Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain.

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Neurogenesis in the adult Drosophila brain.

Neurodegenerative diseases such as Alzheimer's and Parkinson's currently affect ∼25 million people worldwide. The global incidence of traumatic brain injury (TBI) is estimated at ∼70 million/year. Both neurodegenerative diseases and TBI remain without effective treatments. We are utilizing adult Drosophila melanogaster to investigate the mechanisms of brain regeneration with the long-term goal of identifying targets for neural regenerative therapies. We specifically focused on neurogenesis, i.e., the generation of new cells, as opposed to the regrowth of specific subcellular structures such as axons. Like mammals, Drosophila have few proliferating cells in the adult brain. Nonetheless, within 24 hours of a penetrating traumatic brain injury (PTBI) to the central brain, there is a significant increase in the number of proliferating cells. We subsequently detect both new glia and new neurons and the formation of new axon tracts that target appropriate brain regions. Glial cells divide rapidly upon injury to give rise to new glial cells. Other cells near the injury site upregulate neural progenitor genes including asense and deadpan and later give rise to the new neurons. Locomotor abnormalities observed after PTBI are reversed within 2 weeks of injury, supporting the idea that there is functional recovery. Together, these data indicate that adult Drosophila brains are capable of neuronal repair. We anticipate that this paradigm will facilitate the dissection of the mechanisms of neural regeneration and that these processes will be relevant to human brain repair.

Modeling Neurodegenerative Disorders in Drosophila melanogaster.

Drosophila melanogaster provides a powerful genetic model system in which to investigate the molecular mechanisms underlying neurodegenerative diseases. In this review, we discuss recent progress in Drosophila modeling Alzheimer's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease, Ataxia Telangiectasia, and neurodegeneration related to mitochondrial dysfunction or traumatic brain injury. We close by discussing recent progress using Drosophila models of neural regeneration and how these are likely to provide critical insights into future treatments for neurodegenerative disorders.

A Novel Mutation in Brain Tumor Causes Both Neural Over-Proliferation and Neurodegeneration in Adult Drosophila.

A screen for neuroprotective genes in Drosophila melanogaster led to the identification of a mutation that causes extreme, progressive loss of adult brain neuropil in conjunction with massive brain overgrowth. We mapped the mutation to the brain tumor (brat) locus, which encodes a tripartite motif-NCL-1, HT2A, and LIN-41 (TRIM-NHL) RNA-binding protein with established roles limiting stem cell proliferation in developing brain and ovary. However, a neuroprotective role for brat in the adult Drosophila brain has not been described previously. The new allele, bratcheesehead (bratchs ), carries a mutation in the coiled-coil domain of the TRIM motif, and is temperature-sensitive. We demonstrate that mRNA and protein levels of neural stem cell genes are increased in heads of adult bratchs mutants and that the over-proliferation phenotype initiates prior to adult eclosion. We also report that disruption of an uncharacterized gene coding for a presumptive prolyl-4-hydroxylase strongly enhances the over-proliferation and neurodegeneration phenotypes. Together, our results reveal an unexpected role for brat that could be relevant to human cancer and neurodegenerative diseases.

Expression of the Drosophila homeobox gene, Distal-less, supports an ancestral role in neural development.

BACKGROUND: Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. RESULTS: Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos, we show that Dll is expressed in the embryonic, larval and adult central nervous system and peripheral nervous system (PNS) in embryonic and larval neurons, brain and ventral nerve cord glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. CONCLUSIONS: Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts.

The Drosophila auditory system.

Development of a functional auditory system in Drosophila requires specification and differentiation of the chordotonal sensilla of Johnston's organ (JO) in the antenna, correct axonal targeting to the antennal mechanosensory and motor center in the brain, and synaptic connections to neurons in the downstream circuit. Chordotonal development in JO is functionally complicated by structural, molecular, and functional diversity that is not yet fully understood, and construction of the auditory neural circuitry is only beginning to unfold. Here, we describe our current understanding of developmental and molecular mechanisms that generate the exquisite functions of the Drosophila auditory system, emphasizing recent progress and highlighting important new questions arising from research on this remarkable sensory system.

Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system.

Vertebrate Dlx genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in Dlx genes have been linked to clinical conditions such as epilepsy and autism. Here we show that the single Drosophila Dlx homolog, distal-less, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. We establish that distal-less is necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of distal-less function in an invertebrate nervous system and demonstrate that the Drosophila larval olfactory system is a powerful model in which to understand distal-less functions during neurogenesis.

Development of Johnston's organ in Drosophila.

Hearing is a specialized mechanosensory modality that is refined during evolution to meet the particular requirements of different organisms. In the fruitfly, Drosophila, hearing is mediated by Johnston's organ, a large chordotonal organ in the antenna that is exquisitely sensitive to the near-field acoustic signal of courtship songs generated by male wing vibration. We summarize recent progress in understanding the molecular genetic determinants of Johnston's organ development and discuss surprising differences from other chordotonal organs that likely facilitate hearing. We outline novel discoveries of active processes that generate motion of the antenna for acute sensitivity to the stimulus. Finally, we discuss further research directions that would probe remaining questions in understanding Johnston's organ development, function and evolution.

Linking pattern formation to cell-type specification: Dichaete and Ind directly repress achaete gene expression in the Drosophila CNS.

Mechanisms regulating CNS pattern formation and neural precursor formation are remarkably conserved between Drosophila and vertebrates. However, to date, few direct connections have been made between genes that pattern the early CNS and those that trigger neural precursor formation. Here, we use Drosophila to link directly the function of two evolutionarily conserved regulators of CNS pattern along the dorsoventral axis, the homeodomain protein Ind and the Sox-domain protein Dichaete, to the spatial regulation of the proneural gene achaete (ac) in the embryonic CNS. We identify a minimal achaete regulatory region that recapitulates half of the wild-type ac expression pattern in the CNS and find multiple putative Dichaete-, Ind-, and Vnd-binding sites within this region. Consensus Dichaete sites are often found adjacent to those for Vnd and Ind, suggesting that Dichaete associates with Ind or Vnd on target promoters. Consistent with this finding, we observe that Dichaete can physically interact with Ind and Vnd. Finally, we demonstrate the in vivo requirement of adjacent Dichaete and Ind sites in the repression of ac gene expression in the CNS. Our data identify a direct link between the molecules that pattern the CNS and those that specify distinct cell-types.

Cut mutant Drosophila auditory organs differentiate abnormally and degenerate.

The Drosophila antenna is a sophisticated structure that functions in both olfaction and audition. Previous studies have identified Homothorax, Extradenticle, and Distal-less, three homeodomain transcription factors, as required for specification of antennal identity. Antennal expression of cut is activated by Homothorax and Extradenticle, and repressed by Distal-less. cut encodes the Drosophila homolog of human CAAT-displacement protein, a cell cycle-regulated homeodomain transcription factor. Cut is required for normal development of external mechanosensory structures and Malphigian tubules (kidney analogs). The role of cut in the Drosophila auditory organ, Johnston's organ, has not been characterized. We have employed the FLP/FRT system to generate cut null clones in developing Johnston's organ. In cut mutants, the scolopidial subunits that constitute Johnston's organ differentiate abnormally and subsequently degenerate. Electrophysiological experiments confirm that adult Drosophila with cut null antennae are deaf. We find that cut acts in parallel to atonal, spalt-major, and spalt-related, which encode other transcription factors required for Johnston's organ differentiation. We speculate that Cut functions in conjunction with these factors to regulate transcription of as yet unidentified targets.

Hearing in Drosophila: development of Johnston's organ and emerging parallels to vertebrate ear development.

In this review, I describe recent progress toward understanding the developmental genetics governing formation of the Drosophila auditory apparatus. The Drosophila auditory organ, Johnston's organ, is housed in the antenna. Intriguingly, key genes needed for specification or function of auditory cell types in the Drosophila antenna also are required for normal development or function of the vertebrate ear. These genes include distal-less, spalt and spalt-related, atonal, crinkled, nanchung and inactive, and prestin, and their vertebrate counterparts Dlx, spalt-like (sall), atonal homolog (ath), myosin VIIA, TRPV, and prestin, respectively. In addition, Drosophila auditory neurons recently were shown to serve actuating as well as transducing roles, much like their hair cell counterparts of the vertebrate cochlea. The emerging genetic and physiologic parallels have come as something of a surprise, because conventional wisdom holds that vertebrate and invertebrate hearing organs have separate evolutionary origins. The new findings raise the possibility that auditory organs are more ancient than previously thought and indicate that Drosophila is likely to be a powerful model system in which to gain insights regarding the etiologies of human deafness disorders.

Distal-less functions in subdividing the Drosophila thoracic limb primordium.

The thoracic limb primordium of Drosophila melanogaster is a useful experimental model in which to study how unique tissue types are specified from multipotent founder cell populations. The second thoracic segment limb primordium gives rise to three structures: the wing imaginal disc, the leg imaginal disc, and a larval mechanosensory structure called Keilin's organ. We report that most of the limb primordium arises within neurogenic ectoderm and demonstrate that the neural and imaginal components of the primordium have distinct developmental potentials. We also provide the first analysis of the genetic pathways that subdivide the progenitor cell population into uniquely imaginal and neural identities. In particular, we demonstrate that the imaginal gene escargot represses Keilin's organ fate and that Keilin's organ is specified by Distal-less in conjunction with the downstream achaete-scute complex. This specification involves both the activation of the neural genes cut and couch potato and the repression of escargot. In the absence of achaete-scute complex function, cells adopt mixed identities and subsequently die. We propose that central cells of the primordium previously thought to contribute to the distal leg are Keilin's organ precursors, while both proximal and distal leg precursors are located more peripherally and within the escargot domain.

Drosophila spalt/spalt-related mutants exhibit Townes-Brocks' syndrome phenotypes.

Mutations in SALL1, the human homolog of the Drosophila spalt gene, result in Townes-Brocks' syndrome, which is characterized by hand/foot, anogenital, renal, and ear anomalies, including sensorineural deafness. spalt genes encode zinc finger transcription factors that are found in animals as diverse as worms, insects, and vertebrates. Here, we examine the effect of losing both of the spalt genes, spalt and spalt-related, in the fruit fly Drosophila melanogaster, and report defects similar to those in humans with Townes-Brocks' syndrome. Loss of both spalt and spalt-related function in flies yields morphological defects in the testes, genitalia, and the antenna. Furthermore, spalt/spalt-related mutant antennae show severe reductions in Johnston's organ, the major auditory organ in Drosophila. Electrophysiological analyses confirm that spalt/spalt-related mutant flies are deaf. These commonalities suggest that there is functional conservation for spalt genes between vertebrates and insects.