4 and ptc-Gal4 lines). Another predicted feature of left-right determinants is their temporally restricted, trigger-like function, which is later relayed by mechanisms acting to maintain the initial symmetry-breaking event. To test this, temperature-shift experiments were carried out using a genetically engineered temperature-sensitive Myo31DF allele. Single temperature-shift experiments with 24-h or 6-h resolution indicated that Myo31DF function is required at day 6 of development, between 126 and 132 h. Double temperature-shift experiments allowed to determine that Myo31DF function is required for as little as 3 h within this period. These data provide high temporal resolution, allowing the temporal mapping of a left-right symmetry-breaking event and demonstrating that situs choice depends on a peak of Myo31DF function in the left-right organizer (Spéder, 2006).

Cilia have emerged as important cellular structures for generating left-right asymmetry in vertebrate embryos. To address their possible contribution to invertebrate left-right determination, genital discs were stained with GT335, an antibody that labels glutamylated tubulin in cilia across species, including Drosophila. GT335 did not detect any cilia in genital discs. Additionally, mutations in the conserved Rfx gene, which controls the formation of ciliated neurons in Drosophila, did not affect genitalia rotation. Together, these data suggest that cilia are not involved in dextral development in Drosophila. It is proposed that Drosophila uses primarily the actin cytoskeleton to determine left-right asymmetry. Consistently, the small GTPase Drac1 and the JNK pathway, known regulators of the actin cytoskeleton, showed specific genetic interactions with Myo31DF. Notably, actin is also important for situs choice in snails, suggesting that in invertebrates the actin cytoskeleton has a central role in left-right determination (Spéder, 2006).

To start investigating how Myo31DF might act to determine left-right asymmetry, two-hybrid screening was used to identify Myo31DF interactors or cargo(es). Using the Myo31DF tail domain (amino acids 737-1011) as bait, several positive clones were found encoding a carboxy-terminal fragment containing ARM repeats 6-12 of the Armadillo/β-catenin protein. This interaction was direct, as shown by glutathione S-transferase (GST)-pulldown experiments using purified proteins or S2 cell extracts. Furthermore, endogenous Myo31DF and a functional Myo31DF-GFP (green fluorescent protein) fusion protein colocalized with Armadillo at the adherens junctions in A8, suggesting that the two proteins can interact in vivo. Notably, inversin has been shown to colocalize and interact with β-catenin in vertebrate epithelial cells, indicating that an interaction with β-catenin is a common feature of both known situs inversus genes. These results are consistent with a demonstrated role of N-cadherin in left-right asymmetry (Spéder, 2006).

How could anterior-posterior organization of Myo31DF and interaction with Armadillo account for left-right asymmetry? The actin cable network might serve as a track for Myo31DF to deliver specific cargoes or vesicles at the adherens junction. Indeed, it was found that Myo31DF interacts with dynamin, consistent with the role of rat MyoID in vesicular transport (Huber, 2000). The anterior-posterior boundary itself creates an asymmetric junction that could serve as a scaffold for Myo31DF to assemble a dextral-specific complex on the anterior side of posterior Myo31DF-expressing cells. Anterior-posterior asymmetry of dextral information could later be translated into left-right asymmetry through the remodelling of cell contacts (cell intercalation or rotation), as seen in other epithelia. For example, 90° rotation of epithelial cells relative to the main body axis is observed in the Drosophila eye imaginal disc. A similar, 90° planar rotation of Myo31DF-expressing cells would result in left-right orientation of the dextral junctional complex in the tissue. In this working model, a short pulse of Myo31DF activity would be essential for spatially restricting the dextral junction, as has been observed (Spéder, 2006).

Coupling of apoptosis and L/R patterning controls stepwise organ looping

Handed asymmetry in organ shape and positioning is a common feature among bilateria (for a review see Huber, 2007), yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. This study utilized the directional 360° clockwise rotation of genitalia in Drosophila to study LR-dependent organ looping. Using time-lapse imaging, it was shown that rotation of genitalia by 360° results from an additive process involving two ring-shaped domains, each undergoing 180° rotation. The results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID: Myo31DF). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. A specific pattern of apoptosis at the ring boundaries is revealed, and this study also shows that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180° LR module (Suzanne, 2010).

Left-right (LR) asymmetric development is essential to the morphogenesis of many vital organs, such as the heart. Directional looping of LR organs is a complex morphogenetic process relying on proper coordination of early LR patterning events with late cell-tissue behaviors. In vertebrates, several developmental models have been proposed for gut coiling downstream of the Nodal-Pitx2 regulatory pathway, including intrinsic asymmetric elongation of the gut in Xenopus or extrinsic force generation by mesenchymal tissue in Zebrafish and by dorsal mesentery in the chick and mouse embryos. However, the cellular mechanisms underlying LR organ morphogenesis are mostly unknown (Suzanne, 2010).

In Drosophila, directional clockwise (or dextral) rotation of the genital plate and gut has been shown only recently to be controlled by the LR determinant myosin ID (MyoID). In myoID mutant flies, LR morphological markers are inverted, leading to counterclockwise (or sinistral) looping of the genital plate, spermiduct, gut, and testis. This indicates that myoID is a unique situs inversus gene in Drosophila. Intriguingly, the expression of MyoID is restricted to two rows of cells within the A8 segment of the genital disc (the analia and genitalia precursor), with one row of expression in the anterior compartment (A8a) and the other in the posterior compartment (A8p) (Suzanne, 2010).

Removal of myoID function specifically in the A8 segment is sufficient to provoke the complete inversion of rotation (360° counterclockwise) of the genitalia and sinistral looping of the spermiduct to which it is attached. The A8 segment therefore represents a LR organizer controlling the directional rotation of the whole genitalia in Drosophila (Suzanne, 2010).

Because circumrotation (the process of 360° rotation) may result from a number of different morphogenetic processes, not deducible from the simple observation of the final adult phenotype, a new and innocuous imaging method was developed to follow the rotation in living pupae (Suzanne, 2010).

To be able to analyze the movement of distinct domains in live developing genitalia, time-lapse imaging was coupled with genital disc 'painting' by expressing different fluorophores in various regions of the genitalia precursor. Analysis of wildtype live genitalia through this method revealed their spatial and temporal organization during rotation. It was first determined that rotation begins at around 25 hr after puparium formation (APF) and lasts 12-15 hr. At 25 hr APF, the genital disc is organized into concentric rings, which, from anterior to posterior, include an A8a ring, an A8p ring, and a large central disc composed of A9-A10 tissues. The analysis of rotation in live pupae coupled to manual tracking allowed the identification of two distinct moving domains: a large posterior domain comprising A8p-A9-A10 (hereafter referred to as A8p) and a smaller anterior domain made of A8a. The A8p domain moves first and is followed by A8a, which starts moving later on. During the entire process, cells from the abdomen, to which the genital disc is connected, remain immobile. The finding of two rotating domains, A8a and A8p, was unexpected. It reveals a complex rotational activity of the genitalia and rules out a simple model in which the genital plate would rotate by 360° as a whole. To further understand how rotation occurs, timelapse imaging of the full, 15-hr-long rotation was performed. This analysis revealed that each ring had a different rotational activity. When viewed from the posterior pole, the A8p ring undergoes 360° clockwise rotation, while the A8a ring makes a 180° clockwise rotation. Whereas the rotation of the central part (A8p-A10) of the disc was inferred from the looping of the spermiduct around the gut, the 180° rotation of A8a was not predicted and could only be revealed by time-lapse analysis because this compartment solely gives rise to a tiny and colorless part of the cuticle. Altogether, these in vivo analyses show that rotation of genitalia in Drosophila is a composite process involving two compartments of the A8 segment, A8a and A8p, each expressing a row of MyoID at its anterior boundary and having its own rotational behavior (Suzanne, 2010).

These findings raise the questions of the contribution of each of the two rings to the entire rotation and of how they interact during rotation. In order to address this question, the intrinsic or real rotational activity of A8a and A8p was determined. So far, each ring movement was analyzed relative to the same immobile referential: the abdomen. Although this referential allows the real movement of A8a to be determined, it cannot be used to determine that of A8p, because A8p moves relatively to a mobile referential, i.e., A8a, to which it is attached. To determine the real movement of A8p, it is thus essential to analyze its angular movement relative to A8a, in other words A8a contribution to motion must be subtracted from the apparent A8p movement. To do so, movies were analyzed by setting A8a as a referential and by determining the angular movement of A8p. Reassessing A8p movement through this approach revealed that A8p rotates clockwise only by 180° relative to A8a. The new angular velocity curve of A8p fits almost perfectly with that of A8a, indicating that both movements have similar features. Importantly, these data also indicate that the observed 360° clockwise rotation is the result of a composite process involving two additive 180° clockwise components: a 180° rotation of the A8a relative to the abdomen and an 180° rotation of A8p relative to A8a (Suzanne, 2010).

To further determine the autonomy of each ring relatively to the other, the role of the LR determinant MyoID in this process was dissected by specifically inactivating myoID in either A8a or A8p or in both. By convention, the presence or absence of myoID is represented by a + or - sign, respectively. Accordingly, the wild-type context is noted 'A8a+A8p+' and the myoID mutant 'A8a-A8p-.' Upon specific inactivation of myoID in the A8a domain (A8a-A8p+ context), the adults showed an apparent 'nonrotation phenotype' (0°, no spermiduct looping and genitalia correctly oriented). However, time-lapse imaging revealed that both rings were spinning, although in opposite directions: the A8a domain rotated counterclockwise by 180° (-180°), whereas the A8p domain rotated clockwise by 180° (+180°, real movement). Reciprocally, the inactivation of myoID in the A8p domain (A8a+A8p- context) also led to an apparent nonrotation phenotype. In this context, the behavior of each domain was inverted compared to the previous condition, with the A8a domain rotating clockwise by 180° (+180°) whereas the A8p domain rotated anticlockwise by 180° (-180°, relative or real movement). In both cases, the movement of each ring is consistent with its myoID genotype and the 'dextralizing' activity of this gene. The strict dependence on MyoID for the direction of the rotation is further confirmed in flies where both A8a and A8p were mutants for myoID (myoIDk1). The rotation is often incomplete in this genotype because of the hypomorphic nature of the myoIDk1 allele analyzed; however, both domains show an anticlockwise movement. Therefore, in all genetic contexts analyzed, all parameters of the rotation remain unaffected except the direction of rotation, as illustrated by the perfect mirror image of the angular velocity curves (Suzanne, 2010).

These experiments reveal that each ring adopts an independent 180° movement relative to more anterior structures (A8a relative to the abdomen and A8p relative to A8a): clockwise in the presence of MyoID, anticlockwise in its absence. When both movements are unidirectional, the net rotation is circumrotation (± 360°), whereas upon opposite movements of A8a and A8p, the net rotation is zero (0°), leading to an apparent nonrotation phenotype. Therefore, the net rotation (or apparent rotation = R) can be modeled through a simple equation in which R equals the addition of A8a and A8p movements, with MyoID acting as a sign function (Suzanne, 2010).

It was next of interest to characterize potential cellular mechanisms acting downstream of LR determination during genitalia rotation. In particular, the cellular events responsible for uncoupling rings at the onset of their rotation was determined. Initial insights came from blocking apoptosis, which leads to genitalia rotation defects, but the role of apoptosis in the process is not completely understood. To determine the morphogenetic function of the apoptotic pathway during genitalia rotation, the spatial and temporal requirements for apoptosis were first characterized by analyzing the expression pattern of hid and reaper (rpr) in the genital disc, using two reporter lines. Both reporters were strongly expressed in the A9 and A10 segments. However, in the A8 segment, only hid expression is observed. This coincides with the phenotype of misrotated genitalia observed specifically when hid function is altered but not in rpr mutants. Then the pattern and timing of cell death was determined in the genital disc. To do so, nuclear fragmentation was followed, and an in vivo reporter of caspase activation (the apoliner construct) was used. At the onset of rotation, a large number of apoptotic cells was detected on the most ventral part of the genital disc, first within the A8p ring bordering A8a, coinciding with the beginning of A8p movement. These data indicate an overlap between the apoptotic field and the domain of MyoID expression. These results have been further confirmed by the detection of apoptotic cells by TUNEL staining of fixed pupal genital discs. Later on, a new wave of apoptosis was detected in the most anterior part of the A8a ring, at the junction between A8a and the abdomen. In contrast, only marginal if any apoptosis was detected before and at the end of rotation. Therefore, two waves of cell death are taking place in the A8 segment, coinciding spatially and temporally with the rotation of A8a and A8p rings (Suzanne, 2010).

Given that rings are initially part of the same epithelium and move independently later, it was reasoned that local cell death may be a mechanism to provide the degree of liberty necessary for proper movement. To test this hypothesis, cell death was inhibited in each compartment separately by expressing the caspase inhibitor p35. Interestingly, inactivation of apoptosis in either A8p or A8a leads to a similar phenotype, with flies showing a high proportion of half-rotated genitalia (180° rotation), suggesting that rotation was blocked in the ring deficient for apoptosis. This has been further demonstrated by following the rotation process in vivo, when apoptosis is specifically blocked in the A8a. In this genetic context, the A8a ring stayed mostly still during the whole process, whereas A8p rotated normally. The resulting 180° rotation is thus exclusively due to the movement of one ring, i.e., A8p, in which apoptosis is unaffected. Inhibiting apoptosis in both domains strongly aggravates the phenotype, with 40% of the flies showing nonrotated genitalia (0°), suggestive of an additive phenotype. The rest of the population had 90° rotated genitalia, which may be due to incomplete inhibition of apoptosis. Alternatively, it is possible that some rotation occurs without apoptosis thanks to tissue elasticity. In any case, the results indicate that cell death is required in each ring for separating them from the neighboring tissues and allowing their free rotation. Consistently, nuclei fragmentation and cell death occur normally in a myoID mutant background. Because local cell death is not likely to provide a direct force for rotation, it is proposed that it contributes to the release of rings from neighboring tissues (Suzanne, 2010).

This study has revealed that organ looping can proceed through discrete steps, breaking down circumrotation into the simple building blocks of 180° each. The incremental nature of genitalia rotation is indeed based upon two 180° LR modules, sharing identical angular velocity and range as well as requirement for MyoID and apoptosis. Modularity in morphogenesis provides interesting control mechanisms (through addition or substraction) and therefore plasticity to the process, both at the organism level and during evolution. Entomologists have described different patterns of genitalia rotation in Diptera, ranging from 0° to 360°, that evolved together with changes in mating position. Interestingly, in the Brachycera suborder, to which Drosophilidae belong, we notice that most ancestral species have a nonrotated genitalia (Stratiomyomorpha and Tabanomorpha), whereas 180° and 360° rotation have appeared progressively later in evolution (in Muscomorpha and Cyclorrhapha, respectively). Together with this sequential organization of rotation amplitude in the phylogenetic tree, these data strongly support a model by which the 360° rotation observed in Brachycera ('modern Diptera') would result from the emergence (transition from 0 to 180°) and duplication (transition from 180° to 360°) of a 180° L/R module, thus providing a simple additive model for both the origin of circumrotation and the evolution of genitalia rotation and mating position. However, it should be noted that alternative mechanisms maylead to a similar pattern of genitalia rotation among Diptera (Suzanne, 2010).

The incremental model presented here also offers a solution to the apparent paradox of circumrotation and the question of its elusive utility, illustrated by the fact that both 360° rotation and the absence of rotation lead to the same final posture of genitalia. A facultative role of 360° rotation is further supported by the finding that D. melanogaster males with nonrotating genitalia (A8a-A8p+ or A8a+A8p-) are normally fertile (data not shown). An incremental origin of 360° rotation in which a second half-turn would be added to the existing 180° rotation would well explain this paradox. Thus, circumrotation can be viewed as recapitulating the evolutionary history of genitalia rotation in Brachycera, and its logic would reveal a case of 'retrograde evolution,' in which duplication of a functional module is used to revoke a previous evolutionary step (Suzanne, 2010).

Finally, this analysis of genitalia rotation highlights a new mechanism of morphogenesis relying on a combination of LR patterning and apoptosis. In this process, a new role for apoptosis is revealed as a releasing mechanism allowing the sliding of two parts of an organ. It will be interesting to test in the future whether this releasing role of apoptosis is used more generally, in other morphogenetic movements requiring important cellular rearrangement (Suzanne, 2010).

Chirality in planar cell shape contributes to left-right asymmetric epithelial morphogenesis

Some organs in animals display left-right (LR) asymmetry. To better understand LR asymmetric morphogenesis in Drosophila, LR directional rotation of the hindgut epithelial tube was studied. Hindgut epithelial cells adopt a LR asymmetric (chiral) cell shape within their plane, referred to as planar cell-shape chirality (PCC). Drosophila E-cadherin (DE-Cad) is distributed to cell boundaries with LR asymmetry, which is responsible for the PCC formation. Myosin ID (Myosin 31DF) switches the LR polarity found in PCC and in DE-Cad distribution, which coincides with the direction of rotation. An in silico simulation showed that PCC is sufficient to induce the directional rotation of this tissue. Thus, the intrinsic chirality of epithelial cells in vivo is an underlying mechanism for LR asymmetric tissue morphogenesis (Taniguchi, 2011).

Directional left-right (LR) asymmetry is widely found in animals, such as in the position and structure of the heart, spleen, gut, and lung in vertebrates. The mechanisms of LR axis formation are well understood in some vertebrates, and the cellular basis for LR symmetry breaking, including cell polarities, is beginning to be elucidated. Drosophila shows a directional LR asymmetry of certain organs, including the embryonic hindgut. Although some unique features of Drosophila laterality development have been revealed, such as the involvement of myosin ID (MyoID), the detailed mechanisms of its LR asymmetric development remain largely unknown (Taniguchi, 2011).

The Drosophila embryonic hindgut begins as a symmetric midline structure that curves ventrally at stage 12. It subsequently makes a 90° left-handed rotation, forming a rightward curving structure by stage 13. The hindgut epithelium, but not the overlying visceral muscles, is responsible for this rotation, which is not accompanied by cell proliferation or cell death. Therefore, it was speculated that the hindgut epithelial cells themselves might have LR polarity, which could contribute to the rotation (Taniguchi, 2011).

To analyze LR polarity in the hindgut epithelial cells, the locations of the centrosomes, which reflect cell polarity in other systems, was examined. Each cell's centroid was calculated with respect to its boundaries and the relative position of the centrosome was plotted, labeled with green fluorescent protein (GFP)-centrosomin. In wild-type animals, the relative position of the centrosome was significantly biased to the right-posterior region. These results suggest that hindgut epithelial cells adopt a LR polarity within their plane before the hindgut rotates (Taniguchi, 2011).

It was speculated that this LR polarity would be reflected in the cell shape and participate directly in the left-handed rotation. To address this, the angle was measured between apical cell boundaries and the antero-posterior (AP) axis of the hindgut epithelial tube before rotation (late-stage 12). These apical cell boundaries corresponds to the zonula adherens (ZA). Cell-boundary angles of -90° to 0° to the AP axis were more frequent than those of 0° to 90°, indicating that hindgut epithelial cells have a LR-biased planar cell shape. This LR bias was designated as planar cell-shape chirality (PCC), because the mirror image of the cell's planar shape does not overlap with its original cell shape (Taniguchi, 2011).

Previous studies demonstrated that the hindgut rotates right-handedly in embryos homozygous for Myo31DF, which encodes MyoID. In Myo31DFL152 homozygotes, the distribution of angle x° was reversed from that of wild type, although the LR bias became less prominent. The reversed PCC in Myo31DFL152 was rescued by the overexpression of Myo31DFGFP. Rho family guanosine triphosphatases, including Rho1 and Rac1, regulate the organization of the actin cytoskeleton. It was previously shown that overexpression of a dominant-negative Rho1 (Rho1.N19) or Rac1 (Rac1.N17) in the hindgut epithelium disrupts the hindgut's LR asymmetry. No PCC was observed in these epithelial cells, suggesting that PCC formation depends on the actin cytoskeleton. These results support the suggestion that PCC could determine the subsequent laterality of the hindgut (Taniguchi, 2011).

To identify genes involved in PCC formation, a screen was carried out for mutations affecting LR asymmetry of the hindgut. It was found that shotgun (shg) mutations (shgR758, shgR1232, and shgR69, a null allele) disrupted the laterality of the hindgut. shg encodes DE-Cad, a conserved transmembrane protein that mediates cell-cell adhesion in the epithelium. Genetic analyses suggested that DE-Cad functions downstream of MyoID, and both are required in the hindgut epithelium just before its rotation for normal LR asymmetric development. In shgR69 homozygotes, the angle x° did not demonstrate LR asymmetry, indicating that PCC was not formed in this mutant. This PCC defect in shgR69 homozygotes was rescued by the overexpression of shgDECH (Taniguchi, 2011).

To understand how DE-Cad contributes to PCC formation, whether the distribution of DE-Cad showed LR polarity in hindgut epithelial cells was examined. For this, the mean of DE-Cad's relative intensity at the ZA of each cell boundary in the hindgut epithelium was at late-stage 12 was calculated. In wild type, the mean intensity was significantly greater at the cell boundaries with an angle x° of -90° to 0° than in those with 0 to 90° angles, whereas this situation was reversed in Myo31DFL152 homozygotes. Rose diagrams depicting the intensity of DE-Cad in the cell boundaries bundled for 30° intervals showed that DE-Cad was enriched in cell boundaries with an angle x° of -90° to -30°. Conversely, in Myo31DFL152 homozygotes, this situation was reversed. This reversed bias was restored to the wild-type situation by overexpressing Myo31DFGFP in the hindgut epithelium (Taniguchi, 2011).

It was then asked whether the LR bias of DE-Cad distribution was attributable to a cell-autonomous function of MyoID. To address this, a new system was developed for generating a mosaic hindgut epithelium composed of Myo31DFL152 homozygous cells with (+) or without (-) Myo31DFmEGFP overexpression. In the hindgut epithelium, the cell boundaries were classified into three types according to the cell type on either side: +/+, green; +/-, yellow; -/-, magenta. Cell boundaries of +/+ showed the wild-type LR bias of DE-Cad localization, which was reversed in the -/- boundaries. The +/- cell boundaries did not show a statistically significant LR bias. Thus, the LR asymmetry of DE-Cad distribution at each cell boundary is attributable to the concordance of LR polarity in two adjacent cells (Taniguchi, 2011).

To gain insight into how MyoID reverses the LR asymmetric distribution of DE-Cad, defects were sought in endocytic trafficking, because DE-Cad's localization to the ZA is controlled by its recycling. Rab11-positive recycling endosomes became fewer in the apical-middle part of cells in Myo31DFL152 homozygotes compared with wild type, and this defect was restored by Myo31DFGFP expression. These results may suggest that MyoID is involved in the recycling of DE-Cad (Taniguchi, 2011).

Besides the angle x°, the length was also measured of cell boundaries at the ZA of the hindgut epithelial cells. In wild-type animals, the cell boundaries gradually expanded from late-stage 12 to late-stage 13. In homozygous Myo31DFL152 or shgR69 embryos, the length was greater than in wild type at all stages examined. This increase was rescued by the overexpression of Myo31DFGFP or shgDECH in the respective mutant background. Thus, DE-Cad and MyoID appear to restrict the expansion of these cell boundaries, suggesting that these proteins introduce cortical tension, possibly with LR asymmetry (Taniguchi, 2011).

To evaluate the idea that PCC is involved in the hindgut LR asymmetric development, an in silico simulation model was built of the PCC of the hindgut epithelial cells and the directional rotation of the tube composed of these cells. This model consisted of two epithelial sheets composed of model cells, forming the dorsal and ventral arcs of a tube with boundary cells separating the sheets, as found in vivo. In this simulation, the number of cells along the AP and LR sides was set to mimic the in vivo situation, and a statistical LR shape bias was not introduced initially. In vivo, DE-Cad was enriched at cell boundaries with an angle x of -90° to 0° and might restrict the cell-boundary expansion. Therefore, in this simulation, the constriction of cell boundaries was maximized at -45° to the AP axis of the hindgut epithelial tube, and the maximized value was twofold greater than at -135° or +45°. This parameter introduced PCC in the modeled epithelial cells (corresponding to late-stage 12) (Taniguchi, 2011).

Because DE-Cad and MyoID were required before but not during the left-handed rotation of the hindgut epithelium, no LR bias was added to the cell-boundary constriction during the epithelial remodeling. The removal of LR bias subsequently led the modeled epithelial cells to assume stable cell shapes that were mostly regular hexagons (corresponding to late-stage 13). This progressive transition in cell shape was also observed in vivo. This simulation reproduced the 90° left-handed rotation of the epithelial tube in silico, suggesting that PCC is sufficient to explain this rotation in vivo. In addition, LR asymmetric changes in the cell-boundary length observed in the hindgut epithelium in vivo were recapitulated in the simulation, supporting the validity of the model (Taniguchi, 2011).

This study has reported PCC as a previously unknown mechanism of LR asymmetric morphogenesis. Various mutants of genes encoding the core components of planar cell polarity (PCP), a well-understood type of epithelial planar polarity, did not affect the laterality of the Drosophila embryonic gut, suggesting that PCC is not simply a variant of PCP. Although the importance of single-cell chirality has not been studied in multicellular organisms in vivo, intrinsic cell chirality has been found in the LR-polarized protrusion of neutrophil-like cells in vitro. Therefore, cell chirality may be a general property of animal cells. These findings demonstrate a contribution of such chirality to LR asymmetric morphogenesis (Taniguchi, 2011).

DE-Cadherin regulates unconventional Myosin ID and Myosin IC in Drosophila left-right asymmetry establishment

In bilateria, positioning and looping of visceral organs requires proper left-right (L/R) asymmetry establishment. Recent work in Drosophila has identified a novel situs inversus gene encoding the unconventional type ID myosin (MyoID). In myoID mutant flies, the L/R axis is inverted, causing reversed looping of organs, such as the gut, spermiduct and genitalia. MyoID has been shown to interact physically with β-Catenin, suggesting a role of the adherens junction in Drosophila L/R asymmetry. This study shows that DE-Cadherin co-immunoprecipitates with MyoID and is required for MyoID L/R activity. It was further demonstrated that MyoIC (Myo61F), a closely related unconventional type I myosin, can antagonize MyoID L/R activity by preventing its binding to adherens junction components, both in vitro and in vivo. Interestingly, DE-Cadherin inhibits MyoIC, providing a protective mechanism to MyoID function. Conditional genetic experiments indicate that DE-Cadherin, MyoIC and MyoID show temporal synchronicity for their function in L/R asymmetry. These data suggest that following MyoID recruitment by β-Catenin at the adherens junction, DE-Cadherin has a twofold effect on Drosophila L/R asymmetry by promoting MyoID activity and repressing that of MyoIC. Interestingly, the product of the vertebrate situs inversus gene inversin also physically interacts with β-Catenin, suggesting that the adherens junction might serve as a conserved platform for determinants to establish L/R asymmetry both in vertebrates and invertebrates (Petzoldt, 2012).

This study showns DE-Cadherin has a dual role in promoting MyoID activity. DE-Cadherin directly stabilizes MyoID at the adherens junctions and inhibits the antagonistic activity of MyoIC, thus eliciting MyoID function. MyoID activity and organization are dependent on the level of MyoIC protein. In MyoIC gain of function, MyoID intracellular pattern is modified with a reduced overall signal and the protein level remains unchanged. In MyoIC loss of function, MyoID is not detected by immunohistochemistry and the protein level measured by western blot is reduced. Nevertheless, in MyoIC loss of function, sufficient MyoID activity remains as confirmed by the wild-type rotation phenotype. Thus, MyoID activity is only impaired when MyoIC is in large excess (Petzoldt, 2012).

It is important to note that these results were obtained through two independent approaches, either by direct modulation of MyoIC expression levels or indirectly by affecting DE-cadherin, silencing of which in the A8 segment leads to an increase of MyoIC (and of MyoID) levels. These results indicate that DE-Cadherin negatively regulates both MyoID and MyoIC levels. Taken together, these data indicate that DE-Cadherin controls both the L/R determinant MyoID and its repressor MyoIC activity and protein levels (Petzoldt, 2012).

Specific depletion of DE-Cadherin in the A8 segment, the L/R organizer, leads to a no-rotation phenotype. Could stronger depletion of the adherens junction components lead to a sinistral phenotype? In other words, does DE-Cadherin depletion also affect sinistral development or pathway, which is taking over in absence of MyoID? To tackle this question, both DE-Cadherin and MyoID were depleted. It was found that, unlike the sole depletion of MyoID that leads to a majority of sinistral flies, the double depletion leads to a majority of non-rotated flies. These data therefore indicate that DE-Cadherin acts both on the dextral (MyoID-dependent) and on the sinistral pathways, making it a general regulator of L/R asymmetry in flies (Petzoldt, 2012).

Adherens junctions have previously been connected to MyoID-mediated L/R asymmetry establishment through biochemical analysis, which identified a physical interaction between the MyoID-tail domain and β-Catenin in vitro. This study demonstrates a role of the adherens junction in the Drosophila L/R pathway and places the adherens junction component DE-Cadherin as a regulator of both unconventional myosins MyoIC and MyoID. DE-Cadherin dually promotes MyoID and β-Catenin association at the adherens junctions and inhibits the antagonistic function of MyoIC. Indeed, a partial colocalization of MyoID and DE-Cadherin was observed and the concomitant exclusion of MyoIC from the adherens junction. Further supporting this model is the additional biochemical evidence that MyoID, but not MyoIC, can directly interact with DE-Cadherin and β-Catenin in vitro. Excess of MyoIC is able to disrupt this interaction both in vivo and in vitro, leading to MyoID loss-of-function phenotypes. This disruption can be rescued by MyoID overexpression. It is proposed that MyoID L/R function depends on its physical interaction with adherens junction through both β-Catenin and DE-Cadherin. Upon adherens junction reduction, MyoID does not associate with β-Catenin, MyoIC is no longer repressed and in turn inhibits MyoID activity, leading to L/R defects. Finally, the temporal synchrony of MyoID, MyoIC and DE-Cadherin requirement in genitalia rotation implies a concomitant activity of these proteins in L/R establishment (Petzoldt, 2012).

Interestingly, MyoIC has been reported to affect L/R looping of the embryonic gut, another marker of L/R asymmetry in Drosophila. It was shown that MyoIC overexpression also causes fully penetrant embryonic gut inversion. Thus, the antagonistic function of MyoIC appears to be conserved in Drosophila L/R tissues. Furthermore, it was recently shown that DE-Cadherin is required for L/R asymmetry establishment of the Drosophila hindgut (Taniguchi, 2011). The dextral curving of the hindgut was lost in a myoID and DE-cadherin mutant background accompanied by a loss of L/R biased asymmetric cell shape and a differential DE-Cadherin localization. It was suggested that both factors are implicated in the creation of asymmetric cortical tension prior to asymmetric curving of the hindgut. Taken together, these data indicate that the Drosophila L/R tissues, hindgut and genitalia, show a similar dependency on adherens junction and type I unconventional myosins for L/R determination (Petzoldt, 2012).

Is the role of adherens junctions in L/R asymmetry establishment conserved among species? Interestingly, despite the apparent differences between vertebrates and invertebrates mechanisms of L/R asymmetry establishment, a common theme can be found in the only two molecularly described situs inversus genes to date. In addition to myoID, the mouse inversin gene also causes constant L/R axis inversion in homozygous mutants. Interestingly, the inversin protein was shown to co-precipitate with β-catenin and N-cadherin and to localize to the adherens junction of polarized epithelial cells (Nurnberger, 2002). Therefore, both situs inversus proteins interact with β-catenin and associate with adherens junction molecules, such as N- or E-cadherin. Additionally, N-cadherin was reported to play a role in L/R asymmetry establishment in chicken, as N-cadherin absence at the Hensen's node leads the randomization of L/R asymmetry, similar to the current results showing a role of DE-Cadherin in L/R determination in Drosophila. The Drosophila inversin homologue diego belongs to the planar cell polarity gene family. Depletion of diego, or of any other of PCP gene, in the L/R organizer does not affect the directionality of genitalia rotation, suggesting that Diego does not play a critical role in Drosophila L/R asymmetry. In conclusion, the situs inversus proteins MyoID and Inversin, which play a central and upstream role in the establishment of L/R asymmetry, both require an interaction with the adherens junctions for their function (Petzoldt, 2012).

It is important to note that the L/R axis is established after and, most importantly, relatively to the other two main axes, the anterior-posterior and dorsal-ventral axes. Indeed, L/R determinants have to be oriented along existing spatial coordinates so that the L/R axis is positioned perpendicular to existing axes. It is proposed that the association of MyoID (and more generally of situs inversus proteins) with the adherens junctions is an essential mechanism to orient MyoID activity along the apical-basal axis, which in epithelia is perpendicular to the anterior-posterior axis and is thus equivalent to a dorsal-ventral axis. Therefore, association of MyoID with the adherens junctions would represent a way to orient and polarize its activity. Following polarization of MyoID in epithelia, the intrinsic chirality of myosins, through their directional activity towards one end of the actin filaments, could create de novo an asymmetric axis similar to the F-molecule model proposed by Brown and Wolpert (Brown, 1990; Petzoldt, 2012 and references therein).

In conclusion, the current findings reveal an important link between the adherens junction and L/R asymmetry determinant in Drosophila and suggest an evolutionary conservation in vertebrates and invertebrates of the interactions between the adherens junction and the situs inversus proteins linking cell architecture and polarity to the patterning of the L/R axis. Future work on the molecular targets of MyoID and Inversin will help understand how the L/R axis becomes oriented to lead to stereotyped asymmetric organogenesis (Petzoldt, 2012).

The atypical cadherin Dachsous controls left-right asymmetry in Drosophila

Left-right (LR) asymmetry is essential for organ development and function in metazoans, but how initial LR cue is relayed to tissues still remains unclear. This study proposes a mechanism by which the Drosophila LR determinant Myosin ID (MyoID) transfers LR information to neighboring cells through the planar cell polarity (PCP) atypical cadherin Dachsous (Ds). Molecular interaction between MyoID and Ds in a specific LR organizer controls dextral cell polarity of adjoining hindgut progenitors and is required for organ looping in adults. Loss of Ds blocks hindgut tissue polarization and looping, indicating that Ds is a crucial factor for both LR cue transmission and asymmetric morphogenesis. It was further shown that the Ds/Fat and Frizzled PCP pathways are required for the spreading of LR asymmetry throughout the hindgut progenitor tissue. These results identify a direct functional coupling between the LR determinant MyoID and PCP, essential for non-autonomous propagation of early LR asymmetry (Gonzalez-Morales, 2015).

This work has revealed the existence of an hindgut-specific LR organizer having transient activity. LR information is transferred non-autonomously from this organizing center to the target tissue, involving a unique MyoID-Ds interaction taking place at a PCP signaling boundary (the H1/H2 boundary). Propagation of this initial LR information to the developing hindgut requires both Ds/Ft global and core Fz PCP signaling. Notably, these results suggest that MyoID can act as a directional cue to bias planar cell polarity (Gonzalez-Morales, 2015).

So far, only a role for the core PCP pathway in cilia positioning and LR asymmetry had been reported in mouse, chick, and Xenopus. This study revealed a role of the Fat/Ds PCP pathway in LR asymmetry. The atypical cadherin Ds is essential for early LR planar polarization of hindgut precursors and later on for looping morphogenesis. Ds has a cell-non-autonomous function, allowing transfer of LR information from the H1 domain to H2 hindgut precursor cells. Ds, therefore, represents a critical relay factor acting at the boundary between, and linking, a LR organizer and its target tissue (Gonzalez-Morales, 2015).

In addition to a MyoID-dependent function in H1, the mislooped phenotype induced upon Ds silencing in the H2 domain suggests that Ds also has a MyoID-independent activity in H2 cells, likely through interaction with other PCP genes. Indeed, reducing the activity of PCP global or core gene functions reveals that the two pathways are important in the H2 region for adult hindgut looping. However, the results reveal important differences in the way these pathways control hindgut asymmetry. First, although the adult phenotype is similar upon silencing of one or the other pathway, the early polarization of H2 cells in pupae (10 hr APF) is only affected when knocking down the activity of Ds, Ft, and Fj. These results show that the Ds/Ft pathway, but not the core pathway, is required for establishing early LR polarity. Second, the phenotype is quantitatively different, since silencing of the Ds, Ft, or Fj PCP gene led to a consistent and very strong phenotype, while reducing Fz PCP signaling had a significantly less penetrant one. These data suggest a partly overlapping function of both PCP signaling pathways for late hindgut morphogenesis. Therefore, the following sequential model is proposed: in H1 cells, MyoID interacts with the Ds intracellular domain, which becomes 'biased' toward dextral through a currently unknown mechanism. This initial LR bias is then transmitted across the H1/H2 boundary through Ds/Ft heterophilic interaction. Then, boundary H2 cells relay the initial bias and spread it to the remaining H2 cells through classical Ds/Ft PCP. It is interesting that the local signaling boundary suggested by this model is consistent with recent studies showing that Ds can propagate polarity information in a range of up to eight cells, a distance that is consistent with the size of the H2 domain at 10 hr APF. Once initial polarity has been set up through the Ds/Ft pathway, this is further relayed to and/or amplified by the core pathway. Notably, a similar two-step mechanism has also been proposed for the wing and could apply to other tissues (Gonzalez-Morales, 2015).

The discovery of a coupling between the MyoID dextral factor and Ds is a nice example of crosstalk between existing signaling modules. In the simplest crosstalk model, the role of MyoID would just be to bias or tilt Ds function toward one side, possibly through Ds localization and/or activity polarization along the LR axis. Using both in vitro and in vivo assays, this study has shown that interaction between Ds and MyoID requires Ds intracellular domain, supporting a cytoplasmic interaction between the two proteins. These results, along with recent findings, suggest that Ds may represent a general platform for myosin function in different tissues. In particular, the intracellular domain of Ds was found to bind to the unconventional myosin Dachs, controlling Dachs polarized localization, which is important for subsequent cell rearrangements underlying thorax morphogenesis. However, in contrast to thoracic Dachs, MyoID is not obviously polarized in H1 cells, suggesting that the interaction between myosins and Ds may involve different mechanisms. Additionally, no LR polarized localization of MyoID or Ds was observed in H1 cells, although the existence of subtle asymmetries undetectable by available tools cannot be excluded. Nevertheless, alternative means to generate the LR bias in H1 include: (1) LR polarized expression of an unknown asymmetric factor or (2) LR asymmetric activity of Ds. These interesting possibilities are consistent with recent work showing that some type I myosins can generate directed spiral movement of actin filaments in vitro. It is tempting to speculate that, similarly, MyoID putative chiral activity could be translated into Ds asymmetrical function along the LR axis. Future work will explore this possibility as well as others to unravel the molecular basis of MyoID LR biasing activity in the H1 organizer (Gonzalez-Morales, 2015).

The identification of the H1 domain as a specific adult tissue LR organizer demonstrates the existence of multiple independent tissue and stage-specific LR organizers in flies. This situation echoes what is known in other models, including vertebrates, in which at least two phases of asymmetry establishment can be distinguished. A first pre-gastrula phase, as early as the four-cell stage in Xenopus, involves the generation of asymmetric gradients of ions. Then, a second phase takes place at gastrulation and involves Nodal flow and asymmetric cell migration, eventually leading to asymmetric expression of the nodal gene in the left lateral plate mesoderm. In Drosophila, some interesting common and specific features can be drawn out by comparing the hindgut and terminalia organizers. The first major common feature is the fact that both organizers rely on MyoID function, showing the conserved role of this factor in Drosophila LR asymmetry. Second, the two organizers show temporal disconnection, acting much earlier than LR morphogenesis, which is expected of a structure providing directionality to tissues per se (24 hr for terminalia and ~72 hr for hindgut looping). Such temporal disconnection of MyoID function with late morphogenesis is also observed in the terminalia where a peak of MyoID activity precedes terminalia rotation by 24 hr. Time lag in MyoID function requires LR cue transmission and maintenance in developing tissues until directional morphogenesis. The finding of a role of Ds and PCP in hindgut LR asymmetry provides a simple mechanism by which initial LR information is maintained and transmitted across tissue through long-range PCP self-propagation (Gonzalez-Morales, 2015).

Notably, the two organizers also show distinct features. In terminalia, MyoID has a cell-autonomous function in two adjacent domains. In addition, the terminalia organizer is permanent, developing as an integral component of the adult tissue. In contrast, MyoID in the imaginal ring has a cell-non-autonomous function. Indeed, a striking feature of the hindgut organizer is its transience as it detaches from the hindgut precursors 50 r before full looping morphogenesis prior to its degradation and elimination; hence, the need to transfer LR information to the H2 hindgut primordium. An interesting question then is whether the MyoID-Ds/PCP interaction is conserved in terminalia. This study has shown that the terminali rotation requires the activity of DE-cadherin; however, invalidation of the atypical cadherins Ds or Ft or core PCP signaling in the terminalia organizer did not affect asymmetry. The fact that PCP does not have a general role in Drosophila LR asymmetry is not altogether surprising, as MyoID cell-autonomous function in terminalia and organizer persistence does not require that LR information be transferred to and stored in other parts of the tissue, as is the case in the hindgut. Therefore, despite conservation of the MyoID-dependent upstream dextral cue, significant differences in downstream morphogenetic pathways imply alternative cellular mechanisms controlling cue transmission and maintenance (Gonzalez-Morales, 2015).

The LR signaling module, comprising the dextral determinant MyoID and the still-unknown sinistral determinant, can therefore be coupled to distinct morphogenetic modules, including PCP, as shown in this study. It is suggested that coupling between LR asymmetry and PCP might be observed in processes requiring long-distance patterning of tissues and organ precursors, both in invertebrate and vertebrate models. Understanding organ LR morphogenesis clearly requires studying diverse and complementary models. In this context, the multiplicity of LR organizers discovered in Drosophila represents a powerful model to study the diversity in the coupling of LR organizers with downstream programs responsible for late tissue morphogenesis. In particular, the Drosophila hindgut represents an invaluable model for studying the genetic basis and molecular mechanisms coupling LR asymmetry with PCP patterning (Gonzalez-Morales, 2015).

Plasma membrane localization of apoptotic caspases for non-apoptotic functions

Caspases are best characterized for their function in apoptosis. However, they also have non-apoptotic functions such as apoptosis-induced proliferation (AiP), where caspases release mitogens for compensatory proliferation independently of their apoptotic role. This study reports that the unconventional myosin, Myo1D, which is known for its involvement in left/right development, is an important mediator of AiP in Drosophila. Mechanistically, Myo1D translocates the initiator caspase Dronc to the basal side of the plasma membrane of epithelial cells where Dronc promotes the activation of the NADPH-oxidase Duox for reactive oxygen species generation and AiP in a non-apoptotic manner. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment for caspases. Finally, Myo1D promotes tumor growth and invasiveness of the neoplastic scrib Ras(V12) model. Together, these studies have identified a new function of Myo1D for AiP and tumorigenesis and reveal a mechanism by which cells sequester apoptotic caspases in a non-apoptotic compartment at the plasma membrane (Amcheslavsky, 2018).

Under stress conditions, when a large number of cells are dying, there is a need for compensatory proliferation to replace the lost cells with new cells. Work using several model organisms has shown that, under these conditions, apoptotic cells can release mitogenic signals that induce proliferation of surviving cells for the replacement of dying cells. Because apoptotic cells are actively triggering this type of compensatory proliferation, this process has been termed apoptosis-induced proliferation (AiP) (Amcheslavsky, 2018).

Caspases are Cys proteases that are the main effectors of apoptosis. They are produced as inactive zymogens with a prodomain and after processing a large and small subunit. There are initiator and effector caspases. Initiator caspases carry protein/protein interacting motifs in their prodomains, which mediate their incorporation into large multimeric protein complexes. For example, the mammalian initiator caspase-9 is recruited into the Apaf-1 apoptosome, while its Drosophila ortholog Dronc forms the apoptosome with the Apaf-1 homolog Dark. Effector caspases such as mammalian caspase-3, or Drosophila DrICE and Dcp-1, are proteolytically processed by activated initiator caspases and mediate the apoptotic process (Amcheslavsky, 2018).

In addition to apoptosis, caspases are also mediating AiP. They trigger the release of Wnt, bone morphogenetic protein (BMP)/transforming growth factor β (TGF-β), epidermal growth factor (EGF), and Hedgehog mitogens for AiP. This has been best studied for the Drosophila initiator caspase Dronc using the 'undead' AiP model in which apoptotic signaling is induced by expression of upstream cell death factors such as hid, but the execution of apoptosis is blocked by co-expression of the effector caspase inhibitor p35, thus rendering cells in an undead condition. Because P35 inhibits apoptosis, but not Dronc, Dronc can still mediate non-apoptotic functions such as AiP. When hid and p35 are co-expressed using the ey-Gal4 driver (ey > hid,p35), which is expressed in epithelial cells of eye imaginal discs, Dronc continuously signals for AiP and triggers hyper-proliferation. Consequently, the discs are enlarged and the resulting heads of the adult flies are overgrown . In genetic screens, screening was carried out for suppressors of the overgrowth phenotype of undead (ey > hid,p35) adult heads to identify genes and mechanisms involved in AiP (Amcheslavsky, 2018).

Mechanistically, this study showed that, in undead cells, Dronc stimulates the NADPH-oxidase Duox for the production of extracellular reactive oxygen species (eROS). eROS recruits and activates hemocytes, Drosophila immune cells similar to macrophages, to the undead imaginal disc. In turn, hemocytes release the tumor necrosis factor-like ligand Eiger, which induces JNK activity in epithelial disc cells. JNK promotes the expression of the apoptotic genes reaper and hid, which initiate a positive feedback loop to maintain undead signaling (Fogarty, 2016). In addition, it induces the release of the mitogens Wingless (Wg), a Wnt-like gene in Drosophila, decapentaplegic, a BMP/TGF-β homolog, and Spitz, an EGF ligand, which all promote AiP (Amcheslavsky, 2018).

In addition to undead AiP, there is also 'genuine' AiP, during which dying cells complete the apoptotic process, and the response of the affected tissue to replace the dying cells is examined. In contrast to undead AiP, genuine AiP does not promote overgrowth. Therefore, although most genes identified in undead AiP also have important roles in genuine AiP, there must be differences between the two AiP models. In any case, genuine AiP is used as a model of tissue regeneration, while the hyper-proliferation of undead AiP serves as a tumorigenic model (Amcheslavsky, 2018).

Class I unconventional myosins are conserved actin-based motor proteins, composed of the N-terminal head (motor) region with an ATP binding motif (including P-, switch1-, and switch2 loops) and an actin-binding domain, a neck region characterized by two to three IQ motifs, and a C-terminal tail domain that interacts with phospholipids at membranes. Mammals have eight class I myosins, Drosophila has three, Myosin 1D (Myo1D, also known as Myo31DF), MyoIC (Myo61F), and Myo95E. While Myo1D and Myo1C are involved in left/right (L/R) development of visceral organs, the function of Myo95E is unknown (Amcheslavsky, 2018).

Although Drosophila is a bilateral organism, certain visceral organs such as the gut and the coiling of the spermiducts around the gut, which occurs in a morphogenetic movement termed male terminalia rotation, display L/R asymmetry. In Myo1D mutants, the chirality of these asymmetric organs and movements are reversed. For example, the male terminalia rotation during pupal development, which, in wild-type, occurs for 360° in clockwise (dextral) orientation, proceeds in Myo1D mutants sinistrally, defining Myo1D as dextral determinant. Myo1D engages the actin cytoskeleton and adherens junctions for this movement (Amcheslavsky, 2018).

Overexpression of Myo1C antagonizes the dextral activity of Myo1D by displacing it from adherens junctions. However, the loss-of-function phenotype of Myo1C did not confirm this antagonizing function. Instead, while Myo1C single mutants do not display any L/R defect, the Myo1C Myo1D double mutant has a stronger sinistral male terminalia phenotype than Myo1D mutants indicating that Myo1C has a partially redundant dextral activity with Myo1D (Amcheslavsky, 2018).

It has long been known that genes in the apoptosis pathway, such as hid, dronc, and drICE, are also involved in male terminalia rotation in Drosophila. Indeed, localized apoptotic activity is required for this L/R process. How Myo1D and the apoptosis pathway interact for male terminalia rotation is not very well understood. Interestingly, mutants of the JNK signaling pathway or overexpression of puckered, an inhibitor of JNK activity, also display defects in male terminalia rotation (Amcheslavsky, 2018).

This study reports that Myo1D is an essential component of AiP in the undead model. Genetic inactivation of Myo1D strongly suppresses ey > hid,p35-induced overgrowth of the head capsule, while overexpression of Myo1D enhances it. Myo1D promotes the generation of ROS by Duox for AiP signaling. Further mechanistic analysis reveals that Myo1D is required for membrane localization of Dronc, specifically to the basal side of the plasma membrane of undead epithelial disc and salivary gland cells. Here, Dronc exerts a non-apoptotic function resulting in Duox activation. It is proposed that the basal side of the plasma membrane constitutes a non-apoptotic compartment that allows non-apoptotic processes of Dronc and potentially other caspases to occur. Therefore, in addition to the dextral activity of Myo1D, this study identified a second function of Myo1D for the control of apoptosis-induced proliferation (Amcheslavsky, 2018).

Mechanistically, it was found that Myo1D is involved in the localization of the initiator caspase Dronc to the basal side of the plasma membrane of undead DP disc and SG cells. Myo1D interacts with Dronc, suggesting that it may directly translocate Dronc to the plasma membrane. However, Myo1D does not appear to be a cleavage target of the caspase Dronc (Amcheslavsky, 2018).

The observed localization of Dronc to the basal side of the plasma membrane in undead DP cells is critical for the mechanism of AiP. Undead cells attract hemocytes to the discs in a Dronc- and Duox-dependent manner. However, that occurs at the basal side of DP cells of imaginal discs because the basal side is exposed to the hemolymph that contains circulating hemocytes, while the apical side faces the lumen between the DP and the PM. Consistently, there is also an enrichment of Duox at the basal side of the plasma membrane. Therefore, in order to be able to activate Duox for ROS generation and hemocyte activation, Dronc needs to be specifically present at the basal side of the plasma membrane (Amcheslavsky, 2018).

It has long been known that caspases, including Dronc, have non-apoptotic functions in addition to their well characterized role in apoptosis. This paper reveals one mechanism by which cells may activate a caspase (Dronc) without the detrimental consequences of apoptosis. The sequestration of Dronc to the basal side of the plasma membrane in a Myo1D-dependent manner and the low abundance of Dronc's apoptotic partner Dark at the plasma membrane may ensure localized and controlled apoptosome activity which is sufficient for AiP, but not for killing cells. Alternatively, apoptotic substrates needed for the execution of apoptosis may not be present at the plasma membrane or in insufficient amount to pass the apoptotic threshold (Amcheslavsky, 2018).

While this study addressed the role of membrane localization of Dronc under undead conditions, recently membrane-localized Dronc was shown in SGs under normal conditions, which explains the membrane localization of Dronc at control SGs. Here, membrane-localized Dronc is required for F-actin cytoskeleton dismantling at the end of larval development in a non-apoptotic manner. In addition to the plasma membrane, the outer mitochondrial membrane has been shown to provide a non-apoptotic platform for caspase activation, in this case during sperm maturation. Therefore, membranes in general may provide a local environment for non-apoptotic caspase activities (Amcheslavsky, 2018).

The membrane localization of Dronc in SGs is mediated by Tango7, which has previously been implicated in spermatid maturation. As mentioned above, membrane-localized Dronc is required for dismantling of the cortical F-actin cytoskeleton in SGs of late larvae. However, while Tango7 RNAi blocks actin dismantling, Myo1D RNAi does not , suggesting that the roles of Tango7 and Myo1D for membrane localization of Dronc are different from each other. That also explains why in undead SGs the membrane localization of Dronc strongly increases in a Myo1D-dependent manner. Unfortunately, it was not possible to test if Tango7 is involved in AiP. Tango7 RNAi in eye imaginal discs results in complete loss of the disc. Tango7 encodes the homolog of eukaryotic translation initiation factor 3m (eIF3m), suggesting that it may also have an important requirement for protein translation, explaining the loss of the eye disc by Tango7 RNAi (Amcheslavsky, 2018).

In addition to Myo1D and Tango7, there is at least one other factor, Crinkled (Ck), which directs Dronc to non-apoptotic functions. Ck bridges the interaction between Dronc and the kinase Shaggy/glycogen synthase kinase beta (GSK-β), resulting in the selective activation of Shaggy/GSK-β, which then promotes non-apoptotic activities such as the specification of scutellar bristles, border cell migration, and correct branching of the aristae. Interestingly, Ck encodes another unconventional myosin, a member of the class VII myosin family, potentially suggesting that other myosins may also direct non-apoptotic functions to caspases (Amcheslavsky, 2018).

Myo1D and the apoptotic machinery have been linked to male terminalia rotation, an L/R process during pupal development. Indeed, apoptosis is required for Myo1D-dependent male terminalia rotation. It is unknown how Myo1D interacts with the apoptotic machinery to direct this L/R movement. In future studies, it will be interesting to examine if the Myo1D-dependent mechanism identified here for AiP also applies to male terminalia rotation or whether a separate mechanism exists in this context (Amcheslavsky, 2018).

Myo1D not only localizes Dronc to the plasma membrane, it also stabilizes it. Dronc is activated in undead cells, and activated Dronc is subject of increased protein degradation. Thus, Myo1D prevents degradation of Dronc by changing its subcellular localization to the plasma membrane (Amcheslavsky, 2018).

Myo1D has a very strong requirement for AiP in the undead model, and a requirement in the scrib-/-RasV12 tumorigenesis model, yet it does not appear to play any significant role in genuine AiP. In fact, Myo1D is the first gene identified that is essential for the hyper-proliferation of undead AiP, but not required for the regeneration of genuine AiP. The mechanism revealed in this paper provides an explanation for this behavior. During genuine AiP, cells are allowed to undergo apoptosis, which requires cytosolic Dronc activity. Although ROS are generated during genuine AiP, the origin of these ROS has not been determined and may not require the plasma membrane-localized Duox. Therefore, a key difference between genuine AiP and undead AiP, and potentially between other regenerative versus tumorigenic models, may be the altered localization of Dronc to a non-apoptotic compartment at the plasma membrane, and a shift from balanced apoptosis and proliferation to dominant proliferation. The next big question will be to examine what exactly is prompting Myo1D to drive this re-localization of Dronc under sustained undead conditions, but not under the limited regenerative conditions of the genuine AiP models, and whether that answer provides any insight into the cancer versus wound healing models (Amcheslavsky, 2018).

In conclusion, in addition to its role in L/R development, this study identified a second function of Myo1D for AiP and tumorigenesis. The basal side of the plasma membrane was identified as a non-apoptotic environment for caspase function. In future work, it will be important to identify the mechanisms by which Dronc mediates its non-apoptotic functions at the plasma membrane for AiP and other cellular processes that require membrane localization of Dronc and other caspases (Amcheslavsky, 2018).

Proper direction of male genitalia is prerequisite for copulation in Drosophila, implying cooperative evolution between genitalia rotation and mating behavior

Animal morphology and behavior often appear to evolve cooperatively. However, it is difficult to assess how strictly these two traits depend on each other. The genitalia morphologies and courtship behaviors in insects, which vary widely, may be a good model for addressing this issue. In Diptera, phylogenetic analyses of mating positions suggested that the male-above position evolved from an end-to-end position. However, with this change in mating position, the dorsoventral direction of the male genitalia became upside down with respect to that of the female genitalia. It was proposed that to compensate for this incompatibility, the male genitalia rotated an additional 180 degrees during evolution, implying evolutionary cooperativity between the mating position and genitalia direction. According to this scenario, the proper direction of male genitalia is critical for successful mating. This study tested this hypothesis using a Drosophila Myosin31DF (Myo31DF) mutant, in which the rotation of the male genitalia terminates prematurely, resulting in various deviations in genitalia direction. The proper dorsoventral direction of the male genitalia was found to be a prerequisite for successful copulation, but it did not affect the other courtship behaviors. Therefore, these results suggested that the male genitalia rotation and mating position evolved cooperatively in Drosophila (Inatomi, 2019).

Cooperative evolution between organ morphology and behavior is a well-established concept. Phylogenetic analyses in various animal groups have demonstrated remarkable coincidences between organ morphology and behavior. This study sought to obtain evidence supporting the idea that mating behaviors cooperatively evolved with the rotation of male genitalia in dipteran insects (Inatomi, 2019).

In these insects, the evolution from the end-to-end to the male-above position should result in an incompatibility in the dorsoventral direction of the male genitalia with respect to that of the female genitalia. It was previously proposed that this incompatibility was overcome by the additional 180° rotation of the male genitalia (in total, a 360° rotation called 'circumversion'). This study has shown that males with genitalia resulting from complete circumversion had a great advantage for reproduction over those with deviation in the rotation angle of the genitalia. The results indicated that the proper dorsoventral direction of the male genitalia is a prerequisite for successful copulation in the male-above mating position in Drosophila. Therefore, the results are consistent with the previously proposed idea that the mating position of dipterans evolved cooperatively with changes in the rotation angle of the male genitalia (Inatomi, 2019).

This analysis found that males with genitalia classified as the Left 45° and Right 45° angle deviation still copulated and had offspring. Therefore, males may be able to overcome a small aberration in the dorsoventral direction of their genitalia, although the possiblility cannot be excluded that females also contribute to this adjustment. In D. melanogaster, the males have genitalia sensilla, including bristles on the genital claspers, which detect the position of the mating partner and contribute to the male's proprioception. Acebes (2003) showed that ablating the genital sensilla caused a reduction in mating frequency and decreased the male's ability to detect his position on the female's back. Therefore, in the current experiments, it is possible that the sensitivity of the male genitalia contributed to the adjustment to a small aberration in the dorsoventral direction of the male genitalia. However, Acebes (2003) also showed that ablating the genital sensilla does not affect the male's attempt to mount the female back, demonstrating that the male's attempt to copulate on the female's back is an unalterable component of male-above mating. This idea is consistent with the observation that the males with genitalia classified as Right 45° and 180° (upside down genitalia) angle deviation still used the male-above mating position, and no other mating positions, in all of the video data examined. Upon an alteration in body structure, mating behaviors are also found to be inflexible in other insects. For example, the South American four spot-roach, Eublaberus distanti has a highly ritualized mating behavior, which requires male wing rise. Males in which the wings have been partially or completely surgically removed still perform the full sequence of mating behaviors and complete their courtship ritual. Therefore, in cases in which a particular body structure and mating behavior are cooperatively required to achieve successful mating, the mating behavior is often inflexible to adjust to a change in body structure, as found in this study for the dorsoventral direction of the male genitalia (Inatomi, 2019).

This study obtained Myo31DF mutant male flies with genitalia exhibiting various deviations in the rotation angle. In the classification of the angle deviation, the Right 45° and Left 45° males had a deviation of the same angle, which was left-right inversed. However, there is a 270° difference in the amount of rotation needed to achieve these deviations; the Right 45° and Left 45° angle deviations were achieved by 315° and 45° counterclockwise rotations, respectively. However, no noticeable difference was found between the success rates of reproduction between these two categories. These results suggested that the amount of genital rotation itself does not impact reproduction; rather, the final dorsoventral angle of the genitalia resulting from the rotation is critical for successful copulation. In this context, it has been proposed based on phylogenetic analyses that morphological asymmetry itself is not advantageous for reproduction in spiders and insects, but rather the newly adopted mating position is. That idea is consistent with the current experimental observation that the degree of genitalia rotation did not affect reproduction if the final dorsoventral direction of the male genitalia was the same (Inatomi, 2019).

In the current experiments, males with genitalia demonstrating all classes of angle deviation were obtained from Myo31DF. The males with genitalia classified as 0°, Right 45°, and 180° angle deviation and control males, whose genetic background was unified with Myo31DF through five backcrosses, did not show significant differences in courtship latency or in the percentage of males initiating the courtship. However, although the CI was not significantly different among males with genitalia classified as 0°, Right 45°, and 180° angle deviation, the CI of the w1118/Y control males was lower than those of the males with angle deviations in their genitalia. Therefore, the Myo31DFK2 mutation may affect the CI. In support of this possibility, Myo31DF was recently shown to be involved in the formation of neuromuscular junctions and in TNF signaling. Alternatively, the genetic background of the control males may not have become completely unified with that of Myo31DF. However, all of the current conclusions were based on comparisons between males with genitalia showing the classified angle deviations as well as the normal direction, all of which were on the same Myo31DF genetic background. Therefore, the observed difference in the CI of the control males does not affect the conclusion that the normal dorsoventral direction of the male genitalia was highly preferable for successful copulation (Inatomi, 2019).

Although the current hypothesis is based on the single example of D. melanogaster, it supports the idea that the male-above position evolved cooperatively with the additional 180° rotation of the male genitalia from the ancestral end-to-end position. However, it was also found that Drosophila males have a very limited ability to adjust to a deviation in the direction of their own genitalia. In contrast, the ancestral species that used the end-to-end position might have had high flexibility in the mating position, which gave it the ability to tolerate the future additional 180° rotation of the male genitalia. That is, such flexibility might have subsequently allowed cooperative evolution between the mating position and the rotation of male genitalia. Alternatively, the evolution from the end-to-end to male-above position might have gone through an intermediate step, such as the false-male-above or male-above with flexed male abdomen position. In this scenario, the mating position evolved before the additional 180° rotation of the male genitalia. To evaluate these ideas, further phylogenetic analyses of the relationship between mating behavior and male genitalia rotation are required (Inatomi, 2019).

The Drosophila actin nucleator DAAM is essential for left-right asymmetry

Left-Right (LR) asymmetry is essential for organ positioning, shape and function. Myosin 1D (Myo1D) has emerged as an evolutionary conserved chirality determinant in both Drosophila and vertebrates. However, the molecular interplay between Myo1D and the actin cytoskeleton underlying symmetry breaking remains poorly understood. To address this question, a dual genetic screen was performed to identify new cytoskeletal factors involved in LR asymmetry. The conserved actin nucleator DAAM was identified as an essential factor required for both dextral and sinistral development. In the absence of DAAM, organs lose their LR asymmetry, while its overexpression enhances Myo1D-induced de novo LR asymmetry. These results show that DAAM is a limiting, LR-specific actin nucleator connecting up Myo1D with a dedicated F-actin network important for symmetry breaking (Chougule, 2020).

Left-Right (LR) asymmetry, or chirality, is a universal feature of living organisms. It is essential to organs for their positioning (e.g., heart on the left side), lateralized differentiation (e.g., heart, lungs) and proper directional coiling (e.g., gut, heart tube). The study of LR asymmetry in model organisms has led to the identification of key molecular pathways and symmetry breaking mechanisms. While vertebrates use directional movement of cells (chick), ions (Xenopus) or cilia-dependent nodal flow (mouse) as symmetry breaking processes, invertebrates (snail, nematode, Drosophila) establish LR asymmetry mostly through acto-myosin-based mechanisms. In particular, work in Drosophila identified the conserved myosin1D (myo1D) gene as a major dextral determinant. myo1D establishes LR asymmetry through interaction with the adherens junction, Hox genes, planar cell polarity and cell death pathways. In flies, several organs are chiral and undergo stereotyped looping in the dextral direction (testis, genitalia, gut). Dextral is the wild type orientation and thus corresponds to the situs solitus condition in Drosophila. Loss of myo1D function leads to a sinistral or situs inversus phenotype, making organs undergo looping in the opposite direction. The existence of two opposite phenotypes and previous genetic data suggest that two pathways exists, one dextral and one sinistral, with dextral being 'dominant' over sinistral. To date, the genetic basis of sinistral asymmetry remains uncharacterized in any system, due to the lack of dedicated genetic screens to identify genes with a specific role in sinistral development (Chougule, 2020).

Recent work showed that myo1D is able to induce de novo chirality at all biological scales, from molecular to organismal level. Indeed, ectopic expression of myo1D in naive tissues like the larval epidermis or trachea is sufficient to induce their directional twisting. These results indicate that Myo1D is not only necessary for native LR asymmetry but also sufficient to induce de novo chirality at multiple scales (Chougule, 2020).

Interestingly, recent work showed that myo1D is also involved in LR asymmetry of Xenopus and zebrafish, hence myo1D represents a unique dextral determinant whose function is conserved across phyla. These findings, together with the existence of nodal-independent mechanisms for LR development of the heart, further suggest that myo1D-dependent and actin-based processes may represent a unifying mechanism controlling LR asymmetry in both vertebrates and invertebrates. In further support of this view, recent work identified a mutation in the diaphanous1 (dia1) gene as being important for controlling dextral chirality of snail shell in Lymnaea stagnalis. dia1 belongs to the family of formin genes, encoding conserved factors involved in actin assembly (Chougule, 2020).

While a role of actin and associated factors emerges as a central mechanism for LR asymmetry establishment across phyla, the exact nature of actin factors and their interplay remain largely unknown. To try addressing these questions, this study has undertaken a dedicated genetic screen aiming at identifying novel regulators of dextral and/or sinistral development in Drosophila. In this study, 539 genes involved in cytoskeleton homeostasis were screened and novel candidate genes were identified whose loss-of-function leads to LR asymmetry defects. The role of the formin DAAM (Dishevelled Associated Activator of Morphogenesis), was further characterized, showing that it is a LR-specific actin nucleator essential for myo1D function both in native and de novo LR asymmetry. Of note, DAAM is also playing a critical role in the sinistral pathway, making it a unique common denominator of Drosophila LR pathways. This genetic screen further identified flightless (fli), chickadee (chic; encoding Profilin) and the src family non-receptor tyrosine kinase Tec29, which have previously been identified as regulators of DAAM for actin nucleation and F-actin polymerization. Altogether, these results uncover the DAAM pathway as a key regulator providing a specific F-actin network essential for myo1D-dependent LR asymmetry (Chougule, 2020).

The genetic screen allowed identification of a number of new regulators of LR asymmetry. Based on their known function, candidate genes can be sorted into three main categories: 1) DAAM, dia, fli, chic and Tec29, that are likely to work together to control actin nucleation and F-actin polymerization, 2) rhea/talin and mys, which are involved in cell adhesion through integrins, and, 3) l(2)gl which is involved in adherens junction and apico-basal polarity. These findings are entirely consistent with current knowledge in fly and vertebrate models. First, the zebrafish lgl2 gene has been shown to be important for E-cadherin localization at the adherens junction in the Kupffer vesicle (the LR organizer in fish), with lgl2 mutants showing reduced vesicular lumen and cilia number. Second, the Myo1D protein has been shown to directly interact with &beta'-catenin and DE-cadherin in the genitalia, and a role of Myo1D in the chirality and remodeling of the adherens junction has been established in the embryonic hindgut and adult male genitalia. It is therefore speculated that l(2)gl may be involved in the interaction between Myo1D and the adherens junction for proper LR asymmetry (Chougule, 2020).

The fact that rhea/talin and mys are involved in integrin adhesion suggests an important role of cell-extracellular matrix (ECM) adhesion for genitalia LR asymmetry. Of note, mutations in the Drosophila tenectin gene, encoding a ligand for PS2 integrin, can induce genitalia rotation defects, and recent work showed that genitalia rotation involves cell intercalation and asymmetry of junction remodeling. A role for cell adhesion and ECM has also been shown in vertebrates for LR asymmetry. In particular, work in chick showed that the asymmetrically expressed N-Cadherin is important for Pitx2 expression and heart looping. Additionally, N-cadherin controls the asymmetry of the ECM in the dorsal mesentery that is essential for proper gut looping. Future work will help to characterize the precise role played by the adhesion genes identified in our screen during Drosophila LR asymmetry and chiral morphogenesis (Chougule, 2020).

While genetic screening allowed identifying new factors important for LR asymmetry, it also provided information about factors, gene families or cellular functions that are likely not being involved in the process. In particular, genes involved in microtubule formation or regulation have not been identified as potential regulators of Drosophila chirality. This observation suggests that unlike vertebrates, in which microtubules and associated molecular motors play an important role for cilia-driven flow and asymmetry, Drosophila does not depend on microtubule cytoskeleton for establishing LR asymmetry. Hence, while Drosophila rely exclusively on actin-based processes, vertebrates use both systems (actin and microtubules) for establishing LR asymmetry. Nevertheless, Myo1D is the sole common denominator between invertebrates and vertebrates, and it will be interesting to characterize how Myo1D, actin and microtubules interact for establishing vertebrate LR asymmetry (Chougule, 2020).

This screen and analysis show that the formin DAAM is essential for both sinistral and dextral development. Genetic invalidation of all Drosophila actin nucleators (formins, spire, Arp2/3) individually or in combination with DAAM shows that the formin DAAM is an LR-specific actin nucleating factor. The genetic data also suggest that the long form of DAAM (DAAM-PD) may have a specific function during LR asymmetry, which might be linked to the extra N-terminal domain present in this protein isoform. Finally, this study showed that the FH2 actin assembly domain of DAAM is essential for both normal and de novo laterality (Chougule, 2020).

Based on these results, a model is proposed in which DAAM and associated regulators (Chic, FliI, Tec29) build a specific F-actin network that serves as a substrate for Myo1D function. Because Myo1D has been shown to induce chiral movement of F-actin in an in vitro gliding assays, it is speculated that interaction between Myo1D and the DAAM-dependent F-actin network (daamF-actin) induces a chiral cytoskeleton. Since Myo1D has been shown to bind directly to DE-cadherin and &betq;-catenin, Myo1D could serve as a scaffold and determinant at the adherens junction for assembling a DAAM-complex and a chiral cytoskeleton. As a result of the formation of the tripartite complex (Myo1D, daamF-actin, AJs), cell-cell adhesion would be biased and lead to multiscale chirality of tissues and the whole body as observed in twisted larvae, trachea or genitalia (Chougule, 2020).

Could DAAM and/or formins play a conserved role in LR asymmetry across phyla? It is interesting to note that in chick, Daam2 has been shown to be downstream of Pitx2 and Wnt for cadherin-based adhesion and cell remodeling during gut looping. It would therefore be interesting to know if, in addition to Xenopus and zebrafish, Myo1D plays any role in chick LR asymmetry. Recent work showed a link between Myo1D and Planar Cell Polarity (PCP) in Drosophila, zebrafish and Xenopus, and Daam1 function has been shown to be involved in PCP-mediated renal tubulogenesis, suggesting a conserved Myo1D-DAAM-PCP link. In the pond snail Lymnaea stagnalis, some natural variants show a sinistral phenotype in their shell coiling. Recent genetic linkage and mutant analysis has provided data showing that the Lymnae diaphanous1 (Lsdia1) gene is important for dextral coiling of the snail shell. These data indicate that snail depend on formin activity for their LR asymmetry. It has been recently shown that chiral interaction between Myo1D and F-actin induces chirality at all biological scales, indicating a molecular origin of macroscopic chirality. Interestingly, work using cell culture has revealed that isolated cells can develop intrinsic chirality, leading to the self-organization and polarization of F-actin bundles. In particular, recent work showed that this process of autonomous chirality could be abolished by blocking formins, in particular mDia1. In conclusion, DAAM and Dia may represent genuine formins involved in LR asymmetry in both vertebrates and invertebrates. The identification of DAAM as a LR-specific actin nucleator in vivo provides strong evidence that a specific subset of F-actin is assembled in LR organizers for Myo1D activity. Because DAAM is also required for de novo asymmetry, it is concluded that DAAM is a general molecular effector helping to assemble a specific F-actin substrate for Myo1D activity in both native and de novo chirality (Chougule, 2020).

Functions of Myosin 31DF orthologs in other species

A conserved role of the Unconventional Myosin 1d in laterality determination
Anatomical and functional asymmetries are widespread in the animal kingdom. In vertebrates, many visceral organs are asymmetrically placed. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question. Nodal signaling is widely used, and many, but not all, vertebrates use cilia for symmetry breaking. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs. The role of myo1d in left-right (LR) axis formation was studied in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila, acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, these data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry (Tingler, 2018).


Search PubMed for articles about Drosophila Myosin 31DF

Acebes, A., Cobb, M. and Ferveur, J. F. (2003). Species-specific effects of single sensillum ablation on mating position in. Drosophila. J. Exp. Biol. 206: 3095–3100

Amcheslavsky, A., Wang, S., Fogarty, C. E., Lindblad, J. L., Fan, Y. and Bergmann, A. (2018). Plasma membrane localization of apoptotic caspases for non-apoptotic functions. Dev Cell 45(4): 450-464.e453. PubMed ID: 29787709

Bahler, M. and Rhoads, A. (2002) Calmodulin signaling via the IQ motif. FEBS Lett. 513: 107-113. PubMed ID: 11911888

Brown, N. A. and Wolpert, L. (1990). The development of handedness in left/right asymmetry. Development 109(1): 1-9. PubMed ID: 2209459

Chougule, A., Lapraz, F., Foldi, I., Cerezo, D., Mihaly, J. and Noselli, S. (2020). The Drosophila actin nucleator DAAM is essential for left-right asymmetry. PLoS Genet 16(4): e1008758. PubMed ID: 32324733

Eley, L., et al. (2004). A perspective on inversin. Cell Biol. Int. 28: 119-124. PubMed ID: 14984757

Freeman, G. and Lundelius, J. (1982). The developmental genetics of dextrality and sinistrality in the gastropod Lymnaea peregra. Wilhelm Roux's Archives 191: 69-83

Gillespie, P. G., et al. (2001). Myosin-I nomenclature. J. Cell Biol. 155: 703-704. PubMed ID: 11724811

Gonzalez-Morales, N., Geminard, C., Lebreton, G., Cerezo, D., Coutelis, J.B. and Noselli, S. (2015). The atypical cadherin Dachsous controls left-right asymmetry in Drosophila. Dev Cell 33(6):675-89. PubMed ID: 26073018

Hozumi, S., et al. (2006). An unconventional myosin in Drosophila reverses the default handedness in visceral organs. Nature 440(7085): 798-802. PubMed ID: 16598258

Huber, L. A., et al. (2000). Both calmodulin and unconventional myosin Myr4 regulate membrane trafficking along the recycling pathway of MDCK cells. Traffic 1: 494-503. PubMed ID: 11208135

Huber. B. A., Sinclair, B. J. and Schmitt, M. (2007). The evolution of asymmetric genitalia in spiders and insects. Biol. Rev. Camb. Philos. Soc. 82(4): 647-98. PubMed ID: 17944621

Inatomi, M., Shin, D., Lai, Y. T. and Matsuno, K. (2019). Proper direction of male genitalia is prerequisite for copulation in Drosophila, implying cooperative evolution between genitalia rotation and mating behavior. Sci Rep 9(1): 210. PubMed ID: 30659250

Morgan, D., et al. (1998). Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse. Nature Genet. 20: 149-156. PubMed ID: 9771707

Morgan, D., et al. (2002). The left-right determinant inversin has highly conserved ankyrin repeat and IQ domains and interacts with calmodulin. Hum. Genet. 110: 377-384. PubMed ID: 11941489

Morgan, N. S., Heintzelman, M. B. and Mooseker, M. S. (1995). Characterization of Myosin-IA and Myosin-IB, two unconventional myosins associated with the Drosophila brush border cytoskeleton. Dev. Biol. 172: 51-71. PubMed ID: 7589814

Nurnberger, J., Bacallao, R. L. and Phillips, C. L. (2002). Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells. Mol. Biol. Cell 13: 3096-3106. PubMed ID: 12221118

Petzoldt, A. G., et al., (2012). DE-Cadherin regulates unconventional Myosin ID and Myosin IC in Drosophila left-right asymmetry establishment. Development 139(10): 1874-84. PubMed ID: 22491943

Shibazaki, Y., Shimizu, M. and Kuroda, R. (2004) Body handedness is directed by genetically determined cytoskeletal dynamics in the early embryo. Curr. Biol. 14: 1462-1467. PubMed ID: 15324662

Spéder P, Adám G, Noselli S. (2006). Type ID unconventional myosin controls left-right asymmetry in Drosophila. Nature 440(7085): 803-7. PubMed ID: 16598259

Suzanne, M., et al. (2010). Coupling of apoptosis and L/R patterning controls stepwise organ looping. Curr. Biol. 20(19): 1773-8. PubMed ID: 20832313

Taniguchi, K., et al. (2011). Chirality in planar cell shape contributes to left-right asymmetric epithelial morphogenesis. Science 333(6040): 339-41. PubMed ID: 21764746

Tingler, M., Kurz, S., Maerker, M., Ott, T., Fuhl, F., Schweickert, A., LeBlanc-Straceski, J. M., Noselli, S. and Blum, M. (2018). A conserved role of the Unconventional Myosin 1d in laterality determination. Curr Biol 28(5): 810-816.e813. PubMed ID: 29478852

Ueshima, R. and Asami, T. (2003). Evolution: single-gene speciation by left-right reversal. Nature 425: 679. PubMed ID: 14562091

Watanabe, D., et al. (2003). The left-right determinant inversin is a component of node monocilia and other 9 + 0 cilia. Development 130: 1725-1734. PubMed ID: 12642479

Wood, W. B. (1997). Left-right asymmetry in animal development. Annu. Rev. Cell Dev. Biol. 13: 53-82. PubMed ID: 9442868

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date revised: 10 June 2023

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