A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity.

Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system (CNS), where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single-cell RNA sequencing data of Drosophila glia in 2 well-characterised tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila, and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.

From temporal patterning to neuronal connectivity in Drosophila type I neuroblast lineages.

The development of the central nervous system (CNS) in flies and mammals requires the production of distinct neurons in different locations and times. Here we review progress on how Drosophila stem cells (neuroblasts; NBs) generate distinct neurons over time. There are two types of NBs: type I and type II NBs (defined below); here we focus on type I NBs; type II NBs are reviewed elsewhere in this issue. Type I NBs generate neural diversity via the cascading expression of specific temporal transcription factors (TTFs). TTFs are sequentially expressed in neuroblasts and required for the identity of neurons born during each TTF expression window. In this way TTFs specify the "temporal identity" or birth-order dependent identity of neurons. Recent studies have shown that TTF expression in neuroblasts alter the identity of their progeny, including directing motor neurons to form proper connectivity to the proper muscle targets, independent of their birth-order. Similarly, optic lobe (OL) type I NBs express a series of TTFs that promote proper neuron morphology and targeting to the four OL neuropils. Together, these studies demonstrate how temporal identity is crucial in promoting proper circuit assembly within the Drosophila CNS. In addition, TTF orthologs in mouse are good candidates for specifying neuron types in the neocortex and retina. In this review we highlight the recent advances in understanding the role of TTFs in CNS circuit assembly in Drosophila and reflect on the conservation of these mechanisms in mammalian CNS development.

Transcriptional profiling from whole embryos to single neuroblast lineages in Drosophila.

Embryonic development results in the production of distinct tissue types, and different cell types within each tissue. A major goal of developmental biology is to uncover the "parts list" of cell types that comprise each organ. Here we perform single cell RNA sequencing (scRNA-seq) of the Drosophila embryo to identify the genes that characterize different cell and tissue types during development. We assay three different timepoints, revealing a coordinated change in gene expression within each tissue. Interestingly, we find that the elav and Mhc genes, whose protein products are widely used as markers for neurons and muscles, respectively, show broad pan-embryonic expression, indicating the importance of post-transcriptional regulation. We next focus on the central nervous system (CNS), where we identify genes whose expression is enriched at each stage of neuronal differentiation: from neural progenitors, called neuroblasts, to their immediate progeny ganglion mother cells (GMCs), followed by new-born neurons, young neurons, and the most mature neurons. Finally, we ask whether the clonal progeny of a single neuroblast (NB7-1) share a similar transcriptional identity. Surprisingly, we find that clonal identity does not lead to transcriptional clustering, showing that neurons within a lineage are diverse, and that neurons with a similar transcriptional profile (e.g. motor neurons, glia) are distributed among multiple neuroblast lineages. Although each lineage consists of diverse progeny, we were able to identify a previously uncharacterized gene, Fer3, as an excellent marker for the NB7-1 lineage. Within the NB7-1 lineage, neurons which share a temporal identity (e.g. Hunchback, Kruppel, Pdm, and Castor temporal transcription factors in the NB7-1 lineage) have shared transcriptional features, allowing for the identification of candidate novel temporal factors or targets of the temporal transcription factors. In conclusion, we have characterized the embryonic transcriptome for all major tissue types and for three stages of development, as well as the first transcriptomic analysis of a single, identified neuroblast lineage, finding a lineage-enriched transcription factor.

A novel temporal identity window generates alternating Eve+/Nkx6+ motor neuron subtypes in a single progenitor lineage.

BACKGROUND: Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. In vertebrates, these mechanisms generate "cardinal classes" of neurons that share a transcription factor identity and common morphology. In Drosophila, two cardinal classes are Even-skipped (Eve)+ motor neurons projecting to dorsal longitudinal muscles, and Nkx6+ motor neurons projecting to ventral oblique muscles. Cross-repressive interactions prevent stable double-positive motor neurons. The Drosophila neuroblast 7-1 (NB7-1) lineage uses a temporal transcription factor cascade to generate five distinct Eve+ motor neurons; the origin and development of Nkx6+ motor neurons remains unclear. METHODS: We use a neuroblast specific Gal4 line, sparse labelling and molecular markers to identify an Nkx6+ VO motor neuron produced by the NB7-1 lineage. We use lineage analysis to birth-date the VO motor neuron to the Kr+ Pdm+ neuroblast temporal identity window. We use gain- and loss-of-function strategies to test the role of Kr+ Pdm+ temporal identity and the Nkx6 transcription factor in specifying VO neuron identity. RESULTS: Lineage analysis identifies an Nkx6+ neuron born from the Kr+ Pdm+ temporal identity window in the NB7-1 lineage, resulting in alternation of cardinal motor neuron subtypes within this lineage (Eve>Nkx6 > Eve). Co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage - the first evidence that this TTF combination specifies neuronal identity. Moreover, the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for motor neuron targeting to ventral oblique muscles, thereby revealing a molecular specification pathway from temporal patterning to cardinal transcription factor expression to motor neuron target selection. CONCLUSIONS: We show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm > Nkx6 pathway is necessary and sufficient to promote VO motor neuron targeting to the correct ventral muscle group.

The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila.

The generation of neuronal diversity is essential for circuit formation and behavior. Morphological differences in sequentially born neurons could be due to intrinsic molecular identity specified by temporal transcription factors (henceforth called intrinsic temporal identity) or due to changing extrinsic cues. Here, we have used the Drosophila NB7-1 lineage to address this issue. NB7-1 generates the U1-U5 motor neurons sequentially; each has a distinct intrinsic temporal identity due to inheritance of different temporal transcription factors at its time of birth. We show that the U1-U5 neurons project axons sequentially, followed by sequential dendrite extension. We misexpressed the earliest temporal transcription factor, Hunchback, to create 'ectopic' U1 neurons with an early intrinsic temporal identity but later birth-order. These ectopic U1 neurons have axon muscle targeting and dendrite neuropil targeting that are consistent with U1 intrinsic temporal identity, rather than with their time of birth or differentiation. We conclude that intrinsic temporal identity plays a major role in establishing both motor axon muscle targeting and dendritic arbor targeting, which are required for proper motor circuit development.