József Mihály

The iab-7 Polycomb Response Element Maps to a Nucleosome- Free Region of Chromatin and Requires Both GAGA and Pleiohomeotic for Silencing Activity

ABSTRACT In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogasterbithorax complex (BX-C). Previous studies mapped the iab-7PRE to an 860-bp fragment located just distal to the Fab-7boundary. Located within this fragment is an ∼230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of amini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of theiab-7 PRE in vivo.

The Drosophila formin DAAM regulates the tracheal cuticle pattern through organizing the actin cytoskeleton

Formins are involved in a wide range of cellular processes that require the remodeling of the actin cytoskeleton. Here, we have analyzed a novel Drosophila formin, belonging to the recently described DAAM subfamily. In contrast to previous assumptions, we show 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. Our 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.

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. Here, we show expression data, and use 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, we show that dDAAM is required for filopodia formation at axonal growth cones. Our genetic interaction, immunoprecipitation and protein localization studies argue that dDAAM acts in concert with Rac GTPases, Profilin and Enabled during axonal growth regulation. We also show that mouse Daam1 rescues the CNS defects observed in dDAAM 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.

Dissecting the regulatory landscape of the Abd-B gene of the bithorax complex

The three homeotic genes of the bithorax complex (BX-C), Ubx, abd-A and Abd-B control the identity of the posterior thorax and all abdominal segments. Large segment-specific cis-regulatory regions control the expression of Ubx, abd-A or Abd-B in each of the segments. These segment-specific cis-regulatory regions span the whole 300 kb of the BX-C and are arranged on the chromosome in the same order as the segments they specify. Experiments with lacZ reporter constructs revealed the existence of several types of regulatory elements in each of the cis-regulatory regions. These include initiation elements, maintenance elements, cell type- or tissue-specific enhancers, chromatin insulators and the promoter targeting sequence. In this paper, we extend the analysis of regulatory elements within the BX-C by describing a series of internal deficiencies that affect the Abd-B regulatory region. Many of the elements uncovered by these deficiencies are further verified in transgenic reporter assays. Our results highlight four key features of the iab-5, iab-6 and iab-7 cis-regulatory region of Abd-B. First, the whole Abd-B region is modular by nature and can be divided into discrete functional domains. Second, each domain seems to control specifically the level of Abd-B expression in only one parasegment. Third, each domain is itself modular and made up of a similar set of definable regulatory elements. And finally, the activity of each domain is absolutely dependent on the presence of an initiator element.

The role of the Drosophila TAK homologue dTAK during development

The TAK kinases belong to the MAPKKK group and have been implicated in a variety of signaling events. Originally described as a TGF-beta activated kinase (TAK) it has, however, subsequently been demonstrated to signal through p38, Jun N-terminal kinase (JNK) and Nemo types of MAP kinases, and the NFkappaB inducing kinase. Despite these multiple proposed functions, the in vivo role of TAK family kinases remains unclear. Here we report the isolation and genetic characterization of the Drosophila TAK homologue (dTAK). By employing overexpression and double-stranded RNA interference (RNAi) techniques we have analyzed its function during embryogenesis and larval development. Overexpression of dTAK in the embryonic epidermis is sufficient to induce the transcription of the JNK target genes decapentaplegic and puckered. Furthermore, overexpression of dominant negative (DN) or wild-type forms of dTAK in wing and eye imaginal discs, respectively, results in defects in thorax closure and ommatidial planar polarity, two well described phenotypes associated with JNK signaling activity. Surprisingly, RNAi and DN-dTAK expression studies in the embryo argue for a differential requirement of dTAK during developmental processes controlled by JNK signaling, and a redundant or minor role of dTAK in dorsal closure. In addition, dTAK-mediated activation of JNK in the Drosophila eye imaginal disc leads to an eye ablation phenotype due to ectopically induced apoptotic cell death. Genetic analyses in the eye indicate that dTAK can also act through the p38 and Nemo kinases in imaginal discs. Our results suggest that dTAK can act as a JNKKK upstream of JNK in multiple contexts and also other MAPKs in the eye. However, the loss-of-function RNAi studies indicate that it is not strictly required and thus either redundant or playing only a minor role in the context of embryonic dorsal closure.

Drosophila Src regulates anisotropic apical surface growth to control epithelial tube size

Networks of epithelial and endothelial tubes are essential for the function of organs such as the lung, kidney and vascular system. The sizes and shapes of these tubes are highly regulated to match their individual functions. Defects in tube size can cause debilitating diseases such as polycystic kidney disease and ischaemia. It is therefore critical to understand how tube dimensions are regulated. Here we identify the tyrosine kinase Src as an instructive regulator of epithelial-tube length in the Drosophila tracheal system. Loss-of-function Src42 mutations shorten tracheal tubes, whereas Src42 overexpression elongates them. Surprisingly, Src42 acts distinctly from known tube-size pathways and regulates both the amount of apical surface growth and, with the conserved formin dDaam, the direction of growth. Quantitative three-dimensional image analysis reveals that Src42- and dDaam-mutant tracheal cells expand more in the circumferential than the axial dimension, resulting in tubes that are shorter in length—but larger in diameter—than wild-type tubes. Thus, Src42 and dDaam control tube dimensions by regulating the direction of anisotropic growth, a mechanism that has not previously been described.

Combinatorial signaling by the Frizzled/PCP and Egfr-pathways during planar cell polarity establishment in the Drosophila eye

Frizzled (Fz)/PCP signaling regulates planar, vectorial orientation of cells or groups of cells within whole tissues. Although Fz/PCP signaling has been analyzed in several contexts, little is known about nuclear events acting downstream of Fz/PCP signaling in the R3/R4 cell fate decision in the Drosophila eye or in other contexts. Here we demonstrate a specific requirement for Egfr-signaling and the transcription factors Fos (AP-1), Yan and Pnt in PCP dependent R3/R4 specification. Loss and gain-of-function assays suggest that the transcription factors integrate input from Fz/PCP and Egfr-signaling and that the ETS factors Pnt and Yan cooperate with Fos (and Jun) in the PCP-specific R3/R4 determination. Our data indicate that Fos (either downstream of Fz/PCP signaling or parallel to it) and Yan are required in R3 to specify its fate (Fos) or inhibit R4 fate (Yan) and that Egfr-signaling is required in R4 via Pnt for its fate specification. Taken together with previous work establishing a Notch-dependent Su(H) function in R4, we conclude that Fos, Yan, Pnt, and Su(H) integrate Egfr, Fz, and Notch signaling input in R3 or R4 to establish cell fate and ommatidial polarity.

Characterization of the Biochemical Properties and Biological Function of the Formin Homology Domains of Drosophila DAAM

We 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.

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. Here, we have 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, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our 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 our Drosophila model system. Using combinatorial genetics, we demonstrate 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. Our 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.

Drosophila Rab23 Is Involved in the Regulation of the Number and Planar Polarization of the Adult Cuticular Hairs

The planar coordination of cellular polarization is an important, yet not well-understood aspect of animal development. In a screen for genes regulating planar cell polarization in Drosophila, we identified Rab23, encoding a putative vesicular trafficking protein. Mutations in the Drosophila Rab23 ortholog result in abnormal trichome orientation and the formation of multiple hairs on the wing, leg, and abdomen. We show that Rab23 is required for hexagonal packing of the wing cells. We found that Rab23 is able to associate with the proximally accumulated Prickle protein, although Rab23 itself does not seem to display a polarized subcellular distribution in wing cells, and it appears to play a relatively subtle role in cortical polarization of the polarity proteins. The absence of Rab23 leads to increased actin accumulation in the subapical region of the pupal wing cells that fail to restrict prehair initiation to a single site. Rab23 acts as a dominant enhancer of the weak multiple hair phenotype exhibited by the core polarity mutations, whereas the Rab23 homozygous mutant phenotype is sensitive to the gene dose of the planar polarity effector genes. Together, our data suggest that Rab23 contributes to the mechanism that inhibits hair formation at positions outside of the distal vertex by activating the planar polarity effector system.