futsch expression was analyzed in mutant backgrounds that specifically alter the 22C10 expression pattern. The Zn finger transcription factor Tramtrack acts as a repressor of 22C10 antigen expression, and in tramtrack mutant embryos, high levels of 22C10 antigen are found in the mesoderm (Giesen, 1997). Consistent with Futsch being the 22C10 antigen, the expression of futsch mRNA is also drastically increased in tramtrack mutant embryos. In a second experiment, a ftz-GAL4 driver line and EP(X)1419 were used to drive ectopic futsch expression in a pair-rule expression pattern. Ectopic expression of the 22C10 antigen correlates with ectopic expression of the deduced futsch transcript, further supporting the possibility that futsch encodes the 22C10 antigen (Hummel, 2000).
Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).
Given the axon guidance defects in nerfin-1null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).
Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1null embryos (Kuzin, 2005).
Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1null embryos (Kuzin, 2005).
Mammalian Fragile X mental retardation gene (FMR1) encodes an RNA binding protein that acts as a negative translational regulator. A Drosophila fragile X syndrome model has been developed using loss-of-function mutants and overexpression of the FMR1 homolog (Fmr1). Drosophila Fmr1 nulls display enlarged synaptic terminals, whereas neuronal overexpression results in fewer and larger synaptic boutons. Synaptic structural defects are accompanied by altered neurotransmission, with synapse type-specific regulation in central and peripheral synapses. These phenotypes mimic those observed in mutants of microtubule-associated Futsch. Immunoprecipitation of Drosophila Fmr1 shows association with Futsch mRNA, and Western analyses demonstrate that Fmr1 inversely regulates Futsch expression. Fmr1;futsch double mutants restore normal synaptic structure and function. It is proposed that Fmr1 acts as a translational repressor of Futsch to regulate microtubule-dependent synaptic growth and function (Zhang, 2001).
Evidence is presented that Fmr1 negatively regulates Futsch expression. (1) Fmr1 associates with Futsch mRNA. This interaction is specific, since Fmr1 fails to bind other targets such as alpha-tubulin mRNAs and the interaction is missing in Fmr1 null mutants. (2) In Fmr1 null mutants, Futsch protein level in the nervous system is increased and Fmr1 neuronal overexpression causes Futsch expression to be reduced. These results show that the level of Futsch in the nervous system is inversely regulated by the level of Fmr1. Taken together, the biochemical association between Fmr1 protein and Futsch mRNA and the inverse regulation of Futsch expression by Fmr1 strongly support a hypothesis that Fmr1 acts as a negative regulator of Futsch translation (Zhang, 2001).
Futsch appears to be the major target for Fmr1 in the regulation of synaptic structure and function. Structurally, futsch hypomorphs display fewer and enlarged NMJ synaptic boutons with dispersed, punctate anti-Futsch immunoreactivity, a phenotype indistinguishable from that caused by overexpression of Fmr in dfxrNOE. However, futschNOE causes synaptic overgrowth, a phenotype similar to Fmr1 null mutants. Functionally, all four genotypes (loss and overexpression of either Fmr1 or Futsch) enhance neurotransmission at the larval NMJ, and all four genotypes impair neurotransmission in the adult eye. Thus, the expression alterations of Futsch are sufficient to explain the synaptic phenotypes of Fmr1 mutants (Zhang, 2001).
The most conclusive experimental result is the suppression of Fmr1 synaptic phenotypes by the Fmr1;futsch double mutants. The double mutant develops normal synaptic architecture, including the normal number of arboreal branches and synaptic boutons. Strikingly, the double mutant reduces NMJ transmission to suppress the peripheral synaptic phenotype, while at the same time it increases photoreceptor transmission to suppress the central synaptic phenotype. Based on these results, it is proposed that the major function of Fmr1 is the negative regulation of Futsch in the nervous system, which in turn regulates microtubule-dependent synaptic structure and function. Of course, it remains probable that Fmr1 is translationally regulating multiple proteins. However, the Futsch misregulation is sufficient to explain the synaptic phenotypes in Fmr1 mutants and, by extrapolation, possibly the mental retardation of FraX patients (Zhang, 2001).
In Drosophila, mAb 22C10 has been widely used to label neuronal cells within the PNS and CNS. In all stages of development, 22C10 antigen expression starts in postmitotic neurons shortly before axonogenesis is initiated. In the embryonic nervous system, first mAb 22C10 reactivity is detected in the MP2 neurons. During subsequent development, several interneurons and motoneurons express the 22C10 antigen. Within the developing PNS, neuronal cells express the 22C10 antigen from early stages of neuronal differentiation onward. It is important to note that the 22C10 epitope is found in the growing axon, the cell body, and the dendrite. At the end of embryogenesis, 22C10 expression can be found in all sensory neurons. During late larval development, expression is found in all photoreceptor neurons. In addition to expression in the nervous system, some 22C10 antigen can be detected in the muscle attachment sites of the ventrolateral musculature and the tip cell of the Malphigian tubules. By using confocal microscopy, the 22C10 epitope has been localized in the central axonal compartment of larval neurons associated with the microtubule cytoskeleton (Hummel, 2000 and references therein).
The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. At Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1B-related protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, Baz and Par-6 are also expressed at synapses and their synaptic localization depends on aPKC activity. These findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory (Ruiz-Canada, 2004).
During expansion of the NMJ, parent boutons located at the distal end of a branch give rise to new synaptic boutons by budding. New buds separate from parent boutons by the formation of a neck, and NMJ branches extend by neck elongation and bouton enlargement. Throughout this process, the postsynaptic membrane and underlying cytoskeleton impose a barrier to presynaptic extension, since synaptic boutons and their buds are completely surrounded by the muscle cell membrane and underlying cytoskeleton. During branch elongation, a presynaptic signal may induce the retraction of the postsynaptic cytoskeleton barrier. It is proposed that changes in both the pre- and postsynaptic cytoskeleton during branch elongation mediate these events and that these processes are regulated by aPKC with the collaboration of Baz and Par-6 in both locales (Ruiz-Canada, 2004).
The localization of aPKC to MT-rich domains at the NMJ and the marked reduction in new synaptic bouton formation observed in dapkc mutants prompted an investigation of the cytoskeletal changes during branch elongation. At the presynaptic arbor of wild-type larvae, NMJ expansion is accompanied by the presence of unbundled MTs at the distal tip of NMJ branches and by little if any colocalization between MTs and the MAP1B-related protein Futsch. In vertebrates, MAP1B protects MTs from depolymerization. Similarly in Drosophila, MT integrity in axons and at the NMJ is preserved by Futsch; severe mutations in futsch result in MT fragmentation. This Futsch-dependent protection of MT integrity at presynaptic arbors has an important role during NMJ expansion, since MT fragmentation in futsch and dapkc mutants results in marked reduction in bouton number (Ruiz-Canada, 2004).
In epithelial cells and growth cones, an increase in aPKC activity enhances MT lifetime, although the involvement of MT-associated proteins in this process has not been investigated. The results suggest that aPKC activity enhances MT stability in a process that depends on Futsch. (1) Coimmunoprecipitation experiments indicate that aPKC, Tubulin, and Futsch exist in the same biochemical complex and that the interaction between aPKC and MTs is likely to be through Futsch. (2) Loss of dapkc function results in fragmented MTs at terminal boutons, and an increase in aPKC activity by pre-PKM (PKM is a persistantly active kinase) results in longer than normal MTs and an increased colocalization between MTs and Futsch. (3) Presynaptic expression of PKM in a futsch mutant background results in MT fragmentation similar to futsch mutants alone, suggesting that PKM acts through Futsch to stabilize MTs (Ruiz-Canada, 2004).
Three futsch mutations lead to a pronounced reduction in the level of 22C10 antigen expression including futschM455, futschP28, and futschN94. The futschM455 allele leads to a uniform reduction in expression, while both futschP28 and futschN94 also lead to an abnormal subcellular distribution of Futsch. In futschP28, a granular expression in the cell body and dendrites is observed. Interestingly, in futschN94, there is diffuse staining within the nerve terminal that is not observed in wild type. This aspect of the phenotype has been analyzed in the visual system. In third instar wild-type photoreceptor cells, distinct 22C10-positive fibers extend from the cell body into the axon and dendrites, but no staining is observed in the nerve terminals. In futschN94, Futsch protein is distributed uniformly in the nerve terminal. The mislocalization of the Futsch protein can be clearly seen in the nerve terminals in the lamina where the photoreceptor axons of cells R1-R6 terminate as well as in the growth cones of photoreceptor cells R7 and R8 in the medulla. In addition, futschN94 mutant larvae show ectopic Futsch expression in the so-called optic lobe pioneer cells (Hummel, 2000).
In two mutations, futschK68 and futschP158, 22C10 antigen expression is eliminated. Homozygous futschK68 flies are viable, and overall neuronal development proceeds normally in the embryo. In the larvae, a reduced number of synaptic boutons visualized by the antibody nc46 is noted. In futschP158, the lethality and the absence of 22C10 expression could not be separated by recombination. In wild-type embryos, highest levels of mAb 22C10 reactivity are seen in all PNS neurons. Futsch is prominently expressed in the dendrites of the five lateral chordotonal organs. To analyze the morphology of the PNS neurons in mutant futschP158 embryos, the GAL4 driver line P0163 was used to express the transmembrane protein CD2 specifically in PNS neurons, thereby visualizing their axonal and dendritic morphology. In mutant futschP158 larvae, about 97% of the PNS neurons fail to develop their normal dendritic morphology. Occasionally, however, dendritic processes are detected. Axonal growth is impaired as well, and few axons grew normally into the CNS. Axonal pathfinding is not affected, and axons appeared to simply stop growing after some distance.
Within the ventral nerve cord, Futsch is expressed in many inter- and motor-neurons that also express the Fasciclin II protein (Fas II). In stage 16 futschP158 mutant embryos, the longitudinal connectives are reduced. Similarly, disruptions are observed in the motoneuronal connections. In particular, SNb nerves fail to reach muscle fibers 12 and 13. Motoneurons stall and fail to set up the correct innervation pattern in 89% of the segments. To determine the null phenotype of futsch, the CNS defects associated with the deficiencies Df(1)A94 and Df(1)S39 that remove the futsch locus were analyzed. In both cases, a nervous system phenotype very similar to the futschP158 mutant phenotype was observed. Thus, P158 appears to be an amorphic mutation, whereas the K68 is a hypomorphic mutation of the futsch gene. Loss of futsch function leads to disruption of dendritic and axonal growth.
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date revised: 10 January 2005
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