Dishevelled Associated Activator of Morphogenesis

The expression of DAAM was examined; the mRNA is expressed in the developing embryonic tracheal system, starting from stage 13 of embryogenesis onwards. Similarly, immunostaining experiments (using a polyclonal anti-DAAM serum raised against the C-terminal half of the protein revealed the presence of the DAAM protein in the developing embryonic tracheal cells in stage 13 and older embryos. The protein is absent in eye-antennal imaginal discs, where DAAM is expressed during later stages of development (Matusek, 2006).

Effects of Mutation or Deletion

Isolation of DAAM mutants

To begin the functional analysis of DAAM, loss-of-function mutant alleles were generated by using two transposon insertions, EP(1)1336 and EP(1)1542, in the immediate vicinity of the DAAM locus. Remobilization of these insertions allowed isolation of a set of deletion alleles, two of which were mapped to the 5' region of DAAM, and five of them were mapped to the 3' region. The large 5' deletions affect only 5' UTR exons and intronic sequences, while the 3' alleles carry smaller deletions (965-2538 bp), but affect the DAAM ORF, leading to C-terminal truncations that (depending on the allele) remove up to 457 amino acids of the predicted protein. All 5' and 3' deletion alleles are lethal as hemi- or homozygotes, with the exception of DAAMEx1, the smallest C-terminal deletion allele which is semiviable and fertile (viability is 17% when compared with wild type). The lethal 5' and 3' deletion alleles do not complement each other and the deficiencies uncovering this region [Df(1)AD11, Df(1)AC7 and Df(1)sta]. They are, however, complemented by Dp(1;3)sta and Dp(1;Y)Sz280, two transpositional duplications that carry the 1F2-3 cytological region, including DAAM. All lethal alleles are viable over DaamEx1, the weakest and homozygous viable allele, with the exception of the two largest C-terminal deficiency alleles (DaamEx68 and DaamEx36). To demonstrate that the lethality associated with these alleles is solely due to the lack of DAAM, transgenic flies carrying UAS-FL-DAAM (containing the full length RE67944 cDNA clone) were generated and tested in rescue experiments. When UAS-FL-DAAM was expressed under the control of Act-Gal4 or tub-Gal4 in a DAAM mutant background lethality was rescued to adult viability, even in case of the largest deletion allele DaamEx68. Taken together, these results demonstrate that all lethal alleles represent DAAM alleles and these mutations do not affect other essential genetic elements (Matusek, 2006).

Based on biochemical assays and tissue culture experiments it was suggested that a human DAAM family member, DAAM1, is required for non-canonical Wnt/Fz signaling, and might function as a bridging factor between Dsh and RhoA. Xenopus embryo experiments supported this view, since the Xenopus ortholog xDaam1 has been implicated in PCP signaling during the early steps of gastrulation (Habas, 2001). To test whether the Drosophila DAAM is involved in PCP signaling, a loss-of-function analysis of the gene was carried out. DAAM mutant clones were induced in the wing and compound eye, two PCP organization model tissues. Although several alleles were tested, none of them exhibited PCP defects in the eye or wing. The same was observed in adult escapers of the DAAMEx1/DAAMEx249 allelic combination (Matusek, 2006).

Whether the PCP gain-of-function phenotypes induced by overexpression of PCP proteins can be modified by DAAM mutant alleles was tested. To achieve this, the effects of Fz and Dsh overexpression in the wing or eye were compared in wild-type and DAAMEx68 heterozygous mutant backgrounds. No significant difference between the wild-type and the DAAM mutant backgrounds was observed. Thus, the loss-of-function analysis of DAAM argues that the Drosophila DAAM ortholog is either not required for PCP signaling or at best plays a redundant role during PCP establishment (Matusek, 2006).

DAAM regulates the formation of taenidial folds in the cuticle of the trachea

Since DAAM alleles are lethal, it encodes a gene with essential function(s). To identify these, DAAM mutant larvae were examined; these animals display severe trachea defects. These include the collapse and flattening of the tracheal tubes, in both the main airways and the side branches. Wild-type tracheal cells secrete a cuticle on their apical (luminal) surface, which is continuous with the epidermal cuticle. Nevertheless, it is easily distinguished by the presence of cuticle ridges called taenidial folds that project into the lumen. These ridges form annular rings or run a helical course around the lumen of the tubes. Tracheal tubes of DAAM mutant larvae fail to secrete such a highly ordered cuticle. Instead, although some local order is visible, short and curvy apical ridges were observed that rarely run perpendicular to the tube axis. These striking cuticle pattern defects are detected throughout the tracheal system independent of tube types, with the exception of the fusion cells that secrete a different type of cuticle characterized by a dotty pattern instead of taenidial folds. To confirm that the trachea phenotype is caused by DAAM loss of function, a UAS-FL-DAAM construct was expressed under the control of a trachea specific btl-Gal4 driver. This significantly rescued the trachea disruptions, just as a general driver (e.g. Act-Gal4) (Matusek, 2006).

Next, the expression of DAAM was examined and it was confirmed that the mRNA is expressed in the developing embryonic tracheal system, starting from stage 13 of embryogenesis onwards. Similarly, immunostaining experiments (using a polyclonal anti-DAAM serum raised against the C-terminal half of the protein revealed the presence of the DAAM protein in the developing embryonic tracheal cells in stage 13 and older embryos. In DAAMEx68 embryos, the antibody failed to detect protein in the trachea. Similarly, detectable protein was absent from mutant clonal tissue in eye-antennal imaginal discs, where DAAM is expressed during later stages of development. This confirmed that the antibody is specific to DAAM, and that DAAMEx68 is likely to be a protein null. Consistently, the trachea phenotype of DAAMEx68 homo- or hemizygous larvae is very similar to that of DAAMEx68/Df(1)AD11 larvae (Matusek, 2006).

Actin organization in wild type and DAAM mutant tracheal tubes

Since it is well established that formins regulate the actin cytoskeleton, actin organization was investigated in wild-type and DAAM mutant tracheal cells. This revealed that in wild-type tracheal cells apically localized actin is organized into parallel bundles running perpendicular to the axis of the tubes, strongly resembling the organization of teanidial folds in the cuticle. Significantly, the number of actin rings and taenidial folds in a given tracheal region is equal to each other. To determine precisely the time when such actin rings or spirals become apparent, an Actin-GFP (green fluorescent protein) fusion protein was expressed in the trachea of living embryos (using the btl-Gal4 tracheal driver line). This demonstrated that actin rings are first visible in late stage 15 embryos, just before the onset of cuticle secretion (Matusek, 2006).

The embryonic tracheal cells are rather small, and thus it was difficult to study the formation of these actin rings at the subcellular level. Nevertheless, it is clear that these actin rings are not only present when cuticle secretion begins, but are also detected through the third larval instar stage. In contrast to the remarkable actin organization found in wild-type tracheal cells, in DAAMEx68 mutants this organization is completely abolished. Whereas the absence of DAAM does not appear to reduce the apical F-actin level of the tracheal cells, actin cables formed are much shorter and thinner than in wild type. Additionally, actin bundles often appear to be crosslinked to each other instead of running parallel. Taken together, in DAAMEx68 the actin bundles in the tracheal cells display abnormal morphology and fail to be organized into parallel running actin rings under the apical surface of the cells (Matusek, 2006).

Although the global actin organization is severely impaired, local order can often be detected in small patches within a cell. The cuticle pattern of DAAMEx68 mutants displays a similar phenotype, indicating that actin cables direct the run of taenidial folds, even in the DAAM mutant situation. Thus, it appears that the major function of DAAM in Drosophila tracheal cells is to organize an array of actin rings that directs cuticle patterning by specifying the site of taenidial fold formation. Despite the fact that the apical actin level is not significantly reduced in DAAM mutants, the possibility that in the wild-type situation, DAAM also contributes to actin assembly, a well characterized formin function, cannot be excluded. Consistent with this possibility, DAAM largely colocalizes with the apically enriched actin bundles in the embryonic trachea cells. Nevertheless, the potential involvement in actin polymerization appears be a redundant function. The recently characterized form3 (Tanaka, 2004) is the best candidate to be functionally redundant with DAAM for actin assembly, because it is the only other Drosophila formin known to be expressed in the tracheal system (Matusek, 2006).

Formin proteins of the DAAM subfamily play a role during axon growth

The regulation of growth cone actin dynamics is a critical aspect of axonal growth control. Among the proteins that are directly involved in the regulation of actin dynamics, actin nucleation factors play a pivotal role by promoting the formation of novel actin filaments. However, the essential nucleation factors in developing neurons have so far not been clearly identified. This study shows expression data, and uses true loss-of-function analysis and targeted expression of activated constructs to demonstrate that the Drosophila formin DAAM plays a critical role in axonal morphogenesis. In agreement with this finding, it was shown that DAAM is required for filopodia formation at axonal growth cones. Genetic interaction, immunoprecipitation and protein localization studies argue that DAAM acts in concert with Rac GTPases, Profilin and Enabled during axonal growth regulation. It was also shown that mouse Daam1 rescues the CNS defects observed in DAAM mutant flies to a high degree, and vice versa, that Drosophila DAAM induces the formation of neurite-like protrusions when expressed in mouse P19 cells, strongly suggesting that the function of DAAM in developing neurons has been conserved during evolution (Matusek, 2008).

Characterization of the biochemical properties and biological function of the formin homology domains of Drosophila DAAM

This study characterized the properties of Drosophila melanogaster DAAM-FH2 and DAAM-FH1-FH2 fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. The results show that although the DAAM-FH2 fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the DAAM-FH1-FH2 fragment, a profilin-dependent increase in the formation of actin structures is observed. The trachea-specific expression of DAAM-FH1-FH2 also induces phenotypic effects, leading to the collapse of the tracheal tube and lethality in the larval stages. In vitro, both DAAM fragments catalyze actin nucleation but severely decrease both the elongation and depolymerization rate of the filaments. Profilin acts as a molecular switch in DAAM function. DAAM-FH1-FH2, remaining bound to barbed ends, drives processive assembly of profilin-actin, whereas DAAM-FH2 forms an abortive complex with barbed ends that does not support profilin-actin assembly. Both DAAM fragments also bind to the sides of the actin filaments and induce actin bundling. These observations show that the D. melanogaster DAAM formin represents an extreme class of barbed end regulators gated by profilin (Barko, 2010).

Dissecting regulatory networks of filopodia formation in a Drosophila growth cone model

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. This study has focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, embryonic Drosophila primary neurons were as one consistent cellular model to study filopodia regulation. The data for loss-of-function of capping proteins, Enabled, different Arp2/3 complex components, the formin DAAM, and profilin, reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of the Drosophila model system. Using combinatorial genetics, this study demonstrated that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM, filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. These genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps (Goncalves-Pimentel, 2011).


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Dishevelled Associated Activator of Morphogenesis: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 March 2016

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