Invertebrate versus vertebrate neurogenesis: variations on the same theme?
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Animals↗
Biomedical subjects
H J Bellen
Publications and source records attributed to H J Bellen.
gutfeeling, a Drosophila gene encoding an antizyme-like protein, is required for late differentiation of neurons and muscles.
The gutfeeling (guf) gene was uncovered in a genetic screen for genes that are required for proper development of the embryonic peripheral nervous system. Mutations in guf cause defects in growth cone guidance and fasciculation and loss of expression of several neuronal markers in the embryonic peripheral and central nervous systems. guf is required for terminal differentiation of neuronal cells. Mutations in guf also affect the development of muscles in the embryo. In the absence or guf activity, myoblasts are formed properly, but myoblast fusion and further differentiation of muscle fibers is severely impaired. The guf gene was cloned and found to encode a 21-kD protein with a significant sequence similarity to the mammalian ornithine decarboxylase antizyme (OAZ). In mammals, OAZ plays a key regulatory role in the polyamine biosynthetic pathway through its binding to, and inhibition of, ornithine decarboxylase (ODC), the first enzyme in the pathway. The elaborate regulation of ODC activity in mammals still lacks a defined developmental role and little is known about the involvement of polyamines in cellular differentiation. GUF is the first antizyme-like protein identified in invertebrates. We discuss its possible developmental roles in light of this homology.
Amino Acid Sequence↗
Drosophila syntaxin is required for cell viability and may function in membrane formation and stabilization.
The role of the Drosophila homologue of syntaxin-1A (syx) in neurotransmission has been extensively studied. However, developmental Northern analyses and in situ hybridization experiments show that SYX mRNA is expressed during all stages and in many tissues. We have isolated new mutations in syx that reveal roles for syx outside the nervous system. In the ovary, SYX is present in the germarium, but it is predominantly localized to nurse cell membranes. Mitotic recombination experiments in the germline show SYX is essential for oogenesis and may participate in membrane biogenesis in the nurse cells. In the early embryo, a large contribution of maternally deposited RNA is present, and the protein is localized at cell membranes during cellularization. After the maternal contribution is depleted, zygotically produced SYX assists secretion events occurring late in embryogenesis, such as cuticle deposition and neurotransmitter release. However, SYX is also required in larval imaginal discs, as certain hypomorphic mutant combinations exhibit rough eyes and wing notch defects indicative of cell death. Furthermore, recombinant clones that lack syx cause cell lethality in the developing eye. We propose that, similar to its roles in cuticle secretion and neurotransmitter release, SYX may mediate membrane assembly events throughout Drosophila development.
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Evolutionary conservation of sequence and expression of the bHLH protein Atonal suggests a conserved role in neurogenesis.
atonal is a Drosophila proneural gene that belongs to the family of basic helix-loop-helix (bHLH)- containing proteins. It is expressed in the chordotonal organs and photoreceptor cells, and flies that lack Atonal protein are ataxic and blind. Here we report the cloning of atonal homologs from red flour beetle, puffer fish, chicken, mouse, and human. The bHLH domain is conserved throughout evolution, while the entire coding region is highly similar in mammals. Both the chicken and the mouse homologs are expressed early in embryogenesis in the hind brain, and specifically in cells predicted to give rise to the external granular layer of the cerebellum. In addition, these genes are expressed throughout the dorsal part of the spinal cord, in patterns different from those found for other genes, like LH-2 and wnt-1. The mouse homolog (Math1) maps to mouse chromosome 6, and the human homolog (HATH1) to human chromosome 4q22. Two neurological mouse mutants, Lc and chp, were found to map to the vicinity of Math1, but are not caused by mutations in Math1. The evolutionary conservation of this gene and its mRNA expression patterns during embryogenesis suggests that it plays a key role in the development of the vertebrate central nervous system.
Amino Acid Sequence↗
Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission.
Cloning and characterization of the Drosophila syntaxin-1A gene, syx-1A, reveal that it is present in several tissues but is predominantly expressed in the nervous system, where it is localized to axons and synapses. We have generated an allelic series of loss-of-function mutations that result in embryonic lethality with associated morphological and secretory defects dependent on the severity of the mutant allele. Electrophysiological recordings from partial loss-of-function mutants indicate absence of endogenous synaptic transmission at the neuromuscular junction and an 80% reduction of evoked transmission. Complete absence of syx-1A causes subtle morphological defects in the peripheral and central nervous systems, affects nonneural secretory events, and entirely abolishes neurotransmitter release. These data demonstrate that syntaxin plays a key role in nonneuronal secretion and is absolutely required for evoked neurotransmission.
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Presynaptic proteins involved in exocytosis in Drosophila melanogaster: a genetic analysis.
Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely upon in vitro evidence. Recently, genetic approaches in mice, C. elegans and the fruitfly, Drosophila melanogaster, have been used to dissect the in vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release in Drosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, making Drosophila an ideal model system to further unravel the intricacies of synaptic transmission.
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Synaptotagmin controls and modulates synaptic-vesicle fusion in a Ca(2+)-dependent manner.
Although numerous electrophysiological and biochemical studies have defined many of the properties of the putative Ca2+ receptor for exocytosis at the synapse, the molecular mechanisms that couple influx of Ca2+ and release of neurotransmitter have remained elusive. Several proteins have emerged recently as putative Ca2+ sensors. Interestingly, one of these proteins, synaptotagmin, shares many properties with the putative Ca2+ receptor. Recent genetic experiments in Caenorhabditis elegans, Drosophila and mouse have provided important insights about synaptotagmin's role in neurotransmitter release. These experiments, combined with electrophysiological and biochemical studies, suggest that synaptotagmin is a key Ca2+ sensor, converting the ubiquitously used cellular secretory pathway into a Ca(2+)-regulated exocytotic pathway.
Animals↗
Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.
In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.
Animals↗
FlyBase: a virtual Drosophila cornucopia.
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Animals↗
P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster.
The Drosophila embryonic peripheral nervous system (PNS) is an excellent model system to study the molecular mechanisms governing neural development. To identify genes controlling PNS development, we screened 2000 lethal P-element insertion strains. The PNS of mutant embryos was examined using the neural specific marker MAb 22C10, and 92 mutant strains were retained for further analysis. Genetic and cytological analysis of these strains shows that 42 mutations affect previously isolated genes that are known to be required for PNS development: longitudinals lacking (19), mastermind (15), numb (4), big brain (2), and spitz (2). The remaining 50 mutations were classified into 29 complementation groups and the P-element insertions were cytologically mapped. The mutants were classified in five major classes on the basis of their phenotype: gain of neurons, loss of neurons, organizational defects, pathfinding defects and morphological defects. Herein we report the preliminary phenotypic characterization of each of these complementation groups as well as the embryonic lacZ expression pattern of each P-element strain. Our analysis indicates that in most of the P-element insertion strains, the lacZ reporter gene is not expressed in the developing PNS.
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Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants.
Since the demonstration that Ca2+ influx into the presynaptic terminal is essential for neurotransmitter release, there has been much speculation about the Ca2+ receptor responsible for initiating exocytosis. Numerous experiments have shown that the protein, or protein complex, binds multiple Ca2+ ions, resides near the site of Ca2+ influx, and has a relatively low affinity for Ca2+. Synaptotagmin is an integral membrane protein of synaptic vesicles that contains two copies of a domain known to be involved in Ca(2+)-dependent membrane interactions. Synaptotagmin has been shown to bind Ca2+ in vitro with a relatively low affinity. In addition, synaptotagmin has been shown to bind indirectly to Ca2+ channels, positioning the protein close to the site of Ca2+ influx. Recently, a negative regulatory role for synaptotagmin has been proposed, in which it functions as a clamp to prevent fusion of synaptic vesicles with the presynaptic membrane. Release of the clamp would allow exocytosis. Here we present genetic and electrophysiological evidence that synaptotagmin forms a multimeric complex that can function as a clamp in vivo. However, upon nerve stimulation and Ca2+ influx, all synaptotagmin mutations dramatically decrease the ability of Ca2+ to promote release, suggesting that synaptotagmin probably plays a key role in activation of synaptic vesicle fusion. This activity cannot simply be attributed to the removal of a barrier to secretion, as we can electrophysiologically separate the increase in rate of spontaneous vesicle fusion from the decrease in evoked response. We also find that some syt mutations, including those that lack the second Ca(2+)-binding domain, decrease the fourth-order dependence of release on Ca2+ by approximately half, consistent with the hypothesis that a synaptotagmin complex functions as a Ca2+ receptor for initiating exocytosis.
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Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission.
Synaptotagmin is an integral synaptic vesicle protein proposed to be involved in Ca(2+)-dependent exocytosis during synaptic transmission. Null mutations in synaptotagmin have been made in Drosophila, and the protein's in vivo function has been assayed at the neuromuscular synapse. In the absence of synaptotagmin, synaptic transmission is dramatically impaired but is not abolished. In null mutants, evoked vesicle release is decreased by a factor of 10. Moreover, the fidelity of excitation-secretion coupling is impaired so that a given stimulus generates a more variable amount of secretion. However, this residual evoked release shows Ca(2+)-dependence similar to normal release, suggesting either that synaptotagmin is not the Ca2+ sensor or that a second, independent Ca2+ sensor exists. While evoked transmission is suppressed, the rate of spontaneous vesicle fusion is increased by a factor of 5. We conclude that synaptotagmin is not an absolutely essential component of the Ca(2+)-dependent secretion pathway in synaptic transmission but is necessary for normal levels of transmission. Our data support a model in which synaptotagmin functions as a negative regulator of spontaneous vesicle fusion and acts to increase the efficiency of excitation-secretion coupling during synaptic transmission.
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rop, a Drosophila homolog of yeast Sec1 and vertebrate n-Sec1/Munc-18 proteins, is a negative regulator of neurotransmitter release in vivo.
The mammalian homolog of the yeast Sec1p, n-Sec1/Munc-18 has been demonstrated to bind the presynaptic membrane protein syntaxin, a putative synaptic vesicle docking protein. To determine the role of n-Sec1/Munc-18 in neurotransmitter release in vivo, we have overexpressed the Drosophila homolog, rop, in third instar larvae and measured the electrophysiological consequences at the neuromuscular junction. A 3- to 5-fold induction of the rop protein causes a dramatic decrease in neurotransmitter release, suggesting rop may restrict the ability of vesicles to dock or of docked vesicles to fuse. Consistent with this hypothesis, rop overexpression also reduces the number of spontaneous vesicle fusions by more than 50%, and repetitive stimulation results in significant decreases in evoked responses similar to those observed in rab3a mutant mice. However, rop overexpression does not alter significantly the Ca2+ dependence of neurotransmitter release. We propose that the Drosophila n-Sec1/Munc-18 homolog plays a negative role in neurotransmitter release in vivo, in addition to its previously identified positive function, possibly by modulation of docking of synaptic vesicles or activation of a pre-fusion complex at the active zone.
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Mutations affecting the pattern of the PNS in Drosophila reveal novel aspects of neuronal development.
Through a systematic genetic screen, we have identified 55 mutations that affect the development of the PNS of Drosophila embryos. These mutations specify 13 novel and 5 previously characterized genes and define new phenotypes for 2 other known genes. Five classes of mutant phenotypes were identified in the screen: gain of neurons, loss of neurons, abnormal position of chordotonal neurons, aberrant neuronal trajectories, and abnormal morphology of neurons. Phenotypic analyses of mutations identified in this study revealed three novel aspects of PNS development. First, we have identified a novel gene that may be required to define glial versus neuronal cell identity. Second, our data indicate that neuronal migration plays an important role in pattern formation in the embryonic PNS. Third, we have identified mutations that cause a lack of sensory organs, but unlike mutations in proneural genes, do not affect the formation of sensory organ precursors. These genes may be required for key aspects of neuronal differentiation. Our studies suggest that approximately 70 essential genes are required for proper PNS development in Drosophila embryos.
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Genetic and phenotypic analysis of thirteen essential genes in cytological interval 22F1-2; 23B1-2 reveals novel genes required for neural development in Drosophila.
In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aberrations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.
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Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca(2+)-activated neurotransmitter release.
Synaptotagmin (syt), a synaptic vesicle-specific protein known to bind Ca2+ in the presence of phospholipids, has been proposed to mediate Ca(2+)-dependent neurotransmitter release. We have addressed the role of syt in neurotransmitter release in vivo by generating mutations in synaptotagmin (syt) in the fruitfly and assaying the subsequent effects on neurotransmission. Most embryos that lack syt fail to hatch and exhibit very reduced, uncoordinated muscle contractions. Larvae with partial lack-of-function mutations show almost no evoked excitatory junctional potentials (EJPs) in 0.4 mM Ca2+ and a 15-fold reduction in EJP amplitude in 1.0 mM Ca2+ when compared with heterozygous controls. In contrast, we observe an increase in the frequency of spontaneous miniature EJPs in the mutants. These results provide in vivo evidence that syt plays a key role in Ca2+ activation of neurotransmitter release and indicate the existence of separate pathways for evoked and spontaneous neurotransmitter release.
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Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse.
Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin's cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. We have initiated the study of Synaptotagmin's expression during Drosophila neurogenesis in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin (syt) message is present in the cell bodies of all peripheral nervous system neurons and many, if not all, central nervous system neurons during neurite outgrowth and synapse formation, and in mature neurons. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes the discrete regions of the neurophil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, we propose that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms underlying these processes.
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The Drosophila couch potato gene: an essential gene required for normal adult behavior.
Through enhancer detection screens we have isolated 14 insertions in an essential gene that is expressed in embryonic sensory mother cells (SMC), in most cells of the mature embryonic peripheral nervous system (PNS), and in glial cells of the PNS and the central nervous system (CNS). Embryos homozygote for amorphic alleles die, but show no obvious defects in their cuticle, PNS or CNS. The gene has been named couch potato (cpo) because several insertional alleles alter adult behavior. Homozygous hypomorphic cpo flies recover slowly from ether anaesthesia, show aberrant flight behavior, fail to move toward light and do not exhibit normal negative behavior. However, the flies are able to groom and walk, and some are able to fly when prodded, indicating that not all processes required for behavior are severely affected. A molecular analysis shows that the 14 insertions are confined to a few hundred nucleotides which probably contain key regulatory sequences of the gene. The orientation of these insertions and their position within this DNA fragment play an important role in the couch potato phenotype. In situ hybridization to whole mount embryos suggest that some insertions affect the levels of transcription of cpo in most cells in which it is expressed.
Alleles↗