Different modes of APC/C activation control growth and neuron-glia interaction in the developing Drosophila eye.

The development of the nervous system requires tight control of cell division, fate specification and migration. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that affects different steps of cell cycle progression, as well as having postmitotic functions in nervous system development. It can therefore link different developmental stages in one tissue. The two adaptor proteins, Fizzy/Cdc20 and Fizzy-related/Cdh1, confer APC/C substrate specificity. Here, we show that two distinct modes of APC/C function act during Drosophila eye development. Fizzy/Cdc20 controls the early growth of the eye disc anlage and the concomitant entry of glial cells onto the disc. In contrast, fzr/cdh1 acts during neuronal patterning and photoreceptor axon growth, and subsequently affects neuron-glia interaction. To further address the postmitotic role of Fzr/Cdh1 in controlling neuron-glia interaction, we identified a series of novel APC/C candidate substrates. Four of our candidate genes are required for fzr/cdh1-dependent neuron-glia interaction, including the dynein light chain Dlc90F Taken together, our data show how different modes of APC/C activation can couple early growth and neuron-glia interaction during eye disc development.

Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila.

Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3'UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.

Switch in FGF signalling initiates glial differentiation in the Drosophila eye.

The formation of a complex nervous system requires the intricate interaction of neurons and glial cells. Glial cells generally migrate over long distances before they initiate their differentiation, which leads to wrapping and insulation of axonal processes. The molecular pathways coordinating the switch from glial migration to glial differentiation are largely unknown. Here we demonstrate that, within the Drosophila eye imaginal disc, fibroblast growth factor (FGF) signalling coordinates glial proliferation, migration and subsequent axonal wrapping. Glial differentiation in the Drosophila eye disc requires a succession from glia-glia interaction to glia-neuron interaction. The neuronal component of the fly eye develops in the peripheral nervous system within the eye-antennal imaginal disc, whereas glial cells originate from a pool of central-nervous-system-derived progenitors and migrate onto the eye imaginal disc. Initially, glial-derived Pyramus, an FGF8-like ligand, modulates glial cell number and motility. A switch to neuronally expressed Thisbe, a second FGF8-like ligand, then induces glial differentiation. This switch is accompanied by an alteration in the intracellular signalling pathway through which the FGF receptor channels information into the cell. Our findings reveal how a switch from glia-glia interactions to glia-neuron interactions can trigger formation of glial membrane around axonal trajectories. These results disclose an evolutionarily conserved control mechanism of axonal wrapping, indicating that Drosophila might serve as a model to understand glial disorders in humans.

Transcriptional characterization of iPSC-derived microglia as a model for therapeutic development in neurodegeneration.

Microglia are the resident immune cells in the brain that play a key role in driving neuroinflammation, a hallmark of neurodegenerative disorders. Inducible microglia-like cells have been developed as an in vitro platform for molecular and therapeutic hypothesis generation and testing. However, there has been no systematic assessment of similarity of these cells to primary human microglia along with their responsiveness to external cues expected of primary cells in the brain. In this study, we performed transcriptional characterization of commercially available human inducible pluripotent stem cell (iPSC)-derived microglia-like (iMGL) cells by bulk and single cell RNA sequencing to assess their similarity with primary human microglia. To evaluate their stimulation responsiveness, iMGL cells were treated with Liver X Receptor (LXR) pathway agonists and their transcriptional responses characterized by bulk and single cell RNA sequencing. Bulk transcriptome analyses demonstrate that iMGL cells have a similar overall expression profile to freshly isolated human primary microglia and express many key microglial transcription factors and functional and disease-associated genes. Notably, at the single-cell level, iMGL cells exhibit distinct transcriptional subpopulations, representing both homeostatic and activated states present in normal and diseased primary microglia. Treatment of iMGL cells with LXR pathway agonists induces robust transcriptional changes in lipid metabolism and cell cycle at the bulk level. At the single cell level, we observe heterogeneity in responses between cell subpopulations in homeostatic and activated states and deconvolute bulk expression changes into their corresponding single cell states. In summary, our results demonstrate that iMGL cells exhibit a complex transcriptional profile and responsiveness, reminiscent of in vivo microglia, and thus represent a promising model system for therapeutic development in neurodegeneration.

Spinster controls Dpp signaling during glial migration in the Drosophila eye.

The development of multicellular organisms requires the well balanced and coordinated migration of many cell types. This is of particular importance within the developing nervous system, where glial cells often move long distances to reach their targets. The majority of glial cells in the peripheral nervous system of the Drosophila embryo is derived from the CNS and migrates along motor axons toward their targets. In the developing Drosophila eye, CNS-derived glial cells move outward toward the nascent photoreceptor cells, but the molecular mechanisms coupling the migration of glial cells with the growth of the eye imaginal disc are mostly unknown. Here, we used an enhancer trap approach to identify the gene spinster, which encodes a multipass transmembrane protein involved in endosome-lysosome trafficking, as being expressed in many glial cells. spinster mutants are characterized by glial overmigration. Genetic experiments demonstrate that Spinster modulates the activity of several signaling cascades. Within the migrating perineurial glial cells, Spinster is required to downregulate Dpp (Decapentaplegic) signaling activity, which ceases migratory abilities. In addition, Spinster affects the growth of the carpet cell, which indirectly modulates glial migration.

MicroRNA-9: functional evolution of a conserved small regulatory RNA.

The functional significance of microRNA-9 (miR-9) during evolution is evidenced by its conservation at the nucleotide level from flies to humans but not its diverse expression patterns. Recent studies in several model systems reveal that miR-9 can regulate neurogenesis through its actions in neural or non-neural cell lineages. In vertebrates, miR-9 exerts diverse cell-autonomous effects on the proliferation, migration, and differentiation of neural progenitor cells by modulating different mRNA targets. In some developmental contexts, miR-9 suppresses apoptosis and is misregulated in several types of cancer cells, influencing proliferation or metastasis formation. Moreover, downregulation of miR-9 in postmitotic neurons is also implicated in some neurodegenerative diseases. Thus, miR-9 is emerging as an important regulator in development and disease through its ability to modulate different targets in a manner dependent on the developmental stage and the cellular context.

Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD.

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

Insights into C9ORF72-Related ALS/FTD from Drosophila and iPSC Models.

GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of ALS and FTD. An important issue is how repeat RNAs and their translation products, various dipeptide repeat (DPR) proteins, cause neurodegeneration. Drosophila has been widely used to model G4C2 repeat RNA and DPR protein toxicity. Overexpression of disease molecules in flies has revealed important molecular insights. These have been validated and further explored in human neurons differentiated from induced pluripotent stem cells (iPSCs), a disease-relevant model in which expanded G4C2 repeats are expressed in their native molecular context. Approaches that combine the genetic power of Drosophila and the disease relevance of iPSC-derived patient neurons will continue to unravel the underlying pathogenic mechanisms and help identify potential therapeutic targets in C9ORF72-ALS/FTD.

Antagonistic feedback loops involving Rau and Sprouty in the Drosophila eye control neuronal and glial differentiation.

During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, we found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor required two mutually antagonistic feedback loops. We identified a positive feedback loop mediated by the Ras association (RA) domain-containing protein Rau that sustained Ras activity and counteracted the negative feedback loop mediated by Sprouty. Rau has two RA domains that together showed a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerized and was found in large-molecular weight complexes. Deletion of rau in flies decreased the differentiation of retinal wrapping glia and induced a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty was repressed and that of rau was increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, our findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye.

Long-range signaling systems controlling glial migration in the Drosophila eye.

The Drosophila compound eye comprises about 750 individual ommatidia arranged into an almost crystalline array. The eye is not needed for viability and thus served as a favorite model organ to decipher many signaling systems controlling diverse aspects such as cell fate allocation or cell-cycle control. Here, we review that the Drosophila eye can also serve to study the interaction between neurons and glial cells. In the Drosophila eye, all glial cells originate from the brain lobes and need to migrate onto the larval eye disc as neurogenesis is initiated during the third instar stage. Although we do have a relatively good understanding of the sequential progression of neurogenesis in the eye disc, we are still at the beginning in our dissection of the molecular pathways orchestrating the coordinated development of neurons and glial cells.

The eye imaginal disc as a model to study the coordination of neuronal and glial development.

A complex nervous system comprises two distinct cell types, neurons and glial cells, whose development, differentiation and function is mutually interdependent. Many studies contributed to uncovering the basic mechanisms determining neuronal and glial fate and we are progressing enormously towards an understanding of how neurons interconnect to form intricate neuronal networks. However, the mechanisms used to couple neuronal and glial development remain largely obscure. Here we advocate the usefulness of the developing Drosophila compound eye as a new model to study the complex relationship between glial and neuronal cells.