Kaye Suyama

Costal2, a Novel Kinesin-Related Protein in the Hedgehog Signaling Pathway

The Hedgehog (HH) signaling proteins control cell fates and patterning during animal development. In Drosophila, HH protein induces the transcription of target genes encoding secondary signals such as DPP and WG proteins by opposing a repressor system. The repressors include Costal2, protein kinase A, and the HH receptor Patched. Like HH, the kinase Fused and the transcription factor Cubitus interruptus (CI) act positively upon targets. Here we show that costal2 encodes a kinesin-related protein that accumulates preferentially in cells capable of responding to HH. COS2 is cytoplasmic and binds microtubules. We find that CI associates with COS2 in a large protein complex, suggesting that COS2 directly controls the activity of CI.

Thirty-One Flavors of Drosophila Rab Proteins

Rab proteins are small GTPases that play important roles in transport of vesicle cargo and recruitment, association of motor and other proteins with vesicles, and docking and fusion of vesicles at defined locations. In vertebrates, >75 Rab genes have been identified, some of which have been intensively studied for their roles in endosome and synaptic vesicle trafficking. Recent studies of the functions of certain Rab proteins have revealed specific roles in mediating developmental signal transduction. We have begun a systematic genetic study of the 33 Rab genes in Drosophila. Most of the fly proteins are clearly related to specific vertebrate proteins. We report here the creation of a set of transgenic fly lines that allow spatially and temporally regulated expression of Drosophila Rab proteins. We generated fluorescent protein-tagged wild-type, dominant-negative, and constitutively active forms of 31 Drosophila Rab proteins. We describe Drosophila Rab expression patterns during embryogenesis, the subcellular localization of some Rab proteins, and comparisons of the localization of wild-type, dominant-negative, and constitutively active forms of selected Rab proteins. The high evolutionary conservation and low redundancy of Drosophila Rab proteins make these transgenic lines a useful tool kit for investigating Rab functions in vivo.

naked cuticle encodes an inducible antagonist of Wnt signalling.

During animal development, cells have to respond appropriately to localized secreted signals. Proper responses to Hedgehog, transforming growth factor-β, epidermal growth factor and fibroblast growth factor/Ras signals require cognate inducible antagonists such as Patched, Dad, Argos and Sprouty. Wnt signals are crucial in development and neoplasia. Here we show that naked cuticle (nkd), a Drosophila segment-polarity gene, encodes an inducible antagonist for the Wnt signal Wingless (Wg). In fly embryos and imaginal discs nkd transcription is induced by Wg. In embryos, decreased nkd function has an effect similar to excess Wg; at later stages such a decrease appears to have no effect. Conversely, overproduction of Nkd in Drosophila and misexpression of Nkd in the vertebrate Xenopus laevis result in phenotypes resembling those of loss of Wg/Wnt function. nkd encodes a protein with a single EF hand (a calcium-binding motif) that is most similar to the recoverin family of myristoyl switch proteins. Nkd may therefore link ion fluxes to the regulation of the potency, duration or distribution of Wnt signals. Signal-inducible feedback antagonists such as nkd may limit the effects of Wnt proteins in development and disease.

Planar Cell Polarity Enables Posterior Localization of Nodal Cilia and Left-Right Axis Determination during Mouse and Xenopus Embryogenesis

Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP) in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cells apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP) is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2) in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.

Asymmetric Distribution of Prickle-Like 2 Reveals an Early Underlying Polarization of Vestibular Sensory Epithelia in the Inner Ear

Vestibular hair cells have a distinct planar cell polarity (PCP) manifest in the morphology of their stereocilia bundles and the asymmetric localization of their kinocilia. In the utricle and saccule the hair cells are arranged in an orderly array about an abrupt line of reversal that separates fields of cells with opposite polarity. We report that the putative PCP protein Prickle-like 2 (Pk2) is distributed in crescents on the medial sides of vestibular epithelial cells before the morphological polarization of hair cells. Despite the presence of a line of polarity reversal, crescent position is not altered between hair cells of opposite polarity. Frizzled 6 (Fz6), a second PCP protein, is distributed opposite Pk2 along the lateral side of vestibular support cells. Similar to Pk2, the subcellular localization of Fz6 does not differ between cells located on opposite sides of the line of reversal. In addition, in Looptail/Van Gogh-like2 mutant mice Pk2 is distributed asymmetrically at embryonic day 14.5 (E14.5), but this localization is not coordinated between adjacent cells, and the crescents subsequently are lost by E18.5. Together, these results support the idea that a conserved PCP complex acts before stereocilia bundle development to provide an underlying polarity to all cells in the vestibular epithelia and that cells on either side of the line of reversal are programmed to direct the kinocilium in opposite directions with respect to the polarity axis defined by PCP protein distribution.

A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis.

Niemann-Pick type C (NPC) disease is a fatal autosomal-recessive neurodegenerative disorder characterized by the inappropriate accumulation of unesterified cholesterol in aberrant organelles. The disease is due to mutations in either of two genes, NPC1, which encodes a transmembrane protein related to the Hedgehog receptor Patched, and NPC2, which encodes a secreted cholesterol-binding protein. Npc1 mutant mice can be partially rescued by treatment with specific steroids. We have created a Drosophila NPC model by mutating dnpc1a, one of two Drosophila genes related to mammalian NPC1. Cells throughout the bodies of dnpc1a mutants accumulated sterol in a punctate pattern, as in individuals with NPC1 mutations. The mutants developed only to the first larval stage and were unable to molt. Molting after the normal first instar period was restored to various degrees by feeding the mutants the steroid molting hormone 20-hydroxyecdysone, or the precursors of ecdysone biosynthesis, cholesterol and 7-dehydrocholesterol. dnpc1a is normally highly expressed in the ecdysone-producing ring gland. Ring gland-specific expression of dnpc1a in otherwise mutant flies allowed development to adulthood, suggesting that the lack of ecdysone in the mutants is the cause of death. We propose that dnpc1a mutants have sterols trapped in aberrant organelles, leading to a shortage of sterol in the endoplasmic reticulum and/or mitochondria of ring gland cells, and, consequently, inadequate ecdysone synthesis.

Rab35 Controls Actin Bundling by Recruiting Fascin as an Effector Protein

Fascin-Actin Rab Bristles Rab proteins have diverse functions in directing intracellular traffic and may also affect development. Zhang et al. (p. 1250) show that during Drosophila development Rab35 influences the development of bristles, neurosensory structures built upon bundled actin. Rab35 also caused massive actin-rich filopodia protrusions from cultured cells. Activated Rab35 interacted directly with fascin, an actin filament bundling protein, to colocalize near the plasma membrane. When Rab35 was engineered to interact with the surface of mitochondria, it stimulated localized actin assembly in a fascin-dependent manner. Thus, fascin is a Rab35 effector protein that links membrane trafficking regulation to cytoskeleton assembly during development. Development of sensory bristles in flies depends on controlling the colocalization of actin assembly on membranes. Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. We found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly.

Jelly belly: A Drosophila LDL Receptor Repeat-Containing Signal Required for Mesoderm Migration and Differentiation

Inductive interactions subdivide the Drosophila mesoderm into visceral, somatic, and heart muscle precursors. The muscle precursors form organs by executing tissue-specific migrations and cell fusions. We identified a novel gene, jelly belly (jeb), which is required for visceral mesoderm development. jeb encodes a secreted protein that contains an LDL receptor repeat. In jeb mutants, visceral mesoderm precursors form, but they fail to migrate or differentiate normally; no visceral muscles develop. Jeb protein is produced in somatic muscle precursors and taken up by visceral muscle precursors. jeb reveals a signaling process in which somatic muscle precursors support the proper migration and differentiation of visceral muscle cells. Later in embryogenesis, jeb is transcribed in neurons and Jeb protein is found in axons.

A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin signaling and control body size

Growth and body size are regulated by the CNS, integrating the genetic developmental program with assessments of an animals current energy state and environmental conditions. CNS decisions are transmitted to all cells of the animal by insulin/insulin-like signals. The molecular biology of the CNS growth control system has remained, for the most part, elusive. Here we identify NS3, a Drosophila nucleostemin family GTPase, as a powerful regulator of body size. ns3 mutants reach <60% of normal size and have fewer and smaller cells, but exhibit normal body proportions. NS3 does not act cell-autonomously, but instead acts at a distance to control growth. Rescue experiments were performed by expressing wild-type ns3 in many different cells of ns3 mutants. Restoring NS3 to only 106 serotonergic neurons rescued global growth defects. These neurons are closely apposed with those of insulin-producing neurons, suggesting possible communication between the two neuronal systems. In the brains of ns3 mutants, excess serotonin and insulin accumulate, while peripheral insulin pathway activation is low. Peripheral insulin pathway activation rescues the growth defects of ns3 mutants. The findings suggest that NS3 acts in serotonergic neurons to regulate insulin signaling and thus exert global growth control.

Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused

The mechanism by which the secreted signaling molecule Hedgehog (Hh) elicits concentration-dependent transcriptional responses from cells is not well understood. In the Drosophila wing imaginal disc, Hh signaling differentially regulates the transcription of target genes decapentaplegic (dpp), patched (ptc) and engrailed (en) in a dose-responsive manner. Two key components of the Hh signal transduction machinery are the kinesin-related protein Costal2 (Cos2) and the nuclear protein trafficking regulator Suppressor of Fused [Su(fu)]. Both proteins regulate the activity of the transcription factor Cubitus interruptus (Ci) in response to the Hh signal. We have analyzed the activities of mutant forms of Cos2 in vivo and found effects on differential target gene transcription. A point mutation in the motor domain of Cos2 results in a dominant-negative form of the protein that derepresses dpp but not ptc. Repression of ptc in the presence of the dominant-negative form of Cos2 requires Su(fu), which is phosphorylated in response to Hh in vivo. Overexpression of wild-type or dominant-negative cos2 represses en. Our results indicate that differential Hh target gene regulation can be accomplished by differential sensitivity of Cos2 and Su(Fu) to Hh.