Nuria Paricio

Dishevelled Activates JNK and Discriminates between JNK Pathways in Planar Polarity and wingless Signaling

Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling. Dishevelled is required in both signaling pathways. Here, we use genetic and overexpression assays to show that Dishevelled activates JNK cascades. Rescue analysis reveals different protein domain requirements in Dishevelled for the two pathways; the C-terminal DEP domain is essential to rescue planar polarity defects and induce JNK signaling. Furthermore, the planar polarity-specific dsh1 allele is mutated in the DEP domain. Our results indicate that different Wnt/Fz signals activate distinct intracellular pathways, and Dishevelled discriminates among them by distinct domain interactions.

The Drosophila STE20‐like kinase Misshapen is required downstream of the Frizzled receptor in planar polarity signaling

The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss‐ and gain‐of‐function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20‐like kinase in Fz/Dsh‐mediated signaling. This demonstrates that seven‐pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun‐N‐terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen‐activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn.

Drosophila as a Model of Wound Healing and Tissue Regeneration in Vertebrates

Understanding the molecular basis of wound healing and regeneration in vertebrates is one of the main challenges in biology and medicine. This understanding will lead to medical advances allowing accelerated tissue repair after wounding, rebuilding new tissues/organs and restoring homeostasis. Drosophila has emerged as a valuable model for studying these processes because the genetic networks and cytoskeletal machinery involved in epithelial movements occurring during embryonic dorsal closure, larval imaginal disc fusion/regeneration, and epithelial repair are similar to those acting during wound healing and regeneration in vertebrates. Recent studies have also focused on the use of Drosophila adult stem cells to maintain tissue homeostasis. Here, we review how Drosophila has contributed to our understanding of these processes, primarily through live‐imaging and genetic tools that are impractical in mammals. Furthermore, we highlight future research areas where this insect may provide novel insights and potential therapeutic strategies for wound healing and regeneration. Developmental Dynamics 240:2739–2404, 2011.

Drosophila Models of Parkinson's Disease: Discovering Relevant Pathways and Novel Therapeutic Strategies

Parkinsons disease (PD) is the second most common neurodegenerative disorder and is mainly characterized by the selective and progressive loss of dopaminergic neurons, accompanied by locomotor defects. Although most PD cases are sporadic, several genes are associated with rare familial forms of the disease. Analyses of their function have provided important insights into the disease process, demonstrating that three types of cellular defects are mainly involved in the formation and/or progression of PD: abnormal protein aggregation, oxidative damage, and mitochondrial dysfunction. These studies have been mainly performed in PD models created in mice, fruit flies, and worms. Among them, Drosophila has emerged as a very valuable model organism in the study of either toxin-induced or genetically linked PD. Indeed, many of the existing fly PD models exhibit key features of the disease and have been instrumental to discover pathways relevant for PD pathogenesis, which could facilitate the development of therapeutic strategies.

Molecular Characterization and Evolution of the Protein Phosphatase 2A B′ Regulatory Subunit Family in Plants

Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation events in plants and other organisms. PP2A proteins are oligomeric complexes constituted by a catalytic subunit and several regulatory subunits that modulate the activity of these phosphatases. The analysis of the complete genome of Arabidopsis allowed us to characterize four novel genes, AtB′ε, AtB′ζ,AtB′η, and AtB′θ, belonging to the PP2A B′ regulatory subunit family. Because four genes of this type had been described previously, this family is composed of eight members. Reverse transcriptase-polymerase chain reaction experiments showed thatAtB′ε mRNAs are present in all Arabidopsis tissues analyzed, and their levels do not respond significantly to heat stress. Expressed sequence tags corresponding to AtB′ζ,AtB′η, and AtB′θ have been identified, indicating that the new genes are actively transcribed. The genomic organization of this family of PP2A regulatory subunits is reported, as well as its chromosomal location. An extensive survey of the family has been carried out in plants, characterizing B′ subunits in a number of different species, and performing a phylogenetic study that included several B′ regulatory proteins from animals. Our results indicate that the animal and plant proteins have evolved independently, that there is a relationship between the number of B′ isoforms and the complexity of the organism, and that there are at least three main subfamilies of regulatory subunits in plants, which we have named α, η, and κ.

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.

Antioxidant compound supplementation prevents oxidative damage in a Drosophila model of Parkinson's disease.

Loss-of-function mutations in the DJ-1 gene are linked to rare autosomal recessive forms of parkinsonism. In Drosophila, two DJ-1 orthologs have been identified, DJ-1α and DJ-1β. Several studies have shown that DJ-1β mutant flies are viable and fertile but exhibit age-dependent locomotor defects, shortened life span, and enhanced sensitivity to toxins that induce oxidative stress response compared to control flies. We also demonstrated that long-term dietary supplementation with antioxidant compounds was effective at increasing life-span values of DJ-1β mutants. These results, together with high levels of oxidative stress markers detected in newly eclosed DJ-1β mutant flies compared to controls, led to the proposal that the life-span phenotype was in part due to defects in the oxidative stress response. To further demonstrate this assumption, we analyzed in detail several markers of oxidative stress in control and DJ-1β mutant flies, either untreated or treated with antioxidant compounds. First, we quantified global reactive oxygen species (ROS) as well as H2O2 production; next we measured the activity of several enzymes that respond to oxidative stress such as catalase and superoxide dismutase; and finally we determined protein oxidative damage. Our results showed that DJ-1β mutants exhibit elevated ROS production and protein oxidative damage as well as decreased antioxidant enzyme activity compared to control flies of the same age, which is consistent with the proposed protective role of DJ-1β against oxidative stress. We found that supplementation with either α-tocopherol or the general antioxidant compound ascorbic acid (vitamin C) increased catalase activity and decreased H2O2 and oxidized protein levels in DJ-1β mutants and control flies, but it led to decreased superoxide dismutase activity, maybe as a consequence of a global reduction in oxidative stress. However, α-tocopherol supplementation specifically reduced global ROS production in DJ-1β mutant flies. This study confirms the important role of DJ-1β in oxidative stress response in Drosophila, especially at the level of H2O2 detoxification, and provides evidence that early antioxidant supplementation is an effective treatment to suppress phenotypes in DJ-1β mutants partly by reducing oxidative damage.

Dual sex-specific functions of Drosophila Upstream of N-ras in the control of X chromosome dosage compensation.

Dosage compensation in Drosophila melanogaster involves the assembly of the MSL-2-containing dosage compensation complex (DCC) on the single X chromosome of male flies. Translational repression of msl-2 mRNA blocks this process in females. Previous work indicated that the ubiquitous protein Upstream of N-ras (UNR) is a necessary co-factor for msl-2 repression in vitro. Here, we explore the function of UNR in vivo. Hypomorphic Unr mutant flies showed DCC assembly on high-affinity sites in the female X chromosomes, confirming that UNR inhibits dosage compensation in female flies. Unexpectedly, male mutant flies and UNR-depleted SL2 cells showed decreased DCC binding to the X chromosome, suggesting a role for UNR in DCC assembly or targeting. Consistent with this possibility, UNR overexpression resulted in moderate loss of DCC from the male X chromosome and predominant male lethality. Immunoprecipitation experiments revealed that UNR binds to roX1 and roX2, the non-coding RNA components of the DCC, providing possible targets for UNR function in males. These results uncover dual sex-specific functions of UNR in dosage compensation: to repress DCC formation in female flies and to promote DCC assembly on the male X chromosome.

Identification and analysis of cabut orthologs in invertebrates and vertebrates

Cabut (cbt) is a Drosophila melanogaster gene involved in epidermal dorsal closure (DC). Its expression is dependent on the Jun N-terminal kinase (JNK) cascade, and it functions downstream of Jun regulating dpp expression in the leading edge cells. The Cbt protein contains three C2H2-type zinc fingers and a serine-rich domain, suggesting that it functions as a transcription factor. We have identified single cbt orthologs in other Drosophila species, as well as in other insects and invertebrate organisms like ascidians and echinoderms, but not in nematodes. Gene structure and protein sequence are highly conserved among Drosophilidae, but are more diverged in the other species of invertebrates analyzed. According to this, we demonstrate that cbt expression is detected in the embryonic lateral epidermis in several Drosophila species, as it occurs in D. melanogaster, thus suggesting that the cbt orthologs may have a conserved role in these species during DC. We have also analyzed the genomes of several vertebrate species, finding that the cbt orthologous genes in these organisms encode proteins that belong to the TIEG family of Sp1-like/Krüppel-like transcription factors. Phylogenetic analysis of the invertebrate and vertebrate proteins identified indicates that they mainly follow the expected phylogeny of the species, and that the cbt gene was duplicated during vertebrate evolution. Because we were not able to identify cbt orthologous genes neither in yeast nor in plants, our results suggest that this gene has been probably conserved throughout metazoans and that it may play a fundamental role in animal biology.

Molecular Evolution of P Transposable Elements in the Genus Drosophila. II. The obscura Species Group

Abstract. A phylogenetic analysis of P transposable elements in the Drosophila obscura species group is described. Multiple P sequences from each of 10 species were obtained using PCR primers that flank a conserved region of exon 2 of the transposase gene. In general, the P element phylogeny is congruent with the species phylogeny, indicating that the dominant mode of transmission has been vertical, from generation to generation. One manifestation of this is the distinction of P elements from the Old World obscura and subobscura subgroups from those of the New World affinis subgroup. However, the overall distribution of elements within the obscura species group is not congruent with the phylogenetic relationships of the species themselves. There are at least four distinct subfamilies of P elements, which differ in sequence from each other by as much as 34%, and some individual species carry sequences belonging to different subfamilies. P sequences from D. bifasciata are particularly interesting. These sequences belong to two subfamilies and both are distinct from all other P elements identified in this survey. Several mechanisms are postulated to be involved in determining phylogenetic relationships among P elements in the obscura group. In addition to vertical transmission, these include retention of ancestral polymorphisms and horizontal transfer by an unknown mating-independent mechanism.