Research group Heinrich Reichert
Drosophila neuroblasts: neural stem cells in normal brain development and in brain tumor formation
The vast arrays of different neural cell types that characterize the complex circuits of the brain are generated by neural stem cells. In Drosophila, neural stem cells called neuroblasts are similar to vertebrate neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. The numerous cell types that make up the brain of Drosophila derive from a set of approximately 200 neuroblasts, each of which generates its own lineage-specific unit of neural progeny during embryonic and postembryonic development. The goals of our lab are to analyze the developmental mechanisms by which these proliferating brain neuroblasts generate the lineage-specific units of the brain and to analyze the cellular and molecular mechanisms by which the deregulated proliferation of these neural stem cells leads to the formation of brain tumors.
Previous work on Drosophila neuroblasts indicates a causative link between impaired neural stem cell proliferation and brain tumor formation in this genetic model system and supports the hypothesis that impaired cell-fate determination is a major cause of cancerous overgrowth (reviewed in Reichert, 2011; Saini and Reichert, 2012), Notably the amplifying neural stem cells that produce the neural cells of higher brain centres in the fly are prone to tumorigenic dysregulation, and this makes them excellent models for neural stem cell-derived tumors (reviewed in Boyan and Reichert, 2011). We focus on these neural stem cells in the Drosophila central brain and use a comprehensive set of genetic, genomic and transgenic methods to investigate how the balance between stem cell self-renewal, neural differentiation and programmed cell death is precisely controlled to ensure normal brain development as well as to study how dysregulation of this balance leads to neural stem cell derived tumorigenesis and formation of metastasis.
An important advance in the analysis of neural stem cell biology in Drosophila is based on the availability of methods for isolating large numbers of pure neural stem cells and differentiating neurons. With the development and implementation of a novel FACS-based method for isolating neuroblasts and differentiating neurons that retain both cell-cycle and lineage characteristics from the Drosophila brain, this has been achieved (Berger et al., 2012). This FACS purification method does not affect viability, proliferation properties, or lineage characteristics of the isolated neuroblasts and of their progeny in vitro or in vivo. Importantly, with this methodological advance, it is now possible to obtain high-quality neural stem cell-specific gene expression data. Based on transcriptional profiles derived by mRNA sequencing of pure populations of isolated cells a total of 28 predicted neural stem cell-specific transcription factors are identified and arranged into a transcriptional network which reveals hubs for Notch signalling, growth control, and chromatin regulation. In addition to their identification, the functional relevance of these individual transcription factors in brain tumor formation is tested in genetic gain-of-function and loss-of-function studies.
In addition to previously known factors, this functional analysis uncovers the Klumpfuss transcription factor, a member of the EGR family of transcriptional regulators which has been connected to human cancers, as a novel regulator of self-renewal. Targeted overexpression of Klumpfuss in neural stem cells results in tumorigenesis and metastasis after transplantation into a host environment. This demonstrates that the obtained transcriptional data provides a useful basis for a more targeted search for functional interactions between redundantly acting factors. Thus, both the method for isolation of pure neural stem cells and the data on the transcription factor network that underlies self-renewal in these stem cells will be valuable resources for further work.
Previous studies based on genome-wide RNAi analysis have uncovered additional candidate genes with potential tumor suppressor function. The tumor suppressor function of these candidate genes is analysed in terms of stem cell tumor formation potential using an in vivo transplantation model (Laurenson et al., 2012). These investigations comprise transplantation of transgenic brain tissue, in which RNAi knockdown is targeted to genetically labeled neural stem cells, qualitative and quantitative assays of tumor formation after transplantation into wildtype host, and investigation of the metastatic potential of the transplanted tissue using an ovarian micro-metastasis assay. These experiments demonstrate that loss-of-function of candidate genes such as alpha-Adaptin, ap2-sigma, Brahma, Moira, and Daughterless in neural stem cells results in (transplantable) tumor formation as well as in metastases which are invariably lethal. Continuing work on additional candidate tumor suppressor genes and oncogenes is revealing insight into the genetic network that underlies brain tumor formation in the Drosophila model system.
Insight into the neural stem-cell dependent mechanisms that operate during normal brain development is obtained for the class of Drosophila brain neuroblasts that amplify proliferation through intermediate neural progenitors (Jiang and Reichert, 2012a; Viktorin et al., 2011). These studies show that substantial neural overproliferation occurs normally in these neural stem cell lineages and that elimination of excess neurons through programmed cell death during postembryonic development is required for the formation of correct innervation in the developing brain. Thus, amplification of proliferation through intermediate progenitors is counterbalanced by reduction of neuronal number through programmed cell death, and both operate during the normal development of these neural stem cell lineages. While the triggering mechanisms for programmed cell death in the amplifying neuroblast lineages remains currently unknown, key molecular signals for initiation of programmed cell death in other identified lineages are now revealed, and a novel neuroblast-specific role of Hox genes during normal brain development is documented (Kuert et al., 2012). This work focuses on identified lineages in the tritocerebral and subesophageal brain neuromeres, which represent the postembryonic expression domains of the Antp-class Hox genes. Thus, for example, the Hox gene labial is required in two identified neuroblast lineages of the tritocerebral brain neuromere for correct and cell autonomous termination of proliferation through programmed cell death. This Hox-dependent programmed cell death is stage- and lineage-specific, and in its absence identified ectopic neuroblast lineages are formed which never occur in the normal wildtype brain. Remarkably similar findings obtained in the developing mammalian hindbrain for the murine homolog Hoxa1 gene support the notion of highly conserved mechanisms of brain development.
In addition, a comprehensive study of the neuroblast lineages generated during normal embryogenesis in the larval olfactory system is carried out, which sheds light on the lineal relationship between larval and adult neurons of the brain (Das et al., 2012). This work reveals an unexpected level of complexity of the different projection neurons and local interneurons in the simple larval olfactory system. Moreover, it establishes the lineages of origin for the large diversity of larval neuron cell types in the olfactory system and, in doing so, identifies a novel fifth neuroblast lineage of olfactory interneurons. Remarkably, in this system each different cell type appears to be represented by a small number of neural cells, and in many cases, by a single identified neuron. Thus, although the larval olfactory circuitry may be reduced in terms of neuronal number, it shows a surprising diversity of interneuronal cell types that is comparable to that of the highly complex adult olfactory system which derives from the same set of neuroblasts.
Finally, we are pioneering novel investigations into the diversity and evolution of brain development by studying non-model systems organisms that belong to so called lesser phyla. For this we are analysing the central nervous systems in Cycliophora, Loricifera, Kinorhyncha, and Tardigrada using both state of the art imaging and 3D ultrastructural techniques as well as RNA-seq based gene expression studies.
The general significance of these and related findings is the subject of several invited reviews (Saini and Reichert, 2012; Jiang and Reichert, 2012b; Bailly et al., 2012).