The Interactive Fly

Genes involved in tissue and organ development

Handed asymmetry in organ shape and positioning

LR-dependent looping of Drosophila Genitalia

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

The atypical cadherin Dachsous controls left-right asymmetry in Drosophila

Single-minded 2 is required for left-right asymmetric stomach morphogenesis

LR-dependent looping of Drosophila Genitalia

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 (Figure S3), 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).

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. This study found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 and Wnt4, which encode a receptor and ligand of Wnt signaling respectively, are required for the LR asymmetric development of the AMG. arrow, an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 are expressed in the visceral muscles of the midgut. Consistent with these results, it was observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. These results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates (Kuroda, 2012).

This study found that Wnt-signaling components Wnt4 and Fz2 are required for LR asymmetric development of the AMG, although contribution of other Wnt ligands and receptors to this process could not be excluded. For example, it is known that Wnt4 binds to Fz and Fz2, and that fz and fz2 function redundantly in the segmentation of Drosophila embryos. This study found that the AMG of embryos homozygous for fz showed similar LR defects to those of fz2, although at a lower frequency. Therefore, it is possible that Fz acts redundantly as the receptor for canonical Wnt/β-catenin signaling, although the expression of fz in the midgut could not be detected by anti-Fz antibody staining. In contrast, analysis of embryos homozygous for derailed (drl) suggested that Wnt5 may not be involved in the LR asymmetric development of the AMG. Drl is a member of the RYK subfamily of receptor tyrosine kinases and is a receptor for Wnt5. The laterality of the AMG was normal in embryos homozygous for drl (Kuroda, 2012).

Wnt4 is one of the few Wnt ligands whose function has been revealed in Drosophila. This study found that Wnt4–Fz2 activates the canonical Wnt/β-catenin signaling pathway for normal LR asymmetric development of the AMG. Consistent with this finding, Wnt4 activates the canonical Wnt/β-catenin signaling pathway in salivary glands through Fz or Fz2, which is required for the glands’ proper migration. However, the Wnt4–Fz2 pathway is also known to activate non-canonical Wnt signaling in other systems. Wnt4 plays an essential role in the cell movement required for formation of the ovariolar sheath cells. In addition, Wnt4 expressed in the developing ventral lamina is required for ventral projection of the retinal axon. In both of these cases, Fz2 acts as a receptor of Wnt4, and the Wnt4–Fz2 pathway activates non-canonical Wnt signaling. Therefore, although the same combination of Wnt ligand and receptor, Wnt4–Fz2, is involved, the downstream cascades of Wnt signaling may be context-dependent, although the factors acting as molecular switches for these downstream pathways remain unknown (Kuroda, 2012).

The first indication of LR symmetric morphogenesis in the AMG is observed as the LR asymmetric rearrangement of circular visceral muscle (CVMU) cells. These rearrangements can be monitored by measuring the major axial angle of the nuclei in the CVMU cells to the midline of the AMG (Kuroda, 2012).

This study found that the LR asymmetry of the rearranged CVMU cells in the ventral AMG became bilaterally symmetric in embryos homozygous for a Wnt4 mutation. This result was consistent with the AMG’s random LR laterality in these embryos. However, unexpectedly, the CVMU cells were rearranged LR asymmetrically in the dorsal AMG in Wnt4 mutant homozygotes, even though the arrangement of these dorsal cells is bilaterally symmetric in wild-type embryos. This result suggests that Wnt signaling may counteract the LR asymmetric morphogenesis in the dorsal side of the AMG, in addition to its role in introducing a LR bias by inducing the rearrangement of CVMU cells in the ventral AMG, via the Wnt4–Fz2 pathway. In embryos homozygous for loss-of-function mutations of Wnt4, arr, or fz2, the LR asymmetric development of the posterior embryonic gut was largely normal. Thus, in wild-type embryos, the Wnt4–Fz2 signal may function to suppress the influence of the LR asymmetric morphogenic signals from the posterior midgut on the AMG (Kuroda, 2012).

The present analyses clarified the requirement for Wnt4–Fz2 signaling in the LR asymmetric morphogenesis of the AMG, but the precise molecular functions of this signal are still unclear. Because Wnt4–Fz2 activates canonical Wnt/β-catenin signaling, it will be important to identify the target genes responsible for LR asymmetric morphogenesis of the AMG (Kuroda, 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 (González-Morales, 2015).

Single-minded 2 is required for left-right asymmetric stomach morphogenesis

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, transcriptome analyses was performed of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut. sim2 was shown to function downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. These results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in non-laterality-related birth defects (Wyatt, 2021).


González-Morales, N., Géminard, 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

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

Kuroda, J., et al. (2012). Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut. Mech Dev. 128(11-12): 625-39. PubMed ID: 22198363

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

Wyatt, B. H., Amin, N. M., Bagley, K., Wcisel, D. J., Dush, M. K., Yoder, J. A. and Nascone-Yoder, N. M. (2021). Single-minded 2 is required for left-right asymmetric stomach morphogenesis. Development 148(17). PubMed ID: 34486651.

Genes involved in tissue and organ development

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