The mTOR pathway component Unkempt regulates neural stem cell and neural progenitor cell cycle in the Drosophila central nervous system.

The formation of a complex nervous system requires the coordinated action of progenitor cell proliferation, differentiation and maturation. The Drosophila postembryonic central nervous system provides a powerful model for dissecting the cellular and molecular mechanisms underpinning neurogenesis. We previously identified the conserved zinc finger/RING protein Unkempt (Unk) as a key temporal regulator of neuronal differentiation in the Drosophila developing eye and showed that Unk acts downstream of the mechanistic target of rapamycin (mTOR) pathway together with its binding partner Headcase (Hdc). Here we investigate the role of Unk in Drosophila postembryonic thoracic neurogenesis. The Drosophila central nervous system contains neural stem cells, called neuroblasts, and neural progenitors, known as ganglion mother cells (GMCs). Unk is highly expressed in the central brain and ventral nerve cord but is not required to maintain neuroblast numbers or for the regulation of temporal series factor expression in neuroblasts. However, loss of Unk increases the number of neuroblasts and GMCs in S-phase of the cell cycle, resulting in the overproduction of neurons. We also show that Unk interacts with Hdc through its zinc finger domain. The zinc finger domain is required for the synergistic activity of Unk with Hdc during eye development but is not necessary for the activity of Unk in thoracic neurogenesis. Overall, this study shows that Unk and Hdc are novel negative regulators of neurogenesis in Drosophila and indicates a conserved role of mTOR signalling in nervous system development.

Glial enriched gene expression profiling identifies novel factors regulating the proliferation of specific glial subtypes in the Drosophila brain.

Glial cells constitute a large proportion of the central nervous system (CNS) and are critical for the correct development and function of the adult CNS. Recent studies have shown that specific subtypes of glia are generated through the proliferation of differentiated glial cells in both the developing invertebrate and vertebrate nervous systems. However, the factors that regulate glial proliferation in specific glial subtypes are poorly understood. To address this we have performed global gene expression analysis of Drosophila post-embryonic CNS tissue enriched in glial cells, through glial specific overexpression of either the FGF or insulin receptor. Analysis of the differentially regulated genes in these tissues shows that the expression of known glial genes is significantly increased in both cases. Conversely, the expression of neuronal genes is significantly decreased. FGF and insulin signalling drive the expression of overlapping sets of genes in glial cells that then activate proliferation. We then used these data to identify novel transcription factors that are expressed in glia in the brain. We show that two of the transcription factors identified in the glial enriched gene expression profiles, foxO and tramtrack69, have novel roles in regulating the proliferation of cortex and perineurial glia. These studies provide new insight into the genes and molecular pathways that regulate the proliferation of specific glial subtypes in the Drosophila post-embryonic brain.

Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control.

Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. We previously identified the insulin receptor (InR)/mTOR pathway as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. Subsequently, this pathway has been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway we screened transcriptional targets of mTOR for neuronal differentiation phenotypes in photoreceptor neurons. We identified the conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. We also identified the gene headcase (hdc) as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, we show that Unkempt-like is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals.