Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

The Blimp-1 transcription factor acts in non-neuronal cells to regulate terminal differentiation of the Drosophila eye.

The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. Here, we show that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.

Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene.

The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns.

An exon junction complex-independent function of Barentsz in neuromuscular synapse growth.

The exon junction complex controls the translation, degradation, and localization of spliced mRNAs, and three of its core subunits also play a role in splicing. Here, we show that a fourth subunit, Barentsz, has distinct functions within and separate from the exon junction complex in Drosophila neuromuscular development. The distribution of mitochondria in larval muscles requires Barentsz as well as other exon junction complex subunits and is not rescued by a Barentsz transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We find that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles, and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex.

Drosophila Sidekick is required in developing photoreceptors to enable visual motion detection.

The assembly of functional neuronal circuits requires growth cones to extend in defined directions and recognize the correct synaptic partners. Homophilic adhesion between vertebrate Sidekick proteins promotes synapse formation between retinal neurons involved in visual motion detection. We show here that Drosophila Sidekick accumulates in specific synaptic layers of the developing motion detection circuit and is necessary for normal optomotor behavior. Sidekick is required in photoreceptors, but not in their target lamina neurons, to promote the alignment of lamina neurons into columns and subsequent sorting of photoreceptor axons into synaptic modules based on their precise spatial orientation. Sidekick is also localized to the dendrites of the direction-selective T4 and T5 cells, and is expressed in some of their presynaptic partners. In contrast to its vertebrate homologs, Sidekick is not essential for T4 and T5 to direct their dendrites to the appropriate layers or to receive synaptic contacts. These results illustrate a conserved requirement for Sidekick proteins in establishing visual motion detection circuits that is achieved through distinct cellular mechanisms in Drosophila and vertebrates.

Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye.

Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye.

The Drosophila Epidermal Growth Factor Receptor does not act in the nucleus.

Mammalian members of the ErbB family, including the epidermal growth factor receptor (EGFR), can regulate transcription, DNA replication and repair through nuclear entry of either the full-length proteins or their cleaved cytoplasmic domains. In cancer cells, these nuclear functions contribute to tumor progression and drug resistance. Here, we examined whether the single Drosophila EGFR can also localize to the nucleus. A chimeric EGFR protein fused at its cytoplasmic C-terminus to DNA-binding and transcriptional activation domains strongly activated transcriptional reporters when overexpressed in cultured cells or in vivo However, this activity was independent of cleavage and endocytosis. Without an exogenous activation domain, EGFR fused to a DNA-binding domain did not activate or repress transcription. Addition of the same DNA-binding and transcriptional activation domains to the endogenous Egfr locus through genome editing led to no detectable reporter expression in wild-type or oncogenic contexts. These results show that, when expressed at physiological levels, the cytoplasmic domain of the Drosophila EGFR does not have access to the nucleus. Therefore, nuclear EGFR functions are likely to have evolved after vertebrates and invertebrates diverged.

COP9 signalosome subunits protect Capicua from MAPK-dependent and -independent mechanisms of degradation.

The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. Here, we show that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitylation by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation.

Endocytic trafficking of signaling receptors is an important mechanism for limiting signal duration. Components of the Endosomal Sorting Complexes Required for Transport (ESCRT), which target ubiquitylated receptors to intra-lumenal vesicles (ILVs) of multivesicular bodies, are thought to terminate signaling by the epidermal growth factor receptor (EGFR) and direct it for lysosomal degradation. In a genetic screen for mutations that affect Drosophila eye development, we identified an allele of Vacuolar protein sorting 4 (Vps4), which encodes an AAA ATPase that interacts with the ESCRT-III complex to drive the final step of ILV formation. Photoreceptors are largely absent from Vps4 mutant clones in the eye disc, and even when cell death is genetically prevented, the mutant R8 photoreceptors that develop fail to recruit surrounding cells to differentiate as R1-R7 photoreceptors. This recruitment requires EGFR signaling, suggesting that loss of Vps4 disrupts the EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and other receptors accumulate in endosomes and EGFR target genes are not expressed; epistasis experiments place the function of Vps4 at the level of the receptor. Surprisingly, Vps4 is required for EGFR signaling even in the absence of Shibire, the Dynamin that internalizes EGFR from the plasma membrane. In ovarian follicle cells, in contrast, Vps4 does not affect EGFR signaling, although it is still essential for receptor degradation. Taken together, these findings indicate that Vps4 can promote EGFR activity through an endocytosis-independent mechanism.

Trafficking of the EGFR ligand Spitz regulates its signaling activity in polarized tissues.

Epidermal growth factor receptor (EGFR) ligands undergo a complex series of processing events during their maturation to active signaling proteins. Like its mammalian homologs, the predominant Drosophila EGFR ligand Spitz is produced as a transmembrane pro-protein. In the secretory pathway, Spitz is cleaved within its transmembrane domain to release the extracellular signaling domain. This domain is modified with an N-terminal palmitate group that tethers it to the plasma membrane. We found that the pro-protein can reach the cell surface in the absence of proteolysis, but that it fails to activate the EGFR. To address why the transmembrane pro-protein is inactive, whereas membrane association through the palmitate group promotes activity, we generated a panel of chimeric constructs containing the Spitz extracellular region fused to exogenous transmembrane proteins. Although the orientation of the EGF domain and its distance from the plasma membrane varies in these chimeras, they are all active in vivo. Thus, tethering Spitz to the membrane via a transmembrane domain at either terminus does not prevent activity. Conversely, removing the N-terminal palmitate group from the C-terminally tethered pro-protein does not render it active. Furthermore, we show that the Spitz transmembrane pro-protein can activate the EGFR in a tissue culture assay, indicating that its failure to signal in vivo is not due to structural features. In polarized imaginal disc cells, unprocessed Spitz pro-protein localizes to apical puncta, whereas the active chimeric Spitz constructs are basolaterally localized. Taken together, our data support the model that localized trafficking of the pro-protein restricts its ability to activate the receptor in polarized tissues.

Apical cell expansion maintained by Dusky-like establishes a scaffold for corneal lens morphogenesis.

The biconvex shape of the Drosophila corneal lens, which enables it to focus light onto the retina, arises by organized assembly of chitin and other apical extracellular matrix components. We show here that the Zona Pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells, and in its absence, these cells undergo apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other Zona Pellucida-domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. Artificially inducing apical constriction with constitutively active Myosin light chain kinase is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape for the morphogenesis of overlying apical extracellular matrix structures.

The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila.

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. We have found that the transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. We show that two LIM-homeodomain proteins, Arrowhead and Lim1, are expressed in the region of the eye-antennal disc affected in Chip mutants, and that both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue.

Two distinct mechanisms of Plexin A function in Drosophila optic lobe lamination and morphogenesis.

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of Semaphorin function indicates that Semaphorin 1a, provided by cells that include Tm5 neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A does not disrupt the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles. Summary statement: The axon guidance molecule Plexin A has two functions in Drosophila medulla development; morphogenesis of the neuropil requires its cytoplasmic domain, but establishing synaptic layers through Semaphorin 1a does not.

The receptor protein tyrosine phosphatase LAR promotes R7 photoreceptor axon targeting by a phosphatase-independent signaling mechanism.

Receptor protein tyrosine phosphatases (RPTPs) control many aspects of nervous system development. At the Drosophila neuromuscular junction (NMJ), regulation of synapse growth and maturation by the RPTP LAR depends on catalytic phosphatase activity and on the extracellular ligands Syndecan and Dally-like. We show here that the function of LAR in controlling R7 photoreceptor axon targeting in the visual system differs in several respects. The extracellular domain of LAR important for this process is distinct from the domains known to bind Syndecan and Dally-like, suggesting the involvement of a different ligand. R7 targeting does not require LAR phosphatase activity, but instead depends on the phosphatase activity of another RPTP, PTP69D. In addition, a mutation that prevents dimerization of the intracellular domain of LAR interferes with its ability to promote R7 targeting, although it does not disrupt phosphatase activity or neuromuscular synapse growth. We propose that LAR function in R7 is independent of its phosphatase activity, but requires structural features that allow dimerization and may promote the assembly of downstream effectors.

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin.

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. We show here that genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. We propose that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions.

Sidekick dynamically rebalances contractile and protrusive forces to control tissue morphogenesis.

Contractile actomyosin and protrusive branched F-actin networks interact in a dynamic balance, repeatedly contracting and expanding apical cell contacts to organize the epithelium of the developing fly retina. Previously we showed that the immunoglobulin superfamily protein Sidekick (Sdk) contributes to contraction by recruiting the actin binding protein Polychaetoid (Pyd) to vertices. Here we show that as tension increases during contraction, Sdk progressively accumulates at vertices, where it toggles to recruit the WAVE regulatory complex (WRC) to promote actin branching and protrusion. Sdk alternately interacts with the WRC and Pyd using the same C-terminal motif. With increasing protrusion, levels of Sdk and the WRC decrease at vertices while levels of Pyd increase paving the way for another round of contraction. Thus, by virtue of dynamic association with vertices and interchangeable associations with contractile and protrusive effectors, Sdk is central to controlling the balance between contraction and expansion that shapes this epithelium.

Three Drosophila liprins interact to control synapse formation.

Liprin-α proteins are adaptors that interact with the receptor protein tyrosine phosphatase leukocyte common antigen-related (LAR) and other synaptic proteins to promote synaptic partner selection and active zone assembly. Liprin-ß proteins bind to and share homology with Liprin-α proteins, but their functions at the synapse are unknown. The Drosophila genome encodes single Liprin-α and Liprin-ß homologs, as well as a third related protein that we named Liprin-γ. We show that both Liprin-ß and Liprin-γ physically interact with Liprin-α and that Liprin-γ also binds to LAR. Liprin-α mutations have been shown to disrupt synaptic target layer selection by R7 photoreceptors and to reduce the size of larval neuromuscular synapses. We have generated null mutations in Liprin-ß and Liprin-γ to investigate their role in these processes. We find that, although Liprin-α mutant R7 axons terminate before reaching the correct target layer, Liprin-ß mutant R7 axons grow beyond their target layer. Larval neuromuscular junction size is reduced in both Liprin-α and Liprin-ß mutants, and further reduced in double mutants, suggesting independent functions for these Liprins. Genetic interactions demonstrate that both Liprin proteins act through the exchange factor Trio to promote stable target selection by R7 photoreceptor axons and growth of neuromuscular synapses. Photoreceptor and neuromuscular synapses develop normally in Liprin-γ mutants; however, removing Liprin-γ improves R7 targeting in Liprin-α mutants, and restores normal neuromuscular junction size to Liprin-ß mutants, suggesting that Liprin-γ counteracts the functions of the other two Liprins. We propose that context-dependent interactions between the three Liprins modulate their functions in synapse formation.

Sensory systems: seeing the world in a new light.

Most terminally differentiated sensory neurons express a single sensory receptor molecule. A Drosophila photoreceptor organ breaks this rule by switching to expressing a different type of Rhodopsin as it metamorphoses from larva to adult.

Palmitoylation of the EGFR ligand Spitz by Rasp increases Spitz activity by restricting its diffusion.

Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp and Porcupine, can enhance their secretion, transport, or activity. We show here that rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function.

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13.

Wnt target gene transcription is mediated by nuclear translocation of stabilized beta-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of beta-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.