Frank Hirth

The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila

Brain development in Drosophila is characterized by two neurogenic periods, one during embryogenesis and a second during larval life. Although much is known about embryonic neurogenesis, little is known about the genetic control of postembryonic brain development. Here we use mosaic analysis with a repressible cell marker (MARCM) to study the role of the brain tumor (brat) gene in neural proliferation control and tumour suppression in postembryonic brain development of Drosophila. Our findings indicate that overproliferation in brat mutants is due to loss of proliferation control in the larval central brain and not in the optic lobe. Clonal analysis indicates that the brat mutation affects cell proliferation in a cell-autonomous manner and cell cycle marker expression shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes cell cycle exit and differentiation of brat mutant cells, thereby abrogating brain tumour formation. Taken together, our results provide evidence that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila, and suggest that Prospero acts as a key downstream effector of brat in cell fate specification and proliferation control.

Hexanucleotide Repeats in ALS/FTD Form Length-Dependent RNA Foci, Sequester RNA Binding Proteins, and Are Neurotoxic

Summary The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila

We have studied the roles of the homeobox genes orthodenticle (otd) and empty spiracles (ems) in embryonic brain development of Drosophila. The embryonic brain is composed of three segmental neuromeres. The otd gene is expressed predominantly in the anterior neuromere; expression of ems is restricted to the two posterior neuromeres. Mutation of otd eliminates the first (protocerebral) brain neuromere. Mutation of ems eliminates the second (deutocerebral) and third (tritocerebral) neuromeres. otd is also necessary for development of the dorsal protocerebrum of the adult brain. We conclude that these homeobox genes are required for the development of specific brain segments in Drosophila, and that the regionalized expression of their homologs in vertebrate brains suggests an evolutionarily conserved program for brain development.

A Pulse of the Drosophila Hox Protein Abdominal-A Schedules the End of Neural Proliferation via Neuroblast Apoptosis

Postembryonic neuroblasts are stem cell-like precursors that generate most neurons of the adult Drosophila central nervous system (CNS). Their capacity to divide is modulated along the anterior-posterior body axis, but the mechanism underlying this is unclear. We use clonal analysis of identified precursors in the abdomen to show that neuron production stops because the cell death program is activated in the neuroblast while it is still engaged in the cell cycle. A burst of expression of the Hox protein Abdominal-A (AbdA) specifies the time at which apoptosis occurs, thereby determining the final number of progeny that each neuroblast generates. These studies identify a mechanism linking the Hox axial patterning system to neural proliferation, and this involves temporal regulation of precursor cell death rather than the cell cycle.

Deep Homology of Arthropod Central Complex and Vertebrate Basal Ganglia

Of Flies and Men Similarities of brain structure, function, and behavior are usually ascribed to convergent evolution. In their review, Strausfeld and Hirth (p. 157) identify multiple commonalities shared by vertebrate basal ganglia and a system of forebrain centers in arthropods called the central complex. The authors conclude that circuits essential to behavioral choice originated very early across phyla. The arthropod central complex and vertebrate basal ganglia derive from embryonic basal forebrain lineages that are specified by an evolutionarily conserved genetic program leading to interconnected neuropils and nuclei that populate the midline of the forebrain-midbrain boundary region. In the substructures of both the central complex and basal ganglia, network connectivity and neuronal activity mediate control mechanisms in which inhibitory (GABAergic) and modulatory (dopaminergic) circuits facilitate the regulation and release of adaptive behaviors. Both basal ganglia and central complex dysfunction result in behavioral defects including motor abnormalities, impaired memory formation, attention deficits, affective disorders, and sleep disturbances. The observed multitude of similarities suggests deep homology of arthropod central complex and vertebrate basal ganglia circuitries underlying the selection and maintenance of behavioral actions.

Evolution of Nervous Systems

Based on classical neuroanatomy, the bilaterians have been divided into two major groups: the Gastroneuralia, such as arthropods and annelids, characterized by a ventral central nervous system (CNS) and the Notoneuralia, such as chordates, characterized by a dorsal CNS. In contrast, molecular genetic studies based on the expression and function of conserved developmental control genes in neurogenesis have revealed striking similarities in the developmental organization of the brain in animals as diverse as flies and mammals. Comparison of the expression, function, and regulation of genes and genetic networks involved in anteroposterior, dorsoventral, and midline patterning of the insect and vertebrate CNS suggests that orthologous genes were already involved in neural specification in a common ancestor, indicating that insect and vertebrate brains evolved from an ancestral urbilaterian brain. The notion that the brains of diverse animal groups are homologous is supported by molecular genetic data that evoke the hypothesis of a dorsoventral body and neuraxis inversion in animal evolution and revive the historic debate about a common Bauplan underlying development in bilaterians. Whereas the evolutionary occurrence and mode of nervous system centralization are still debated, available data suggest that one ancestral and complex nervous system type was at the origin of bilaterian CNS evolution.

An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila.

Studies on expression and function of key developmental control genes suggest that the embryonic vertebrate brain has a tripartite ground plan that consists of a forebrain/midbrain, a hindbrain and an intervening midbrain/hindbrain boundary region, which are characterized by the specific expression of the Otx, Hox and Pax2/5/8 genes, respectively. We show that the embryonic brain of the fruitfly Drosophila melanogaster expresses all three sets of homologous genes in a similar tripartite pattern. Thus, a Pax2/5/8 expression domain is located at the interface of brain-specific otd/Otx2 and unpg/Gbx2 expression domains anterior to Hox expression regions. We identify this territory as the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain. Mutational inactivation of otd/Otx2 and unpg/Gbx2 result in the loss or misplacement of the brain-specific expression domains of Pax2/5/8 and Hox genes. In addition, otd/Otx2 and unpg/Gbx2 appear to negatively regulate each other at the interface of their brain-specific expression domains. Our studies demonstrate that the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain displays developmental genetic features similar to those observed for the midbrain/hindbrain boundary region in vertebrate brain development. This suggests that a tripartite organization of the embryonic brain was already established in the last common urbilaterian ancestor of protostomes and deuterostomes.

Conserved genetic programs in insect and mammalian brain development

In recent years it has become evident that the developmental regulatory genes involved in patterning the embryonic body plan are conserved throughout the animal kingdom. Striking examples are the orthodenticle (otd/Otx) gene family and the Hox gene family, both of which act in the specification of anteroposterior polarity along the embryonic body axis. Studies carried out in Drosophila and mouse now demonstrate that these genes are also involved in the formation of the insect and mammalian brain; the otd/Otx genes are involved in rostral brain development and the Hox genes are involved in caudal brain development. These studies also show that the genes of the otd/Otx family can functionally replace each other in cross‐phylum rescue experiments and indicate that the genetic mechanisms underlying pattern formation in insect and mammalian brain development are evolutionarily conserved. BioEssays 21:677–684, 1999.

Drosophila melanogaster in the Study of Human Neurodegeneration

Human neurodegenerative diseases are devastating illnesses that predominantly affect elderly people. The majority of the diseases are associated with pathogenic oligomers from misfolded proteins, eventually causing the formation of aggregates and the progressive loss of neurons in the brain and nervous system. Several of these proteinopathies are sporadic and the cause of pathogenesis remains elusive. Heritable forms are associated with genetic defects, suggesting that the affected protein is causally related to disease formation and/or progression. The limitations of human genetics, however, make it necessary to use model systems to analyse affected genes and pathways in more detail. During the last two decades, research using the genetically amenable fruitfly has established Drosophila melanogaster as a valuable model system in the study of human neurodegeneration. These studies offer reliable models for Alzheimer’s, Parkinson’s, and motor neuron diseases, as well as models for trinucleotide repeat expansion diseases, including ataxias and Huntington’s disease. As a result of these studies, several signalling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and target of rapamycin (TOR), c-Jun N-terminal kinase (JNK) and bone morphogenetic protein (BMP) signalling, have been shown to be deregulated in models of proteinopathies, suggesting that two or more initiating events may trigger disease formation in an age-related manner. Moreover, these studies also demonstrate that the fruitfly can be used to screen chemical compounds for their potential to prevent or ameliorate the disease, which in turn can directly guide clinical research and the development of novel therapeutic strategies for the treatment of human neurodegenerative diseases.

Differential expression and function of the Drosophila Pax6 genes eyeless and twin of eyeless in embryonic central nervous system development

We analyzed the expression and function of eyeless (ey) and twin of eyeless (toy) in the embryonic central nervous system (CNS) of Drosophila. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain which is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain.