The Fat pathway controls both planar cell polarity (PCP) and organ growth [1, 2]. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP [1], whereas their slope can influence growth [3, 4]. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling [5-7]. Here we show that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, we show that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. We also identify a biochemical mechanism that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat.

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Here, we show that three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. We also identify dachs as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. Our observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway.

Recent studies in Drosophila melanogaster of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. Here, we characterize functional relationships among D. melanogaster tumor suppressors and identify the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded, hippo, salvador and mob as tumor suppressor also share multiple Fat pathway phenotypes but regulate Warts activity independently. Our results functionally link what had been four disparate groups of D. melanogaster tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals.

Fringe proteins are beta1,3-N-acetylglucosaminyltransferases that modulate signaling through Notch receptors by modifying O-linked fucose on epidermal growth factor domains. Fringe is highly conserved, and comparison among 18 different Fringe proteins from 11 different species identifies a core set of 84 amino acids that are identical among all Fringes. Fringe is only distantly related to other glycosyltransferases, but analysis of the predicted Drosophila proteome identifies a set of four sequence motifs shared among Fringe and other putative beta1,3-glycosyltransferases. To gain functional insight into these conserved sequences, we genetically and molecularly characterized 14 point mutations in Drosophila fringe. Most nonsense mutations act as recessive antimorphs, raising the possibility that Fringe may function as a dimer. Missense mutations identify two distinct motifs that are conserved among beta1,3-glycosyltransferases, and that can be modeled onto key motifs in the crystallographic structures of bovine beta1,4-galactosyltransferase 1 and human glucuronyltransferase I. Other missense mutations map to amino acids that are conserved among Fringe proteins, but not among other glycosyltransferases, and thus may identify structural motifs that are required for unique aspects of Fringe activity.

The conserved Drosophila tumor suppressors Fat and Expanded have both recently been implicated in regulating the activity of the Warts tumor suppressor. However, there has been disagreement as to the nature of the links among Fat, Expanded, and Warts and the significance of these links to growth control. We report here that mutations in either expanded or fat can be rescued to viability simply by overexpressing Warts, indicating that their essential function is their influence on Warts rather than reported effects on endocytosis or other pathways. These rescue experiments also separate the transcriptional from the planar cell polarity branches of Fat signaling and reveal that Expanded does not directly affect polarity. We also investigate the relationship between expanded and fat and show, contrary to prior reports, that they have additive effects on imaginal disk growth and development. Although mutation of fat can cause partial loss of Expanded protein from the membrane, mutation of fat promotes growth even when Expanded is overexpressed and accumulates at its normal subapical location. These observations argue against recent proposals that Fat acts simply as a receptor for the Hippo signaling pathway and instead support the proposal that Fat and Expanded can act in parallel to regulate Warts through distinct mechanisms.

The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. We show here that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. Our observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance our understanding of the requirement for Dco in Fat signaling.

fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. We describe studies of fringe function during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe localizes the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. Our results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establish a function for the polar cells in separating germline cysts into individual follicles.

Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.

Intracellular post-translational modifications such as phosphorylation and ubiquitylation have been well studied for their roles in regulating diverse signalling pathways, but we are only just beginning to understand how differential glycosylation is used to regulate intercellular signalling. Recent studies make clear that extracellular post-translational modifications, in the form of glycosylation, are essential for the Notch signalling pathway, and that differences in the extent of glycosylation are a significant mechanism by which this pathway is regulated.

Members of the mammalian beta1,4-galactosyltransferase family are among the best studied glycosyltransferases, but the requirements for all members of this family within an animal have not previously been determined. Here, we describe analysis of two Drosophila genes, beta4GalNAcTA (CG8536) and beta4GalNAcTB (CG14517), that are homologous to mammalian beta1,4-galactosyltransferases. Like their mammalian homologs, these glycosyltransferases use N-acetylglucosamine as an acceptor substrate. However, they transfer N-acetylgalactosamine rather than galactose. This activity, together with amino acid sequence similarity, places them among a group of recently identified invertebrate beta1,4-N-acetylgalactosaminyltransferases. To investigate the biological functions of these genes, null mutations were generated by imprecise excision of a transposable element (beta4GalNAcTA) or by gene-targeted homologous recombination (beta4GalNAcTB). Flies mutant for beta4GalNAcTA are viable and fertile but display behavioral phenotypes suggestive of essential roles for GalNAc-beta1,4-GlcNAc containing glycoconjugates in neuronal and/or muscular function. beta4GalNAcTB mutants are viable and display no evident morphological or behavioral phenotypes. Flies doubly mutant for both genes display only the behavioral phenotypes associated with mutation of beta4GalNAcTA. Thus Drosophila homologs of the mammalian beta4GalT family are essential for neuromuscular physiology or development but are not otherwise required for viability, fertility, or external morphology.

The Drosophila homeotic gene Ultrabithorax (Ubx) encodes transcriptional regulatory proteins (UBX) that specify thoracic and abdominal segmental identities. Ubx autoregulation was examined by manipulating UBX levels, both genetically and with an inducible transgene, and monitoring the effect of these manipulations on the expression of Ubx and Ubx-lacZ reporter genes. Positive autoregulation by Ubx is restricted to the visceral mesoderm, while in other tissues Ubx negatively autoregulates. In some cases, negative autoregulation stabilizes UBX levels, while in others it modulates the spatial and temporal patterns of UBX expression. This modulation of UBX expression may enable Ubx to specify distinct identities in different segments. The upstream control region of Ubx contains multiple autoregulatory elements for both positive and negative autoregulation.

Multiple mechanisms are involved in positioning and restricting specialized dorsal-ventral border cells in the Drosophila wing, including modulation of Notch signaling by Fringe, autonomous inhibition by Notch ligands, and inhibition of Notch target genes by Nubbin. Recent studies have revealed that Fringe also modulates a Notch-mediated signaling process between dorsal and ventral cells in the Drosophila eye, establishing an organizer of eye growth and patterning along the dorsal-ventral midline. Fringe-dependent modulation of Notch signaling also plays a key role in Drosophila leg segmentation and growth. Lunatic Fringe has been shown to be required for vertebrate somitogenesis, where it appears to act as a crucial link between a molecular clock and the regulation of Notch signaling.

Developing organisms may contain billions of cells destined to differentiate in numerous different ways. One strategy organisms use to simplify the orchestration of development is the separation of cell populations into distinct functional units. Our expanding knowledge of boundary formation and function in different systems is beginning to reveal general principles of this process. Fields of cells are subdivided by the interpretation of morphogen gradients, and these subdivisions are then maintained and refined by local cell-cell interactions. Sharp and stable separation between cell populations requires special mechanisms to keep cells segregated, which in many cases appear to involve the regulation of cell affinity. Once cell populations become distinct, specialized cells are often induced along the borders between them. These boundary cells can then influence the patterning of surrounding cells, which can result in progressively finer subdivisions of a tissue. Much has been learned about the signaling pathways that establish boundaries, but a key challenge for the future remains to elucidate the cellular and molecular mechanisms that actually keep cell populations separated.

In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal-ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal-ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.

Metazoan cells are exposed to a multitude of signals, which they integrate to determine appropriate developmental or physiological responses. Although the Hippo pathway was only discovered recently, and our knowledge of Hippo signal transduction is far from complete, a wealth of interconnections amongst Hippo and other signaling pathways have already been identified. Hippo signaling is particularly important for growth control, and I describe how integration of Hippo and other pathways contributes to regulation of organ growth. Molecular links between Hippo signaling and other signal transduction pathways are summarized. Different types of mechanisms for signal integration are described, and examples of how the complex interconnections between pathways are used to guide developmental and physiological growth responses are discussed. Features of Hippo signaling appear to make it particularly well suited to signal integration, including its responsiveness to cell-cell contact and the mediation of its transcriptional output by transcriptional co-activator proteins that can interact with transcription factors of other pathways.

Wing formation in Drosophila requires interactions between dorsal and ventral cells. We describe a new gene, fringe, which is expressed in dorsal cells and encodes for a novel protein that is predicted to be secreted. Wing margin formation and distal wing outgrowth can be induced by the juxtaposition of cells with and without fringe expression, whether at the normal wing margin, at the boundaries of fringe mutant clones in the dorsal wing, or at sites of fringe misexpression in the ventral wing. By contrast, both loss of fringe expression and uniform fringe expression cause wing loss. These observations suggest that fringe encodes a boundary-specific cell-signaling molecule that is responsible for dorsal-ventral cell interactions during wing development.

Notch is a key signaling protein mediating cell-fate decisions during development. In this issue, Acar et al. (2008) describe a new gene called rumi that is required for Notch signaling in Drosophila. This gene encodes an O-glucosyltransferase that attaches glucose sugars to serine residues in the multiple EGF domains of the extracellular region of Notch. This modification by Rumi likely influences Notch folding and trafficking.

After the onset of gastrulation, the Drosophila germband undergoes a morphological change in which its length along the anterior-posterior axis increases over two-and-a-half fold while its width along the dorsal-ventral axis simultaneously narrows. The behavior of individual cells during germband extension was investigated by epi-illumination and time-lapse video microscopy of living embryos. Cells intercalate between their dorsal and ventral neighbors during extension, increasing the number of cells along the anterior-posterior axis while decreasing the number of cells along the dorsal-ventral axis. Mutations that reduce segmental subdivision of the embryo along the anterior-posterior axis decrease both germband extension and its associated cell intercalation. In contrast, cell intercalation and germband extension are still detected in embryos that lack dorsal-ventral polarity. Characterization of germband extension and cell intercalation in mutant embryos with altered segmentation gene expression indicates that these processes are regionally autonomous and are dependent upon the establishment of striped expression patterns for certain pair-rule genes. Based on these observations, we propose a model for germband extension in which cell intercalation results from the establishment of adhesive differences between stripes of cells by pair-rule genes.

Ultrabithorax (Ubx) is a Drosophila homeotic gene that determines the segmental identities of parts of the thorax and abdomen. Appropriate Ubx transcription requires a long upstream control region (UCR) that is defined genetically by the bithoraxoid (bxd) and postbithorax (pbx) subfunction mutations. We have directly analyzed UCR functions by the examination of beta-galactosidase expression in flies containing Ubx-lacZ fusion genes. 35 kb of UCR DNA confers upon beta-galactosidase an expression pattern that closely parallels normal Ubx expression throughout development. In contrast, 22 kb of UCR DNA confers fewer features of normal Ubx expression, and with 5 kb of UCR DNA the expression pattern has no resemblance to Ubx expression except in the visceral mesoderm. We have also shown that bxd chromosome breakpoint mutants form a comparable 5' deletion series in which the severity of the effect on Ubx expression correlates with the amount of upstream DNA remaining in the mutant. In Ubx-lacZ fusions containing 22 kb of UCR DNA, and in comparable bxd mutants, there is a persistent pair-rule pattern of metameric expression in early development, demonstrating that there are distinct mechanisms with different sequence requirements for the initial activation of Ubx in different metameres. The correction of this pair-rule pattern later in embryogenesis shows that there are also distinct mechanisms for the activation of Ubx at different times during development.

The atypical cadherin Fat acts as a receptor for a signaling pathway that regulates growth, gene expression, and planar cell polarity. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. We show that four-jointed encodes a protein kinase that phosphorylates serine or threonine residues within extracellular cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi and is the first molecularly defined kinase that phosphorylates protein domains destined to be extracellular. An acidic sequence motif (Asp-Asn-Glu) within Four-jointed was essential for its kinase activity in vitro and for its biological activity in vivo. Our results indicate that Four-jointed regulates Fat signaling by phosphorylating cadherin domains of Fat and Dachsous as they transit through the Golgi.

Raine syndrome is caused by mutations in FAM20C, which had been reported to encode a secreted component of bone and teeth. We found that FAM20C encodes a Golgi-localized protein kinase, distantly related to the Golgi-localized kinase Four-jointed. Drosophila also encode a Golgi-localized protein kinase closely related to FAM20C. We show that FAM20C can phosphorylate secreted phosphoproteins, including both Casein and members of the SIBLING protein family, which modulate biomineralization, and we find that FAM20C phosphorylates a biologically active peptide at amino acids essential for inhibition of biomineralization. We also identify autophosphorylation of FAM20C, and characterize parameters of FAM20C's kinase activity, including its Km, pH and cation dependence, and substrate specificity. The biochemical properties of FAM20C match those of an enzymatic activity known as Golgi casein kinase. Introduction of point mutations identified in Raine syndrome patients into recombinant FAM20C impairs its normal localization and kinase activity. Our results identify FAM20C as a kinase for secreted phosphoproteins and establish a biochemical basis for Raine syndrome.

The formation of boundaries between groups of cells is a universal feature of metazoan development. Drosophila fringe modulates the activation of the Notch signal transduction pathway at the dorsal-ventral boundary of the wing imaginal disc. Three mammalian fringe-related family members have been cloned and characterized: Manic, Radical and Lunatic Fringe. Expression studies in mouse embryos support a conserved role for mammalian Fringe family members in participation in the Notch signaling pathway leading to boundary determination during segmentation. In mammalian cells, Drosophila fringe and the mouse Fringe proteins are subject to posttranslational regulation at the levels of differential secretion and proteolytic processing. When misexpressed in the developing Drosophila wing imaginal disc the mouse Fringe genes exhibit conserved and differential effects on boundary determination.