The role of the neuroepithelial-glial niche in Drosophila larval neurogenesis: a transcriptomic analysis

Abstract

Adequate neuronal development is essential for communication between organs and, consequently, for the vital functions of animals. Neurogenesis is a process by which neurons are generated from stem cells (SCs) or other progenitor cells, and it is during the initial stages of development of an organism when this process is most active. SCs of the nervous system are derived from neuroepithelial cells (NECs), which are rapidly and symmetrically amplified during the early stages of embryonic development in order to increase the number of SCs. Regulation of proliferation and the transition from NECs to SCs is extremely important. Alterations in the control of this process can have serious consequences for the final size of the adult brain. These problems can lead to brain diseases such as hyper- and hypoplasia, microcephaly and tumour generation, among others, which can also lead to severe neurodegenerative diseases. Previous studies have shown that internal dysregulation of progenitor cells can be caused by factors from their environment, in other words, from a cellular niche. The environment created by other cells close to the progenitors can influence, via extrinsic factors, the intrinsic factors that promote the transition from NECs to SCs.

Studies done by our laboratory using Drosophila melanogaster as a model for brain development have described a glial niche that controls neurogenesis in larval stages. In this research, a type of glial cell defined by the expression of the microRNA miR-8, the homolog of miR-200 in vertebrates, was identified and characterized, and it was determined that it control the neuroepithelium-stem cell transition by regulating the TGF-α/EGFR pathway. The similarity between the NEC-neuroblast transition (the fruit fly neuronal SC) and the NEC-radial glia transition (the mammalian neuronal SC) evidence a conservation in the cellular elements that control the neurogenesis. In addition, this aspect highlights that deregulation of gliogenesis, the generation of glial cells, may have side effects on the formation of neuronal cells and can cause the development of gliomas which are one of the most common and aggressive tumours related to glia in the central nervous system (CNS).

The aim of this doctoral thesis is to contribute to the knowledge of the mechanisms that regulate brain maturation and early neurogenesis through the aforementioned glial niche. However, at the beginning of this study, the laboratory identified a new marker for niche glial cells (cg25c+) that was more specific than the one used before (miR-8+). Consequently, the first part of my work focuses on characterizing this new marker, along with the marker for NECs (c855a+), using the Gal4/UAS expression system and fluorescence microscopy techniques. To address the main objective of this thesis, I used a multidisciplinary genetic approach that includes several state-of-the-art techniques. Firstly, I carried out the precise separation of the two brain cell populations using fluorescence-activated cell sorting (FACS). Then, I sequenced each population’s transcriptome (RNA-seq) and analysed them bioinformatically during a stay in the laboratory of Dr. Vladimir Benes (EMBL-Heidelberg, Germany).

The 151 genes obtained from the bioinformatics screening are related to ligands and receptors (or other intercellular signalling molecules) and are potentially differentially expressed at different larval ages. The genes were validated in vivo using the binary Gal4/UAS system and transgenic fly lines for regulating the gain (UAS) and loss (RNAi) of function of these genes. From the validation, I found 51 genes that function both as ligands and receptors in different signalling pathways involved in, for example, tissue growth (EGFR, Spitz, Argos, Echinoid, and Crumbs), synapses (Neurexin-IV and Inebriated), oncogenes (Ret oncogene), integrins (Myospheroid and Tiggrin), the Hedgehog pathway (Patched) or the Wnt pathway (Wnt2, Wg, Pebble, and Off-track2). In addition, I found other molecules involved in the neurotransmission of nerve impulses (Inebriated), serotonin (5-HT2B), acetylcholine (nAChRalpha3) and glutamate (Mangetout), among others. These 51 genes were identified as producing anomalies during development, including malformations of the CNS, when under- or overexpressed in the niche cells. The analysis of the genes whose altered expression produced the most interesting anomalies allowed me to identify a series of genes that are important for organism development. Individuals with altered expression of these genes undergo delay or arrest at a specific larval stage, which can cause differences between body and brain size and result in lethality.

There is a group of receptors among those genes that is associated with the signalling pathway of the octopamine (OA) neurohormone, which is equivalent to noradrenaline in mammals. The characterization of these receptors (Oamb, Octβ1R and Octβ3R) by histochemical techniques enabled me to define the impact they have on the cells of the niche, on their progeny (neurons and neural circuits) and on other tissues (imaginal discs, fatty tissue, ring gland, etc.), as well as the signalling pathways by which receptors cause a deregulation of the cell cycle that ultimately affects the whole organism's growth. The receptors of the OA pathway have been commonly studied in octopaminergic neural circuits, where the production of OA itself has also been described. Studies in epithelial cells from the oviduct of Drosophila have demonstrated the function of the OA receptors Oamb and Octβ3R, which control the secretion of activity effector molecules in other nearby cell types to establish an epithelial niche. Although a function of this type of receptors in insect glial cells has been hypothesized for years, few evidences are found on this subject. For instance, some studies in Drosophila show that astrocytes of the CNS have Oct-TyrR receptors and stimulate the activity of multiple sensory behaviours through internal regulation of Ca2+. Thus, the findings obtained in this doctoral thesis can provide new conclusions about the role of the OA pathway in the glial niche that controls the brain growth and the development of the organism.