Regulation of Apterous activity in Drosophila wing development.

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Here, we present evidence that complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. We also report that a difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo.     

Osa modulates the expression of Apterous target genes in the Drosophila wing

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO. Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. We have conducted a modular P-element based EP (enhancer/promoter) misexpression screen to look for genes involved in Apterous activity. We have found Osa, a member of the Brahma chromatin-remodeling complex, as a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. We propose that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression.     

The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing.

Dorso-ventral axis formation in the Drosophila wing requires the localized accumulation of the Apterous LIM/homeodomain protein (Ap) in dorsal cells. Here we report that dLdb/Chip encodes a LIM-binding cofactor that controls Ap activity. Both lack and excess of dLdb/Chip function cause the same phenotype as apterous (ap) lack of function; i.e. dorsal to ventral transformations, generation of new wing margins, and wing outgrowths. These results indicate that the normal function of Ap in dorso-ventral compartmentalization requires the correct amount of the DLDB/CHIP co-factor, and suggest that the Ap and DLDB/CHIP proteins form a multimeric functional complex. In support of this model, we show that the dLdb/Chip excess-of-function phenotypes can be rescued by ap overexpression.     

Interactions between chip and the achaete/scute-daughterless heterodimers are required for pannier-driven proneural patterning

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip was previously identified as a cofactor of the dorsal selector Apterous, and we show here that both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax.     

UNC-73/trio RhoGEF-2 activity modulates Caenorhabditis elegans motility through changes in neurotransmitter signaling upstream of the GSA-1/Galphas pathway

Rho-family GTPases play regulatory roles in many fundamental cellular processes. Caenorhabditis elegans UNC-73 RhoGEF isoforms function in axon guidance, cell migration, muscle arm extension, phagocytosis, and neurotransmission by activating either Rac or Rho GTPase subfamilies. Multiple differentially expressed UNC-73 isoforms contain a Rac-specific RhoGEF-1 domain, a Rho-specific RhoGEF-2 domain, or both domains. The UNC-73E RhoGEF-2 isoform is activated by the G-protein subunit Gαq and is required for normal rates of locomotion; however, mechanisms of UNC-73 and Rho pathway regulation of locomotion are not clear. To better define UNC-73 function in the regulation of motility we used cell-specific and inducible promoters to examine the temporal and spatial requirements of UNC-73 RhoGEF-2 isoform function in mutant rescue experiments. We found that UNC-73E acts within peptidergic neurons of mature animals to regulate locomotion rate. Although unc-73 RhoGEF-2 mutants have grossly normal synaptic morphology and weak resistance to the acetylcholinesterase inhibitor aldicarb, they are significantly hypersensitive to the acetylcholine receptor agonist levamisole, indicating alterations in acetylcholine neurotransmitter signaling. Consistent with peptidergic neuron function, unc-73 RhoGEF-2 mutants exhibit a decreased level of neuropeptide release from motor neuron dense core vesicles (DCVs). The unc-73 locomotory phenotype is similar to those of rab-2 and unc-31, genes with distinct roles in the DCV-mediated secretory pathway. We observed that constitutively active Gαs pathway mutations, which compensate for DCV-mediated signaling defects, rescue unc-73 RhoGEF-2 and rab-2 lethargic movement phenotypes. Together, these data suggest UNC-73 RhoGEF-2 isoforms are required for proper neurotransmitter signaling and may function in the DCV-mediated neuromodulatory regulation of locomotion rate.     

Regulators acting in combinatorial codes also act independently in single differentiating neurons

In the Drosophila ventral nerve cord, a small number of neurons express the LIM-homeodomain gene apterous (ap). These ap neurons can be subdivided based upon axon pathfinding and their expression of neuropeptidergic markers. ap, the zinc finger gene squeeze, the bHLH gene dimmed, and the BMP pathway are all required for proper specification of these cells. Here, using several ap neuron terminal differentiation markers, we have resolved how each of these factors contributes to ap neuron diversity. We find that these factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, we also find that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, we show that single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

Genes required for axon pathfinding and extension in the C. elegans nerve ring.

Over half of the neurons in Caenorhabditis elegans send axons to the nerve ring, a large neuropil in the head of the animal. Genetic screens in animals that express the green fluorescent protein in a subset of sensory neurons identified eight new sax genes that affect the morphology of nerve ring axons. sax-3/robo mutations disrupt axon guidance in the nerve ring, while sax-5, sax-9 and unc-44 disrupt both axon guidance and axon extension. Axon extension and guidance proceed normally in sax-1, sax-2, sax-6, sax-7 and sax-8 mutants, but these animals exhibit later defects in the maintenance of nerve ring structure. The functions of existing guidance genes in nerve ring development were also examined, revealing that SAX-3/Robo acts in parallel to the VAB-1/Eph receptor and the UNC-6/netrin, UNC-40/DCC guidance systems for ventral guidance of axons in the amphid commissure, a major route of axon entry into the nerve ring. In addition, SAX-3/Robo and the VAB-1/Eph receptor both function to prevent aberrant axon crossing at the ventral midline. Together, these genes define pathways required for axon growth, guidance and maintenance during nervous system development.     

MIG-10/lamellipodin and AGE-1/PI3K promote axon guidance and outgrowth in response to slit and netrin

BACKGROUND: The cytoplasmic C. elegans protein MIG-10 affects cell migrations and is related to mammalian proteins that bind phospholipids and Ena/VASP actin regulators. In cultured cells, mammalian MIG-10 promotes lamellipodial growth and Ena/VASP proteins induce filopodia. RESULTS: We show here that during neuronal development, mig-10 and the C. elegans Ena/VASP homolog unc-34 cooperate to guide axons toward UNC-6 (netrin) and away from SLT-1 (Slit). The single mutants have relatively mild phenotypes, but mig-10; unc-34 double mutants arrest early in development with severe axon guidance defects. In axons that are guided toward ventral netrin, unc-34 is required for the formation of filopodia and mig-10 increases the number of filopodia. In unc-34 mutants, developing axons that lack filopodia are still guided to netrin through lamellipodial growth. In addition to its role in axon guidance, mig-10 stimulates netrin-dependent axon outgrowth in a process that requires the age-1 phosphoinositide-3 lipid kinase but not unc-34. CONCLUSIONS: mig-10 and unc-34 organize intracellular responses to both attractive and repulsive axon guidance cues. mig-10 and age-1 lipid signaling promote axon outgrowth; unc-34 and to a lesser extent mig-10 promote filopodia formation. Surprisingly, filopodia are largely dispensable for accurate axon guidance.     

Cytoplasmic domain requirements for Frazzled-mediated attractive axon turning at the Drosophila midline

The conserved DCC ligand-receptor pair Netrin and Frazzled (Fra) has a well-established role in axon guidance. However, the specific sequence motifs required for orchestrating downstream signaling events are not well understood. Evidence from vertebrates suggests that P3 is important for transducing Netrin-mediated turning and outgrowth, whereas in C. elegans it was shown that the P1 and P2 conserved sequence motifs are required for a gain-of-function outgrowth response. Here, we demonstrate that Drosophila fra mutant embryos exhibit guidance defects in a specific subset of commissural axons and these defects can be rescued cell-autonomously by expressing wild-type Fra exclusively in these neurons. Furthermore, structure-function studies indicate that the conserved P3 motif (but not P1 or P2) is required for growth cone attraction at the Drosophila midline. Surprisingly, in contrast to vertebrate DCC, P3 does not mediate receptor self-association, and self-association is not sufficient to promote Fra-dependent attraction. We also show that in contrast to previous findings, the cytoplasmic domain of Fra is not required for axonal localization and that neuronal expression of a truncated Fra receptor lacking the entire cytoplasmic domain (Fra delta C) results in dose-dependent defects in commissural axon guidance. These findings represent the first systematic dissection of the cytoplasmic domains required for Fra-mediated axon attraction in the context of full-length receptors in an intact organism and provide important insights into attractive axon guidance at the midline.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system

Nonstop, which has previously been shown to have homology to ubiquitin proteases, is required for proper termination of axons R1-R6 in the optic lobe of the developing Drosophila eye. Herein, we establish that Nonstop actually functions as an ubiquitin protease to control the levels of ubiquitinated histone H2B in flies. We further establish that Nonstop is the functional homolog of yeast Ubp8, and can substitute for Ubp8 function in yeast cells. In yeast, Ubp8 activity requires Sgf11. We show that in Drosophila, loss of Sgf11 function causes similar photoreceptor axon-targeting defects as loss of Nonstop. Ubp8 and Sgf11 are components of the yeast SAGA complex, suggesting that Nonstop function might be mediated through the Drosophila SAGA complex. Indeed, we find that Nonstop does associate with SAGA components in flies, and mutants in other SAGA subunits display nonstop phenotypes, indicating that SAGA complex is required for accurate axon guidance in the optic lobe. Candidate genes regulated by SAGA that may be required for correct axon targeting were identified by microarray analysis of gene expression in SAGA mutants.     

Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. Here, we identify a Drosophila Cas (DCas), find that Cas proteins are highly expressed in neurons and show that DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events.     

Rnd1 regulates axon extension by enhancing the microtubule destabilizing activity of SCG10

The GTPase Rnd1 affects actin dynamics antagonistically to Rho and has been implicated in the regulation of neurite outgrowth, dendrite development, and axon guidance. Here we show that Rnd1 interacts with the microtubule regulator SCG10. This interaction requires a central domain of SCG10 comprising about 40 amino acids located within the N-terminal-half of a putative alpha-helical domain and is independent of phosphorylation at the four identified phosphorylation sites that regulate SCG10 activity. Rnd1 enhances the microtubule destabilizing activity of SCG10 and both proteins colocalize in neurons. Knockdown of Rnd1 or SCG10 by RNAi suppressed axon extension, indicating a critical role for both proteins during neuronal differentiation. Overexpression of Rnd1 in neurons induces the formation of multiple axons. The effect of Rnd1 on axon extension depends on SCG10. These results indicate that SCG10 acts as an effector downstream of Rnd1 to regulate axon extensions by modulating microtubule organization.     

C. elegans dystroglycan DGN-1 functions in epithelia and neurons, but not muscle, and independently of dystrophin

The C. elegans dystroglycan (DG) homolog DGN-1 is expressed in epithelia and neurons, and localizes to basement membrane (BM) surfaces. Unlike vertebrate DG, DGN-1 is not expressed in muscle or required for muscle function. dgn-1 null mutants are viable but sterile owing to severe disorganization of the somatic gonad epithelium, and show defects in vulval and excretory cell epithelia and in motoneuron axon guidance. The defects resemble those of epi-1 laminin alphaB mutants, suggesting that DGN-1 serves as a receptor for laminin. dgn-1(0)/+ animals are fertile but show gonad migration defects in addition to the defects seen in homozygotes, indicating that DGN-1 function is dosage sensitive. Phenotypic analyses show that DGN-1 and dystrophin-associated protein complex (DAPC) components have distinct and independent functions, in contrast to the situation in vertebrate muscle. The DAPC-independent functions of DGN-1 in epithelia and neurons suggest that vertebrate DG may also act independently of dystrophin/utrophin in non-muscle tissues.