The Drosophila planar polarity gene multiple wing hairs directly regulates the actin cytoskeleton.

The evolutionarily conserved frizzled/starry night (fz/stan) pathway regulates planar cell polarity (PCP) in vertebrates and invertebrates. This pathway has been extensively studied in the Drosophila wing, where it is manifested by an array of distally pointing cuticular hairs. Using in vivo imaging we found that, early in hair growth, cells have multiple actin bundles and hairs that subsequently fuse into a single growing hair. The downstream PCP gene multiple wing hairs (mwh) plays a key role in this process and acts to antagonize the actin cytoskeleton. In mwh mutants hair initiation is not limited to a small region at the distal edge of pupal wing cells as in wild type, resulting in multiple hairs with aberrant polarity. Extra actin bundles/hairs are formed and do not completely fuse, in contrast to wild type. As development proceeded additional hairs continued to form, further increasing hair number. We identified a fragment of Mwh with in vivo rescue activity and that bound and bundled F-actin filaments and inhibited actin polymerization in in vitro actin assays. The loss of these activities can explain the mwh mutant phenotype. Our data suggest a model whereby, prior to hair initiation, proximally localized Mwh inhibits actin polymerization resulting in polarized activation of the cytoskeleton and hair formation on the distal side of wing cells. During hair growth Mwh is found in growing hairs, where we suggest it functions to promote the fusion of actin bundles and inhibit the formation of additional actin bundles that could lead to extra hairs.

The diaphanous gene of Drosophila interacts antagonistically with multiple wing hairs and plays a key role in wing hair morphogenesis.

The Drosophila wing is covered by an array of distally pointing hairs that has served as a key model system for studying planar cell polarity (PCP). The adult cuticular hairs are formed in the pupae from cell extensions that contain extensive actin filaments and microtubules. The importance of the actin cytoskeleton for hair growth and morphogenesis is clear from the wide range of phenotypes seen in mutations in well-known actin regulators. Formin proteins promote the formation of long actin filaments of the sort thought to be important for hair growth. We report here that the formin encoding diaphanous (dia) gene plays a key role in hair morphogenesis. Both loss of function mutations and the expression of a constitutively active Dia led to cells forming both morphologically abnormal hairs and multiple hairs. The conserved frizzled (fz)/starry night (stan) PCP pathway functions to restrict hair initiation and activation of the cytoskeleton to the distal most part of wing cells. It also ensures the formation of a single hair per cell. Our data suggest that the localized inhibition of Dia activity may be part of this mechanism. We found the expression of constitutively active Dia greatly expands the region for activation of the cytoskeleton and that dia functions antagonistically with multiple wing hairs (mwh), the most downstream member of the fz/stan pathway. Further we established that purified fragments of Dia and Mwh could be co-immunoprecipitated suggesting the genetic interaction could reflect a direct physical interaction.

Combover/CG10732, a novel PCP effector for Drosophila wing hair formation.

The polarization of cells is essential for the proper functioning of most organs. Planar Cell Polarity (PCP), the polarization within the plane of an epithelium, is perpendicular to apical-basal polarity and established by the non-canonical Wnt/Fz-PCP signaling pathway. Within each tissue, downstream PCP effectors link the signal to tissue specific readouts such as stereocilia orientation in the inner ear and hair follicle orientation in vertebrates or the polarization of ommatidia and wing hairs in Drosophila melanogaster. Specific PCP effectors in the wing such as Multiple wing hairs (Mwh) and Rho Kinase (Rok) are required to position the hair at the correct position and to prevent ectopic actin hairs. In a genome-wide screen in vitro, we identified Combover (Cmb)/CG10732 as a novel Rho kinase substrate. Overexpression of Cmb causes the formation of a multiple hair cell phenotype (MHC), similar to loss of rok and mwh. This MHC phenotype is dominantly enhanced by removal of rok or of other members of the PCP effector gene family. Furthermore, we show that Cmb physically interacts with Mwh, and cmb null mutants suppress the MHC phenotype of mwh alleles. Our data indicate that Cmb is a novel PCP effector that promotes to wing hair formation, a function that is antagonized by Mwh.

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of "upstream" Planar Cell Polarity proteins.

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.

The Drosophila Fry protein interacts with Trc and is highly mobile in vivo.

BACKGROUND: Cell polarity is a common feature of eukaryotic cells. The NDR kinases have been found to regulate polarized growth in both animal cells and fungi. Drosophila Tricornered is an NDR kinase that is essential for the normal polarized growth of extensions of epidermal cells and for the tiling and branching of dendrites of da sensory neurons. Tricornered function requires interacting with the large Furry protein (3479 amino acid). RESULTS: We constructed a furry (fry) transgene and established that it rescued the lethality of fry null mutations. The encoded protein was tagged at both its amino and carboxy termini and this allowed us to demonstrate that the protein existed as an uncleaved protein in vivo. We used the C terminal GFP tag to follow the protein in vivo and found it to be highly mobile. Interestingly Fry accumulated at the distal tip of growing bristles. We established that Fry and Trc could be co-immunoprecipitated from wing discs. CONCLUSIONS: The mobility of Fry in both bristles and dendrites suggests that it could function in directing/mediating the intracellular transport needed for polarized growth. Our observations that full length Fry and Trc show only partial co-localization in growing bristles while an amino terminal fragment of Fry shows close to complete co-localization with Trc suggests that the interaction between these proteins is transient and regulated.

The Drosophila planar polarity proteins inturned and multiple wing hairs interact physically and function together.

The conserved frizzled (fz) pathway regulates planar cell polarity in both vertebrate and invertebrate animals. This pathway has been most intensively studied in the wing of Drosophila, where the proteins encoded by pathway genes all accumulate asymmetrically. Upstream members of the pathway accumulate on the proximal, distal, or both cell edges in the vicinity of the adherens junction. More downstream components including Inturned and Multiple Wing Hairs accumulate on the proximal side of wing cells prior to hair initiation. The Mwh protein differs from other members of the pathway in also accumulating in growing hairs. Here we show that the two Mwh accumulation patterns are under different genetic control with the early proximal accumulation being regulated by the fz pathway and the latter hair accumulation being largely independent of the pathway. We also establish recruitment by proximally localized Inturned to be a putative mechanism for the localization of Mwh to the proximal side of wing cells. Genetically inturned (in) acts upstream of mwh (mwh) and is required for the proximal localization of Mwh. We show that Mwh can bind to and co-immunoprecipitate with Inturned. We also show that these two proteins can function in close juxtaposition in vivo. An InMwh fusion protein provided complete rescue activity for both in and mwh mutations. The fusion protein localized to the proximal side of wing cells prior to hair formation and in growing hairs as expected if protein localization is a key for the function of these proteins.

Rho1 has multiple functions in Drosophila wing planar polarity.

The frizzled (fz) signaling/signal transduction pathway controls planar cell polarity in both vertebrates and invertebrates. Previous data implicated Rho1 as a component of the fz pathway in Drosophila but it was unclear how it functioned. The existence of a G Protein Binding-Formin Homology 3 (GBD-FH3) domain in Multiple Wing Hairs, a downstream component of the pathway suggested that Rho1 might function by binding to and activating Mwh. We re-examined the role of Rho1 in wing planar polarity and found that it had multiple functions. Aberrant Rho1 activity led to changes in the number of hairs formed, changes in cell shape and F-actin and changes in cellular junctions. Experiments that utilized Rho effector loop mutations argued that these phenotypes were mediated by effects of Rho1 on the cytoskeleton and not by effects on transcription. We found strong positive genetic interactions between Rho1 and mwh, that Rho1 regulated the accumulation of Mwh protein and that these two proteins could be co-immunoprecipitated. The Mwh GBD:FH3 domain was sufficient for co-immunoprecipitation with Rho1, consistent with this domain mediating the interaction. However, further experiments showed that Rho1 function in wing differentiation was not limited to interacting with Mwh. We established by genetic experiments that Rho1 could influence hair morphogenesis in the absence of mwh and that the disruption of Rho1 activity could interfere with the zig zag accumulation pattern of upstream fz pathway proteins. Thus, our results argue that in addition to its interaction with Mwh Rho1 has functions in wing planar polarity that are parallel to and upstream of fz. The upstream function may be an indirect one and associated with the requirement for normal apical basal polarity and adherens junctions for the accumulation of PCP protein complexes.

[Screening and identification of novel genes involved in biosynthesis of ginsenoside in Panax ginseng plant].

The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation organ for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, suppression subtractive hybridization (SSH) between mRNAs of 4- and 1-year-old root tissues was performed, and a subtracted cDNA library specific to 4-year-old roots was constructed. Forty cDNA clones selected randomly from the subtracted cDNA library were sequenced. Sequence information of all clones was evaluated by Nucleotide Blast analysis in GenBank/DDBJ/EMBL. The results showed that six subtracted cDNA clones represented the novel genes (ESTs), because no sequence homology with any known sequences was found in the database. Expression in 4-year-old P. ginseng root tissues was verified by reverse Northern dot hybridization for the six clones. These six novel genes were named GBR1, GBR2, GBR3, GBR4, GBR5, and GBR6, and their Accession numbers of GenBank are AF485334, AF485335, AF485336, AF485337, AF485332, and AF485333, respectively. Finally, Northern blot analysis and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed that these six novel genes were differentially expressed in the defined development stage of P. ginseng plant roots. It is possible that their overexpression may play an important role in the ginsenoside biosynthesis. In addition, most of transcripts of all genes could also be detected in other P. ginseng plant tissues such as stem, leaf and seed. Our results provided a basis for obtaining the full-length cDNA sequences of such six novel genes, and for identifying their function involved in the biosynthesis of ginsenoside.