Fatty acid ß-oxidation is required for the differentiation of larval hematopoietic progenitors in Drosophila.

Cell-intrinsic and extrinsic signals regulate the state and fate of stem and progenitor cells. Recent advances in metabolomics illustrate that various metabolic pathways are also important in regulating stem cell fate. However, our understanding of the metabolic control of the state and fate of progenitor cells is in its infancy. Using Drosophila hematopoietic organ: lymph gland, we demonstrate that Fatty Acid Oxidation (FAO) is essential for the differentiation of blood cell progenitors. In the absence of FAO, the progenitors are unable to differentiate and exhibit altered histone acetylation. Interestingly, acetate supplementation rescues both histone acetylation and the differentiation defects. We further show that the CPT1/whd (withered), the rate-limiting enzyme of FAO, is transcriptionally regulated by Jun-Kinase (JNK), which has been previously implicated in progenitor differentiation. Our study thus reveals how the cellular signaling machinery integrates with the metabolic cue to facilitate the differentiation program.

Stem cells are special precursor cells, found in all animals from flies to humans, that can give rise to all the mature cell types in the body. Their job is to generate supplies of new cells wherever these are needed. This is important because it allows damaged or worn-out tissues to be repaired and replaced by fresh, healthy cells. As part of this renewal process, stem cells generate pools of more specialized cells, called progenitor cells. These can be thought of as half-way to maturation and can only develop in a more restricted number of ways. For example, so-called myeloid progenitor cells from humans can only develop into a specific group of blood cell types, collectively termed the myeloid lineage. Fruit flies, like many other animals, also have several different types of blood cells. The fly's repertoire of blood cells is very similar to the human myeloid lineage, and these cells also develop from the fly equivalent of myeloid progenitor cells. These progenitors are found in a specialized organ in fruit fly larvae called the lymph gland, where the blood forms. These similarities between fruit flies and humans mean that flies are a good model to study how myeloid progenitor cells mature. A lot is already known about the molecules that signal to progenitor cells how and when to mature. However, the role of metabolism ­ the chemical reactions that process nutrients and provide energy inside cells ­ is still poorly understood. Tiwari et al. set out to identify which metabolic reactions myeloid progenitor cells require and how these reactions might shape the progenitors' development into mature blood cells. The experiments in this study used fruit fly larvae that had been genetically altered so that they could no longer perform key chemical reactions needed for the breakdown of fats. In these mutant larvae, the progenitors within the lymph gland could not give rise to mature blood cells. This showed that myeloid progenitor cells need to be able to break down fats in order to develop properly. These results highlight a previously unappreciated role for metabolism in controlling the development of progenitor cells. If this effect also occurs in humans, this knowledge could one day help medical researchers engineer replacement tissues in the lab, or even increase our own bodies' ability to regenerate blood, and potentially other organs.

A method of permeabilization of Drosophila embryos for assays of small molecule activity.

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 °C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis.

Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC). Highly conserved orthologs of BTBBRC and all four ZBRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrupt, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, which revealed that the ZBRC clade includes Zabrupt. All four ZBRC domains, including Z4BRC, which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated ZBRC exons even when they contribute to common developmental processes.

Par-1 and PP2A: Yin-Yang of Bazooka localization.

Apical basal cell polarity is a fundamental feature of all epithelial cells. Identification of the genes involved in the polarization of epithelial cells has begun to reveal the mechanisms underlying the establishment and maintenance of cell polarity. An important issue is to understand the molecular basis for localization of cell polarity proteins in the context of the developing organism. Bazooka (Baz, Drosophila homolog of Par-3) plays a crucial role in organizing cell polarity in several different tissues. In the ovarian follicle epithelium, Par-1 protein kinase regulates Baz localization to the apical cell cortex by excluding phosphorylated Baz from the lateral region. In photoreceptor cells of retinal epithelium, Baz is targeted to the adherens junction (AJ) instead of the apical domain. Our study suggests that in photoreceptors, Par-1 blocks the localization of Baz to AJ whereas protein phosphatase 2A (PP2A) promotes Baz localization by antagonizing the Par-1 effects. In this extra view, we provide a brief overview and perspective of our findings on the antagonistic function of Par-1 and PP2A in Baz localization during photoreceptor morphogenesis.

Functional conservation of zinc-finger homeodomain gene zfh1/SIP1 in Drosophila heart development.

Comparative genetic studies of diverse animal model systems have revealed that similar developmental mechanisms operate across the Metazoa. In many cases, the genes from one organism can functionally replace homologues in other phyla, a result consistent with a high degree of evolutionarily conserved gene function. We investigated functional conservation among the Drosophila zinc-finger homeodomain protein 1 (zfh1) and its mouse functional homologue Smad-interacting protein 1 (SIP1). Northern blot analyses of SIP1 expression patterns detected three novel variants (8.3, 2.7, and 1.9 kb) in addition to the previously described 5.3 kb SIP1 transcript. The two shorter novel SIP1 transcripts were encountered only in developing embryos and both lacked zinc-finger clusters or homeodomain regions. The SIP1 transcripts showed complex embryonic expression patterns consistent with that observed for Drosophila zfh1. They were highly expressed in the developing nervous systems and in a number of mesoderm-derived tissues including lungs, heart, developing myotomes, skeletal muscle, and visceral smooth muscle. The expression of the mammalian 5.3 kb SIP1 transcript in Drosophila zfh1 null mutant embryos completely restored normal heart development in the fly, demonstrating their functional equivalence in cardiogenic pathways. Our present data, together with the previously described heart defects associated with both SIP1 and Drosophila zfh1 mutations, solidify the conclusion that the zfh1 family members participate in an evolutionary conserved program of metazoan cardiogenesis.

Drosophila Dok is required for embryonic dorsal closure.

Embryonic dorsal closure (DC) in Drosophila is a series of morphogenetic movements involving the bilateral dorsal movement of the epidermis (cell stretching) and dorsal suturing of the leading edge (LE) cells to enclose the viscera. The Syk family tyrosine kinase Shark plays a crucial role in this Jun amino-terminal kinase (JNK)-dependent process, where it acts upstream of JNK in LE cells. Using a yeast two-hybrid screen, the unique Drosophila homolog of the downstream of kinase (Dok) family, Ddok, was identified by its ability to bind Shark SH2 domains in a tyrosine phosphorylation-dependent fashion. In cultured S2 embryonic cells, Ddok tyrosine phosphorylation is Src dependent; Shark associates with Ddok and Ddok localizes at the cell cortex, together with a portion of the Shark protein. The embryonic expression pattern of Ddok resembles the expression pattern of Shark. Ddok loss-of-function mutant (Ddok(PG155)) germ-line clones possess DC defects, including the loss of JNK-dependent expression of dpp mRNA in LE cells, and decreased epidermal F-actin staining and LE actin cable formation. Epistatic analysis indicates that Ddok functions upstream of shark to activate JNK signaling during DC. Consistent with these observations, Ddok mutant embryos exhibit decreased levels of tyrosine phosphorylated Shark at the cell periphery of LE and epidermal cells. As there are six mammalian Dok family members that exhibit some functional redundancy, analysis of the regulation of DC by Ddok is expected to provide novel insights into the function of the Dok adapter proteins.

molting defective is required for ecdysone biosynthesis.

20-hydroxyecdysone was discovered as the major biologically active insect steroid hormone half a century ago, yet much remains to be learned about its biosynthesis and its activities. 20-hydroxyecdysone controls many biological processes, including progression between larval stages, entry to pupariation and metamorphosis. A number of genes required for 20-hydroxyecdysone production have been identified, including those encoding enzymes that mediate four of the late steps of biosynthesis. A second smaller group of low ecdysone mutants do not encode enzymes. Here, we report identification of one such gene, which we call molting defective, on the basis of its lethal phenotype. molting defective encodes a nuclear zinc finger protein required for ecdysone biosynthesis.

YY1 DNA binding and PcG recruitment requires CtBP.

We found that mammalian Polycomb group (PcG) protein YY1 can bind to Polycomb response elements in Drosophila embryos and can recruit other PcG proteins to DNA. PcG recruitment results in deacetylation and methylation of histone H3. In a CtBP mutant background, recruitment of PcG proteins and concomitant histone modifications do not occur. Surprisingly, YY1 DNA binding in vivo is also ablated. CtBP mutation does not result in YY1 degradation or transport from the nucleus, suggesting a mechanism whereby YY1 DNA binding ability is masked. These results reveal a new role for CtBP in controlling YY1 DNA binding and recruitment of PcG proteins to DNA.

The Drosophila selenoprotein BthD is required for survival and has a role in salivary gland development.

Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability.

The T-box-encoding Dorsocross genes function in amnioserosa development and the patterning of the dorsolateral germ band downstream of Dpp.

Dpp signals are responsible for establishing a variety of cell identities in dorsal and lateral areas of the early Drosophila embryo, including the extra-embryonic amnioserosa as well as different ectodermal and mesodermal cell types. Although we have a reasonably clear picture of how Dpp signaling activity is modulated spatially and temporally during these processes, a better understanding of how these signals are executed requires the identification and characterization of a collection of downstream genes that uniquely respond to these signals. In the present study, we describe three novel genes, Dorsocross1, Dorsocross2 and Dorsocross3, which are expressed downstream of Dpp in the presumptive and definitive amnioserosa, dorsal ectoderm and dorsal mesoderm. We show that these genes are good candidates for being direct targets of the Dpp signaling cascade. Dorsocross expression in the dorsal ectoderm and mesoderm is metameric and requires a combination of Dpp and Wingless signals. In addition, a transverse stripe of expression in dorsoanterior areas of early embryos is independent of Dpp. The Dorsocross genes encode closely related proteins of the T-box domain family of transcription factors. All three genes are arranged in a gene cluster, are expressed in identical patterns in embryos, and appear to be genetically redundant. By generating mutants with a loss of all three Dorsocross genes, we demonstrate that Dorsocross gene activity is crucial for the completion of differentiation, cell proliferation arrest, and survival of amnioserosa cells. In addition, we show that the Dorsocross genes are required for normal patterning of the dorsolateral ectoderm and, in particular, the repression of wingless and the ladybird homeobox genes within this area of the germ band. These findings extend our knowledge of the regulatory pathways during amnioserosa development and the patterning of the dorsolateral embryonic germ band in response to Dpp signals.

Transcription factor YY1 functions as a PcG protein in vivo.

Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, pleiohomeotic (PHO). YY1 might therefore be a vertebrate PcG protein. We used Drosophila embryo and larval/imaginal disc transcriptional repression systems to determine whether YY1 repressed transcription in a manner consistent with PcG function in vivo. YY1 repressed transcription in Drosophila, and this repression was stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablated YY1 repression, and YY1 could substitute for PHO in repressing transcription in wing imaginal discs. YY1 functionally compensated for loss of PHO in pho mutant flies and partially corrected mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, we found that YY1, as well as Polycomb, required the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1.

atonal is required for exoskeletal joint formation in the Drosophila auditory system.

Hearing relies on the delicate arrangement of mechanoreceptor neurones and an acoustomechanical interface. The concerted action of these neural and non-neural components is essential to audition, raising the question of whether they also develop in a concerted way. Drosophila hears with its antennae. A specialized antennal joint allows the distal part of the antenna to vibrate in response to sound and, thus, to serve as the sound receiver. This receiver's vibration is transduced by a chordotonal sense organ (CHO) that is closely associated with the joint. Here, we report that atonal (ato), the proneural gene for CHOs, is required for the formation of this antennal joint. Biophysical measurements in hemi- and homozygous ato(1) mutant flies show that, in addition to eliminating the auditory CHO, loss of ato function makes the antennal receiver insensitive to sound, impairing its auditory function. Anatomically, the cause for this mechanical effect resides in the deprivation of mobile exoskeletal joint structures. Hence, ato, the homologue of mouse Math1, is required for the formation of both the auditory CHO and joint, providing a genetic link between the very neural and exoskeletal components that together transform fly antennae into ears.

Toxicity of argemone oil: effect on hsp70 expression and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9.

The effect of argemone oil on hsp70 expression and tissue damage was investigated by studying beta-galactosidase activity, Western blotting and hybridization, and trypan blue staining in the larval tissues of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Different concentrations of argemone oil were mixed with food and third-instar larvae were allowed to feed on them for different time intervals (2, 4, 24, and 48 h). Argemone oil was found to induce hsp70 even in the lowest concentration of the adulterant while maximum tissue damage was observed in the higher two treatment groups. Malpighian tubules and midgut tissue reflected maximum damage as evidenced by both high beta-galactosidase activity and trypan blue staining in these tissues. A prior temperature shock treatment to the larvae was enough to protect the larvae from argemone oil-induced tissue damage as evidenced by little or no trypan blue staining. The present study suggests the cytotoxic potential of argemone oil and further strengthens the evidence for the use of hsp70 as a biomarker in risk assessment.

Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts.

Establishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs. A 5' phosphate on the target-complementary strand of the siRNA duplex is required for siRNA function, suggesting that cells check the authenticity of siRNAs and license only bona fide siRNAs to direct target RNA destruction.

The protein 4.1, ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a cytoplasmic component of the septate junction, provides functions essential for embryonic development and imaginal cell proliferation.

Coracle is a member of the Protein 4.1 superfamily of proteins, whose members include Protein 4.1, the Neurofibromatosis 2 tumor suppressor Merlin, Expanded, the ERM proteins, protein tyrosine phosphatases, and unconventional myosins. Recent evidence suggests that members of this family participate in cell signaling events, including those that regulate cell proliferation and the cytoskeleton. Previously, we demonstrated that Coracle protein is localized to the septate junction in epithelial cells and is required for septate junction integrity. Loss of coracle function leads to defects in embryonic development, including failure in dorsal closure, and to proliferation defects. In addition, we determined that the N-terminal 383 amino acids define an essential functional domain possessing membrane-organizing properties. Here we investigate the full range of functions provided by this highly conserved domain and find that it is sufficient to rescue all embryonic defects associated with loss of coracle function. In addition, this domain is sufficient to rescue the reduced cell proliferation defect in imaginal discs, although it is incapable of rescuing null mutants to the adult stage. This result suggests the presence of a second functional domain within Coracle, a notion supported by molecular characterization of a series of coracle alleles.

Molecular analysis of a novel Drosophila diacylglycerol kinase, DGKepsilon.

Diacylglycerol kinase plays a central role in the metabolism of diacylglycerol by converting diacylglycerol into phosphatidic acid thus initiating resynthesis of phosphatidylinositols. Diacylglycerol is a known second messenger reversibly activating protein kinase C. In addition, diacylglycerol is a potential precursor for polyunsaturated fatty acids. We describe the identification and molecular analysis of a novel type III Drosophila diacylglycerol kinase isoform, DGKepsilon. Drosophila DGKepsilon is mapped to the cytological position 49C1-3. DGKepsilon mRNA is 1.9 kb in length and is broadly distributed throughout development in different cells, primordia and organs, including testes. In embryogenesis, the transcripts are enriched in the cells, which are in S-phase or undergoing endoreplication. Comparison of the Drosophila DGKepsilon with the human homologue revealed that the first zinc finger-like motif is specific for the type III isoform. Although the testis-specific diacylglycerol kinase activity is dependent upon the dose of DGKepsilon gene, the deletion of DGKepsilon does not modulate the total cellular diacylglycerol level. In spite of a proposed key role of diacylglycerol kinase in termination of the diacylglycerol signal, overexpression of a DGKepsilon transgene in flies under the control of a yeast upstream activating sequence promoter does not disrupt normal development in Drosophila.

Hmx: an evolutionary conserved homeobox gene family expressed in the developing nervous system in mice and Drosophila.

Three homeobox genes, one from Drosophila melanogaster (Drosophila Hmx gene) and two from mouse (murine Hmx2 and Hmx3) were isolated and the full-length cDNAs and corresponding genomic structures were characterized. The striking homeodomain similarity encoded by these three genes to previously identified genes in sea urchin, chick and human, as well as the recently cloned murine Hmx1 gene, and the low homology to other homeobox genes indicate that the Hmx genes comprise a novel gene family. The widespread existence of Hmx genes in the animal kingdom suggests that this gene family is of ancient origin. Drosophila Hmx was mapped to the 90B5 region of Chromosome 3 and at early embryonic stages is primarily expressed in distinct areas of the neuroectoderm and subsets of neuroblasts in the developing fly brain. Later its expression continues in rostral areas of the brain in a segmented pattern, suggesting a putative role in the development of the Drosophila central nervous system. During evolution, mouse Hmx2 and Hmx3 may have retained a primary function in central nervous system development as suggested by their expression in the postmitotic cells of the neural tube, as well as in the hypothalamus, the mesencephalon, metencephalon and discrete regions in the myelencephalon during embryogenesis. Hmx1 has diverged from other Hmx members by its expression in the dorsal root, sympathetic and vagal nerve (X) ganglia. Aside from their expression in the developing nervous system, all three Hmx genes display expression in sensory organ development, and in the adult uterus. Hmx2 and Hmx3 show identical expression in the otic vesicle, whereas Hmx1 is strongly expressed in the developing eye. Transgenic mouse lines were generated to examine the DNA regulatory elements controlling Hmx2 and Hmx3. Transgenic constructs spanning more than 31 kb of genomic DNA gave reproducible expression patterns in the developing central and peripheral nervous systems, eye, ear and other tissues, yet failed to fully recapitulate the endogenous expression pattern of either Hmx2 or Hmx3, suggesting both the presence and absence of certain critical enhancers in the transgenes, or the requirement of proximal enhancers to work synergistically.

Drosophila arginase is produced from a nonvital gene that contains the elav locus within its third intron.

A Drosophila gene encoding a 351-amino acid-long predicted arginase (40% identity with vertebrate arginases) is reported. Interestingly, the third intron of the arginase gene includes the elav locus, whose coding sequence is on the complementary DNA strand to that of the arginase. Terrestrial vertebrates produce two arginases from duplicated genes. One form, essentially present in the liver, is a key enzyme of the urea cycle and eliminates excess ammonia through the excretion of urea. The function of the extrahepatic arginase, more ubiquitous, is not well understood. In macrophages, arginase competes with nitric-oxide synthase, which converts arginine into nitric oxide. Most organisms, including insects, produce only one type of arginase, whose function is not centered on ammonia detoxification. A Drosophila cDNA encoding a predicted arginase was isolated. It produces a 1.3-kilobase transcript present with highest levels toward the end of embryogenesis and thereafter. During embryogenesis, the arginase transcripts localize to the fat body. The first mutant allele of the Drosophila arginase gene was identified. It is predicted to produce a 199-amino acid-long C-terminally truncated protein, likely to be inactive. Preliminary characterization of the mutation shows that this recessive allele causes a developmental delay but does not affect viability.

The Yin-Yang of TCF/beta-catenin signaling.

Wingless/Wnt signaling directs cell-fate choices during embryonic development. In Drosophila, Wingless signaling mediates endoderm induction and the establishment of segment polarity in the developing embryo. The fly Wingless cascade is strikingly similar to the vertebrate Wnt signaling pathway, which controls a number of key developmental decisions such as dorsal-ventral patterning in Xenopus. Factors of the TCF/LEF HMG domain family (Tcfs) have recently been established as the downstream effectors of the Wingless/Wnt signal transduction pathways. Upon Wingless/Wnt signaling, a cascade is initiated that results in the accumulation of cytoplasmic beta-catenin (or its fly homolog, Armadillo). There is also a concomitant translocation of beta-catenin/Armadillo to the nucleus, where it interacts with a specific sequence motif at the N terminus of Tcfs to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes including Siamois and Nodal related gene-3 in Xenopus, engrailed and Ultrabithorax in Drosophila via the sequence-specific HMG box, and mediates their transcriptional activation by virtue of transactivation domains contributed by beta-catenin/Armadillo. In the absence of Wingless/Wnt signals, a key negative regulator of the pathway, GSK3 beta, is activated, which mediates the downregulation of cytoplasmic beta-catenin/Armadillo via the ubiquitin-proteasome pathway. In the absence of nuclear beta-catenin, the Tcfs recruit the corepressor protein Groucho to the target gene enhancers and actively repress their transcription. An additional corepressor protein, CREB-binding protein (CBP), may also be involved in this repression of Tcf target gene activity. Several other proteins, including adenomatous polyposis coli (APC), GSK3 beta, and Axin/Conductin, are instrumental in the regulation of beta-catenin/Armadillo. In APC-deficient colon carcinoma cell lines, beta-catenin accumulates and is constitutively complexed with nuclear Tcf-4. A proportion of APC wild-type colon carcinomas and melanomas also contains constitutive nuclear Tcf-4/beta-catenin complexes as a result of dominant mutations in the N terminus of beta-catenin that render it insensitive to downregulation by APC, GSK3 beta, and Axin/Conductin. This results in the unregulated expression of Tcf-4 target genes such as c-myc. Based on the established role for Tcf-4 in maintaining intestinal stem cells it is likely that deregulation of c-myc expression as a result of constitutive Tcf-4/beta-catenin activity promotes uncontrolled intestinal cell proliferation. This would readily explain the formation of intestinal polyps during colon carcinogenesis. Similar mechanisms leading to deregulation of Tcf target gene activity are likely to be involved in melanoma and other forms of cancer.