BIOLOGICAL OVERVIEW

Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. This study analyzes a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, it is shown that DAAM plays no essential role in planar cell polarity signaling, but it has striking requirements in organizing apical actin cables that define the taenidial fold pattern of the tracheal cuticle. These observations provide evidence the first time that the function of the taenidial organization is to prevent the collapse of the tracheal tubes. The results indicate that although DAAM is regulated by RhoA, it functions upstream or parallel to the non-receptor tyrosine kinases Src42A and Tec29 to organize the actin cytoskeleton and to determine the cuticle pattern of the Drosophila respiratory system (Matusek, 2006).

Proteins of the formin family play key roles in the regulation of the cytoskeleton. Although the connection between formins and microtubules is less well understood, formins are implicated in a large number of actin-based processes, including cell polarization, division, movement, stress fiber formation and vesicular trafficking. Recent work suggests that formins catalyze the assembly of unbranched actin structures and nucleate actin filaments directly (Matusek, 2006).

Although all members of this highly conserved family are defined by the presence of the formin homology domain 2 (FH2), which is both necessary and sufficient to nucleate actin in vitro, formins are multi-domain proteins that contain several other conserved sequences as well. The FH2 domain is usually flanked on the N-terminal side by a proline-rich FH1 domain that can serve as a docking site for the G-actin-binding protein profilin. In addition, the FH1 domain also binds WW domains and SH3 domains, including those of the Src family. A distinct formin subfamily, the DRF group (Diaphanous related formins: see Drosophila Diaphanous) has the ability to interact with an activated Rho GTPase through an N-terminal GTPase-binding domain (GBD). This binding alleviates the autoinhibitory interaction between the GBD and the short C-terminal DAD domain (Diaphanous autoregulatory domain) (Alberts, 2001). Crystallographic analysis of the N-terminal part of mouse Dia1 provides a structural basis of this regulation and has identified several other functional domains within this part of the protein, including a dimerization domain (DD), (Otomo, 2005a; Rose, 2005). In addition, the crystal structure of C-terminal formin domains reveals that the FH2 domain also forms a dimer required for actin nucleation and processive filament capping (Otomo, 2005b; Shimada, 2004; Xu, 2004), strongly suggesting that native formins act in dimeric forms (Matusek, 2006).

Recently, a novel formin subtype, DAAM (Dishevelled-associated activator of morphogenesis), has been identified and implicated in planar cell polarity signaling during Xenopus gastrulation (Habas, 2001). The polarized orientation of cells within the plane of a tissue, or planar cell polarization (PCP), is an important aspect of cellular differentiation and is often necessary to the formation of functional organs. Genes controlling PCP have been extensively studied in Drosophila, revealing a crucial role for frizzled (fz) signaling through dishevelled (dsh) during the course of PCP establishment. The downstream components of the Fz/PCP pathway include RhoA, Drok, the JNK cascade and several other genes, most of which act tissue specifically. Many of the genes involved in PCP signaling are also required for polarized morphogenetic cell movements such as convergent extension during early vertebrate embryogenesis suggested that the novel FH2 protein Daam1 is required for convergent extension is Xenopus embryos and that Daam1 might function as a bridging factor between Dsh and RhoA. Moreover, Wnt/Fz-mediated activation of RhoA appeared to depend on Dvl (a Dsh homolog) and Daam1 (Habas, 2001). However, in contrast to this model, previous work has provided evidence that formins act as Rho effectors downstream of the Rho GTPases (Matusek, 2006).

To gain further insights into the function of this novel class of FH2 proteins, the single Drosophila member of the DAAM family was analyzed. Phenotypic analysis of DAAM mutants showed that it plays either no role or possibly a redundant role in PCP establishment in Drosophila. However, evidence was found that DAAM is involved in the regulation of the actin cytoskeleton in several different tissues, including the tracheal system. The Drosophila tracheal network is one of the best characterized model systems of branching morphogenesis. During the first phase of tracheal development, the primordial cells invaginate in each embryonic hemisegment from the epidermis, migrate and undergo cell shape changes to form the primary branches. Subsequently, some tracheal branches fuse with an adjacent branch to build up a continuous tubular network. This process is mediated by fusion cells located at the branch tips, which recognize each other in the adjacent hemisegments and become doughnut shaped, forming a lumen that connects the two branches. Before the end of embryogenesis, tracheal cells secrete a cuticle on their apical, luminal surface that protects the larvae from dehydration and infection. The tracheal cuticle is distinguished from the epidermal cuticle by the presence of cuticle ridges, often called taenidial folds that are thought to prevent the collapse of tracheal tubes while allowing them to expand and contract along their length (Matusek, 2006).

Drosophila DAAM is required to organize an array of parallel running actin cables beneath the apical surface of the tracheal cells that define the taenidial fold pattern of the cuticle. The actin ring pattern corresponds exactly to that of the taenidial fold pattern, and it is proposed that the actin rings organized by DAAM define the position of taenidial fold formation. The genetic interaction and epistasis data are consistent with a model that DAAM activity is regulated by RhoA. In addition, DAAM works together with the non-receptor tyrosine kinases Src42A and Tec29 to regulate the actin cytoskeleton of the Drosophila tracheal system (Matusek, 2006).

The basic structure of the insect tracheal system is a highly conserved tubular network in every species. The most important function of this network is to allow oxygen flow to target cells. Thus, tracheal tubes need to be both rigid enough, to ensure continuous air transport, and flexible enough along the axis of the tubes, to prevent the break down of the tube system when body parts or segments move relative to each other. These requirements are mainly ensured by the tracheal cuticle, which covers the luminal surface of the tubes and displays cuticle ridges (making the overall structure similar to the corrugated hose of a vacuum cleaner). Analysis of DAAM mutants provides the first direct evidence that this hypothesis is correct. The data demonstrate that in the absence of DAAM the taenidial fold pattern is severely disrupted, often leading to the collapse of the tubes and to discontinuities in the tubular network. In addition, the analysis revealed that the remarkably ordered cuticle pattern, displayed in the wild-type trachea tubes, depends on DAAM-mediated apical actin organization. Apical actin is organized into parallel-running actin cables, much the same way teanidial folds run in the cuticle. Significantly, the formation of these actin bundles precedes the onset of cuticle secretion, and the number and phasing of the actin rings correspond exactly to that of the taenidial folds in the cuticle. Thus these studies revealed a novel aspect of apical actin organization in the tracheal cells that has not been appreciated before (Matusek, 2006).

The DAAM gene encodes a novel member of the formin family of proteins, involved in actin nucleation and polymerization. Consistent with this, DAAM is colocalized with apical actin in the tracheal cells, and the activated form of DAAM is able to induce actin assembly when expressed in tracheal cells and in other cell types. In DAAM mutant tracheal cells, apical actin is still detected, albeit at a somewhat lower level than in wild type, but the bundles formed in the mutant are much shorter and thinner than in wild type, and often appear to be crosslinked to each other. Most strikingly, global actin organization is almost completely lost, although some local order can still be detected. Remarkably, the cuticle pattern in mutant tracheal cells still follows the underlying aberrant actin pattern. Overall, in DAAM mutants, both the tracheal cuticle and the apical actin pattern resemble a mosaic of locally ordered patches that failed to be coordinated and aligned with each other and the axis of the tracheal tubes. It is thus proposed that the apical actin bundles play a key role in patterning the tracheal cuticle by defining the place of taenidial fold formation. Regarding the function of DAAM, the results suggest that the major role of this formin in the tracheal cells is to organize the actin filaments into parallel running actin rings or spirals instead of simply executing the well characterized formin function related to actin assembly. However, whether this is a direct effect on actin organization, and thus represents a novel formin function, needs to be further elucidated. An alternative model could be that DAAM is primarily required for actin polymerization but tightly coupled to an actin 'organizing' protein. In such scenario, the polymerization activity should be a redundant requirement, whereas the link to the organizing protein would be a DAAM-specific function, thereby explaining the presence of unorganized actin bundles in DAAM mutant tracheal cells (Matusek, 2006).

In the case of the main tracheal airways, which are multicellular along their periphery, it is striking that in wild type the run of the actin bundles is perfectly coordinated across cell boundaries. In addition, the run is always perpendicular to that of the tube axis. How does DAAM ensure the coordination of these two aspects of actin organization? Because the DAAM protein and the apical actin cables are both found at the level of the adherens junctions, it is possible that DAAM regulates the coordination of the actin cables at the cell boundaries through a direct interaction with junctional protein complexes. However, other explanations are also possible, and further experiments will be required to elucidate the molecular mechanism of this regulatory function. The fact that actin cables normally run perpendicular to the tube axis seems to suggest that tracheal cells are able to sense a global orientation cue and align their actin bundles accordingly. The nature and source of this cue is unknown, as is the mechanism by which DAAM is involved in the read-out of this signal. Nevertheless, it is interesting that in DAAM and btl-Gal4/UAS-C-DAAM mutant trachea, the main pattern of the cuticle phenotype is often changing from one segment to the other, suggesting that the effect of the 'global' orientation cue is limited to metameric units (Matusek, 2006).

Sequence comparisons of FH2 proteins suggest a close phylogenetic relationship between the DRF, FRL and DAAM subfamilies (Higgs, 2005). Members of these three subfamilies have a high level of conservation in the FH2 domain, and importantly, also in the region of the GBD and DAD domains, suggesting that the FRL and DAAM family formins are also regulated by autoinhibition and RhoGTPases, like the DRFs. Further evidence is presented in support of this view. First, DAAM and RhoA display a strong genetic interaction. Second, C-DAAM (an N-terminally truncated form of DAAM) behaves like an activated form much the same way DRF family formins behave. Third, epistasis experiments with C-DAAM and RhoA suggest that RhoA acts upstream of DAAM. Thus, the data support the model in which DAAM, at least in the Drosophila tracheal system, is regulated by autoinhibition that can be relieved by RhoGTPases (Matusek, 2006).

This conclusion, however, contradicts the observation that human DAAM1 is required for Wnt/Fz/Dvl dependent RhoA activation in cultured cells and that xDaam1 appears to mediate Wnt-11 dependent RhoA activation in Xenopus embryos (Habas, 2001). These results suggested that DAAM functions upstream of RhoA in non-canonical Wnt/Fz-PCP signaling. An explanation for these distinct conclusions might be related to the fact that DAAM, in contrast to xDaam1, does not appear to be required for Fz/Dsh-PCP signaling. Hence, it is possible that the Drosophila ortholog is regulated in the same way as the DRF formins, while the vertebrate family members can be regulated in a different way, once bound by Dsh/Dvl and recruited into PCP signaling complexes (Matusek, 2006).

Genetic interactions with the hypomorphic DAAM^Ex1 allele identified two non-receptor tyrosine kinases, Src42A and Tec29, as strong interacting partners. Although both of these kinases play multiple roles during embryogenesis, single mutants for both affect the tracheal cuticle pattern in a similar way to DAAM. These results suggest that DAAM and the Src family kinases work together to regulate the actin cytoskeleton and cuticle pattern in tracheal cells. Although it is not known whether DAAM physically binds Src42A and/or Tec29, it has been established that the FH1 region of DRFs and other formins can bind SH3 domains, including those of the Src family kinases. In agreement with these data that DAAM acts upstream of Src42A and Tec29 in tracheal cells, cytoskeleton remodeling and SRF activation mediated by mouse Dia1 and mouse Dia2 requires Src activity. Moreover, a recent report suggests that RhoD and human DIA2C regulate endosome dynamics through Src activation, proposing that Src activity is stimulated via human DIA2C dependent recruitment to early endosomes (Gasman, 2003). Similarly, the Limb deformity protein (a formin) interacts with Src on the perinuclear membranes of primary mouse fibroblasts. Based on these examples, it is speculated that in Drosophila tracheal cells the RhoA/DAAM/Src module may not only be required to organize apical actin bundles, but additionally it might represent a link to secretory vesicles and to the regulation of exocytosis. Future studies will be required to test this hypothesis, and to unravel the mechanisms whereby DAAM family formins and Src family kinases contribute to cytoskeletal remodeling in the Drosophila tracheal system and in other tissues (Matusek, 2006).

The formin DAAM functions as molecular effector of the planar cell polarity pathway during axonal development in Drosophila

Recent studies established that the planar cell polarity (PCP) pathway is critical for various aspects of nervous system development and function, including axonal guidance. Although it seems clear that PCP signaling regulates actin dynamics, the mechanisms through which this occurs remain elusive. This study established a functional link between the PCP system and one specific actin regulator, the formin DAAM, which has previously been shown to be required for embryonic axonal morphogenesis and filopodia formation in the growth cone. DAAM also plays a pivotal role during axonal growth and guidance in the adult Drosophila mushroom body, a brain center for learning and memory. By using a combination of genetic and biochemical assays, it was demonstrated that Wnt5 and the PCP signaling proteins Frizzled, Strabismus, and Dishevelled act in concert with the small GTPase Rac1 to activate the actin assembly functions of DAAM essential for correct targeting of mushroom body axons. Collectively, these data suggest that DAAM is used as a major molecular effector of the PCP guidance pathway. By uncovering a signaling system from the Wnt5 guidance cue to an actin assembly factor, it is proposed that the Wnt5/PCP navigation system is linked by DAAM to the regulation of the growth cone actin cytoskeleton, and thereby growth cone behavior, in a direct way (Gombos, 2015).

This study has shown that DAAM plays an important role in the regulation of axonal growth and guidance of the Drosophila MB neurons. Several lines of evidence suggest that DAAM acts in concert with Wnt5 and the core PCP proteins to ensure correct targeting of the KC axons. DAAM functions downstream of Dsh and Rac1, and its ability to promote actin assembly is absolutely required for neural development in the MB. These data suggest a simple model in which axon guidance cues, such as Wnt5, signal through the PCP pathway to activate DAAM to control actin filament formation in the neuronal growth cone. Thus, PCP signaling appears to be linked to cytoskeleton regulation in a direct way, and these results provide compelling experimental evidence suggesting that, at least in neuronal cells, the major cellular target of PCP signaling is the actin cytoskeleton (Gombos, 2015).

Formins are highly potent actin assembly factors that are under tight regulation in vivo. The major mechanism of controlling the activity of the Diaphanous-related formin (DRF) subfamily involves an intramolecular autoinhibitory interaction between the N-terminal diaphanous inhibitory domain (DID) and the C-terminal Diaphanous autoinhibitory domain (DAD). This inhibition can be relieved upon binding of an activated Rho family GTPase that interacts with the GBD (GTP-ase binding domain)/DID region and also by proteins that bind to the DAD domain. Consistently, this study found that the Rac1 GTPase and the DAD domain binding Dsh protein both play role in DAAM activation in MB neurons. With this regard, it is notable that, despite that dsh¹ is considered a PCP-null allele, the DAAM^Ex1, dsh¹ double hemizygous mutants exhibit a stronger MB phenotype than dsh¹ mutants alone, suggesting that DAAM must receive Dsh-independent regulatory inputs for which Rac1 is a prime candidate. Although previous work indicated that Rho GTPases might function downstream of Dsh in a linear pathway), the data suggest that Dsh and Rac1 act in parallel pathways in the MB. As the impairment of GTPase binding severely, but not completely, abolishes DAAM activity, it is concluded that Rac1 is likely to have a stronger contribution to DAAM activation in vivo; nonetheless, the simultaneous binding of Dsh appears to be required for full activation (Gombos, 2015).

Presumably, the most remarkable feature of the PCP system relies in its ability to create subcellular asymmetries. Therefore, it is a tempting idea that, upon guidance signaling, the PCP proteins are involved in the generation of molecular asymmetries within axonal growth cones, yet recent attempts failed to reveal such polarized distributions in MB neurons. Interestingly, however, it was shown that Fz and Vang display a differential requirement during development of the MBs, with Fz predominantly acting in the dorsal lobes and Vang predominantly acting in the medial lobes (MLs). This study found that, in contrast to Fz and Vang, DAAM plays a crucial role in both lobes of the MBs. Additionally, it was demonstrated that Fz promotes the formation of membrane-associated Dsh-DAAM complexes in S2 cells. This result, together with genetic data, suggests that DAAM acts as the downstream effector of a Fz/Dsh module, which is required for the correct growth and guidance of the dorsal MB axon branches (Gombos, 2015).

In addition to their potential connection to Fz signaling in the dorsal lobe, DAAM and Dsh were linked to Vang- and Wnt5-dependent ML development as well. Wnt5 and Vang have an identical effect on ML development when overexpressed, and this GOF phenotype can be suppressed by the same set of mutations (DAAM, dsh, Rac1). In particular, the putative PCP-null dsh¹ allele and heterozygosity for Rac1 cause an almost equally strong, yet partial, suppression with regard to the ML fusion phenotype. This is best explained by assuming that Wnt5 and Vang signal both in a Dsh-dependent and in a Dsh-independent, but Rac-dependent, manner. With regard to DAAM, this study has shown that DAAM nearly completely suppresses the GOF of Wnt5 and Vang, and Dsh and Rac1 both contribute to DAAM activation. Collectively, these data suggest a model in which Wnt5 and Vang promote β lobe extension by signaling to Dsh and Rac1 that will activate DAAM in parallel to each other. The colocalization of Vang and DAAM, observed in S2 cells, indicates that they may bind each other directly, which would be in good accordance with genetic data suggesting a close functional link between DAAM and Vang during β lobe development. However, formins are not known to bind Vang proteins; therefore, an indirect interaction, mediated by Rac1, which has recently been shown to be bound and redistributed by Vangl2 in epithelial cell lines, appears a more likely possibility (Gombos, 2015).

As discussed above, and contrary to Vang, Fz does not appear to be required for ML development, or if anything, it might play an opposite role, as loss of fz leads to ML fusion in 16.1% of the lobes. This is a surprising observation at first glance as Wnt proteins are thought to activate members of the Fz receptor family, but former analysis of Wnt5 signaling during MB development also failed to reveal a Fz requirement in the β lobes. Instead, Wnt5 has been linked to other type of Wnt receptors, the Ryk/Derailed atypical tyrosine kinase receptors, which are known to be involved in axonal guidance in flies and vertebrates. In light of these results, it will be of future interest to analyze the Wnt5-Vang connection in the MB in more details and identify the Wnt5 receptor in this context (Gombos, 2015).

Consistent with the lack of lobe-specific requirement for dsh and DAAM, the current studies revealed that Dsh, DAAM, and Rac1 are used as common effector elements of a dorsal lobe-specific Fz-dependent signal and a Vang-dependent ML-specific signal. It follows that Dsh and DAAM are likely to take part in two types of PCP complexes. Although, in vitro, Dsh has the ability to interact with both Fz and Vang, the conclusion that Dsh functions downstream of Vang in the β lobes is markedly different from the classical PCP regulatory context in which the Fz/Dsh and Vang/Pk complexes have opposing effects. Thus, this result, together with the Wnt5-Vang data, substantiates the earlier findings that the PCP system operates at least partly differently in neurons than during tissue polarity signaling (Gombos, 2015).

During PCP signaling, the vertebrate DAAM orthologs control convergence and extension movements, polarized cell movements during vertebrate gastrulatio. In contrast, DAAM is dispensable for classical planar polarity establishment in flies, suggesting that the tissue polarity function of DAAM might be restricted only to vertebrates. Despite the lack of direct function in establishing tissue polarization, this study provides evidence that DAAM is linked to the PCP pathway in another important regulatory context, notably directed neuronal development in the adult brain. Consistent with the results, recent studies revealed that PCP signaling and DAAM regulate neural development in planarians and in Xenopus embryos. Given that the vertebrate PCP proteins are known to be involved in multiple aspects of CNS development, and the vertebrate DAAM orthologs are strongly expressed in the CNS, it is conceivable that the PCP/DAAM module represents a highly conserved regulatory system that is used to regulate various aspects of neuronal development throughout evolution (Gombos, 2015).

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).

GENE STRUCTURE

cDNA clone length - 5369 (isoform b)

Bases in 5' UTR - 1168

Exons - 8

Bases in 3' UTR - 739

PROTEIN STRUCTURE

Amino Acids - 1153 (isoform b)

Structural Domains

A recently published phylogenetic analysis (based on sequence comparisons of 101 FH2 domains from 26 eukaryotic species) has concluded that metazoan formins can be grouped into seven major subclasses (Higgs, 2005). This analysis suggested that the DAAM subfamily clearly represents a distinct class. This is consistent with the previous finding that the DAAM subfamily exhibits extensive sequence similarity both within and outside of their highly conserved FH1 and FH2 domains (Habas, 2001). At present, however, very little is known about the in vivo function of the DAAM subfamily. Sequence analysis revealed the presence of a single DAAM ortholog in the Drosophila genome corresponding to the annotated gene CG14622. Although FlyBase predicts that this gene might code for several different transcript classes, this work focused on the predicted CG14622-RB transcript that appears to be encoded by the RE67944 EST clone. Sequencing of this full-length cDNA clone indicated a protein consisting of 1153 amino acids. Further analysis revealed that the ORF contains several conserved domains including an FH1, FH2, GBD and a putative DAD domain characteristic of formins. Based on the overall homology level and the phylogenetic analysis of FH2 domains (Higgs, 2005), the DAAM subfamily appears to be most related to the Dia subfamily, raising the possibility that similar to the DRFs, DAAM formins are also regulated by an autoinhibitory mechanism that can be relieved upon RhoGTP binding (Matusek, 2006).

date revised: 22 November 2022

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