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

  1. Inês Lago-Baldaia
  2. Maia Cooper
  3. Austin Seroka
  4. Chintan Trivedi
  5. Gareth T. Powell
  6. Stephen Wilson
  7. Sarah D. Ackerman
  8. Vilaiwan M. Fernandes

  • Curated by eLife

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    This study presents an atlas of glial cell morphology in Drosophila, from distinct locations at different periods of life. The authors integrate morphological information with the transcriptomic signatures of those cells and find that morphological diversity among glial cells of a given class is not a strong predictor of transcriptional identity. The study is of great value as connecting morphology with scRNA sequencing analysis is rarely done and is a necessary step for understanding the underlying biology of these cells. While the weak morphotype-transcriptomic link in many cases may be due to low sequencing resolution, nonetheless, the data are of very high quality and the study will be a very useful resource for the glial biology field.

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Abstract

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, where glia are categorized into five 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 two well-characterized tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the ventral nerve cord. 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.

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  1. eLife

    Author Response

    Reviewer #1 (Public Review):

    The data on embryonic "ventral nerve cord" glia are generated from whole embryos, and even provided that the ventral nerve cord harbors 75% of all glia and thus the majority is ventral nerve cord, the data should not be called vnc-specific. The vnc-specific data set (adult CNS) that is already published (Allen et al., 2020) is strangely not even mentioned in the current manuscript. The idea of having a comprehensive description of glial transcriptional profiles is great - but I was missing the integration of the midline glial cells, which can be considered as ensheathing glial cells that - as the cortex glia - also express wrapper (Stork et al., 2009).

    • We agree with Reviewer 1 that the embryonic glia dataset represents all glia and not just VNC glia. We have amended the text accordingly.

    • We now cite the Allen et al., 2020. Apologies for this omission.

    • Midline Glia:

    The embryonic glial cells analysed in the previous version of our manuscript included only repo+ glia only and therefore did not include midline glia, which do not express repo (Jacobs, 2000). In the revised manuscript, we reanalysed the complete embryonic dataset and identified the midline glia based on known markers and in vivo validation (Figure 3 – figure supplement 1). We also provide a list of genes that show enriched expression in the midline glial cluster as a supplementary file (Source data file 1).

    We performed hierarchical cluster analysis on midline glia, all embryonic repo+ glial clusters and embryonic neuronal clusters to determine the relationship of midline glia to other glia. Interestingly, midline glia formed an outgroup to both neurons and repo+ glia (Figure 3 – figure supplement 1F), suggesting that they are quite distinct from other (repo+) glial classes. This is expected given their mesectodermal origin (Kosman et al., 1991; Thomas et al., 1988). Indeed, although midline glia express wrapper, otherwise known as a cortex glia marker (Banerjee et al., 2017; Noordermeer et al., 1998; Stork et al., 2009), they do not resemble cortex glia in form or function but instead ensheath commissural axons and play critical roles in axon guidance and VNC morphogenesis (Jacobs, 2000). Midline glia have been characterised extensively by several groups (Hartenstein, 2011; Hidalgo, 2003; Jacobs, 2000; Kearney et al., 2004; Vasenkova et al., 2006; Wheeler et al., 2006), therefore, given their distinct origin and the ambiguity surrounding their functional classification, we instead focused our analyses on repo+ glia in this manuscript.

    Unfortunately, I found most of what is reported in this work not to be entirely new. The classification of glial diversity in the adult brain was presented by the Meinerzhagen and Gaul labs (Edwards and Meinertzhagen, 2010; Edwards et al., 2012; Kremer et al., 2017). The description of two astrocyte-like cell types is a reduction of data that defined three morphologically distinct astrocyte-like cells (Peco et al., 2016), which is not discussed. Some other aspects were ignored, too. Two other morphological distinct types of ensheathing glia exist, ensheathing glia and ensheathing/wrapping or track-associated glia were described but this is not discussed (Kremer et al., 2017; Peco et al., 2016).

    We respectfully disagree with Reviewer 1’s assessment that much of the work presented in not new. This work represents the first Drosophila glial cell atlas with thorough validation of cluster marker expression in vivo. It is also the first systematic exploration of the relationship between glial morphology and transcriptional signature, a controversial topic in the field of glial biology. We fully agree that much of the adult glial morphology had been characterised previously by the Meinerzhagen and Gaul labs among many others and we acknowledge this explicitly in our manuscript and in references to Figures 2 (one out of a total of 9 main figures). Indeed, it is because Drosophila glial morphology has been so well characterised that a comprehensive exploration of the relationship between morphology and transcriptional signature was even feasible. Moreover, our revised manuscript also provides more in-depth morphological characterisation and quantification of glial morphology and defines subclasses and morphologies not described previously (e.g. channel perineurial glia and astrocyte morphologies of the lobula and lobula plate). Indeed, even the channel subperineurial glia, which were identified based on lineage relationships, nuclear position and molecular markers, were not described in morphological terms.

    The 3 distinct astrocyte populations defined in Peco et al., (2016) refer to cell body position and neuropil domains covered by astrocytes. We now include this categorisation in our quantification of astrocyte morphology (See response to (6) and Figure 1 – figure supplement 2) and discuss their relationship to the type 1 and type 2 astrocyte morphologies that we observed.

    As well we now include the ensheathing/wrapping or tract ensheathing glia as a morphological category of ensheathing glia in the manuscript (Figure 1A,N,O).

  2. eLife

    eLife assessment

    This study presents an atlas of glial cell morphology in Drosophila, from distinct locations at different periods of life. The authors integrate morphological information with the transcriptomic signatures of those cells and find that morphological diversity among glial cells of a given class is not a strong predictor of transcriptional identity. The study is of great value as connecting morphology with scRNA sequencing analysis is rarely done and is a necessary step for understanding the underlying biology of these cells. While the weak morphotype-transcriptomic link in many cases may be due to low sequencing resolution, nonetheless, the data are of very high quality and the study will be a very useful resource for the glial biology field.

  3. eLife

    Reviewer #1 (Public Review):

    In the current manuscript, scRNA data of the early "ventral nerve cord" and optic system of the adult brain are compared. The authors generated scRNAseq data for the embryo and integrated existing data sets from other labs and extracted repo-positive glial sets to present a description of the transcriptional landscape of glial cells. The main message of the paper is that morphological diversity among glial cells in a given class is not a strong predictor of transcriptional identity.

    However, the data on embryonic "ventral nerve cord" glia are generated from whole embryos, and even provided that the ventral nerve cord harbors 75% of all glia and thus the majority is ventral nerve cord, the data should not be called vnc-specific. The vnc-specific data set (adult CNS) that is already published (Allen et al., 2020) is strangely not even mentioned in the current manuscript. The idea of having a comprehensive description of glial transcriptional profiles is great - but I was missing the integration of the midline glial cells, which can be considered as ensheathing glial cells that - as the cortex glia - also express wrapper (Stork et al., 2009).

    Unfortunately, I found most of what is reported in this work not to be entirely new. The classification of glial diversity in the adult brain was presented by the Meinerzhagen and Gaul labs (Edwards and Meinertzhagen, 2010; Edwards et al., 2012; Kremer et al., 2017). The description of two astrocyte-like cell types is a reduction of data that defined three morphologically distinct astrocyte-like cells (Peco et al., 2016), which is not discussed. Some other aspects were ignored, too. Two other morphological distinct types of ensheathing glia exist, ensheathing glia and ensheathing/wrapping or track-associated glia were described but this is not discussed (Kremer et al., 2017; Peco et al., 2016).

  4. eLife

    Reviewer #2 (Public Review):

    Ines Lago-Baldaia et al. investigate the connection between transcriptional and morphological diversity of glial cells. This is an important question to answer in the glial biology field and has been amplified by recent advances in single-cell sequencing. It remains unclear if transcriptional diversity that is often reported in scRNA analysis equates to morphologically distinct glia. To explore the correlation between transcriptome and morphology, the authors utilize the strength of the Drosophila model system to demonstrate that although morphotypes of glia can be identified in the nervous system, the morphotypes do not correlate with a distinct transcriptional profile. Overall, the paper is well written and the conclusion matches the results that are presented. This work will be an important contribution to the glial biology field.

  5. eLife

    Reviewer #3 (Public Review):

    Our brain is comprised of both electrically active neurons that transmit information and an equal number of a set of cells called glial cells, which are actually comprised of many different cell types with a variety of functions. Compared to neurons, we know much less about glia and therefore need model systems in which they can be studied.

    This study reports the generation of an atlas of glial cells in the Drosophila fly model. Drosophila glia have many similarities with those of vertebrates and are a useful model system for the interrogation of glia due to their simplicity and ease of genetic manipulation to better understand glial cell biology. This study catalogued the morphology of various types of glia in different areas of both the developing and adult fly, building a repository that will be of immense value to researchers. The study also aimed to determine how the shape of different glia, or even within glia of the same type related to the genes that they expressed, and their molecular state. The study found that while cell morphology was tightly linked to gene expression state in some cases, in others it was not, meaning that cells with very different shapes had very similar gene expression profiles/ molecular states. This latter finding suggests that at least some glial cells' shapes are more likely controlled by their interactions with the environment or molecular events that are independent of gene expression per se. The study is very impressive in its depth of characterisation and will come to represent a very useful resource for the community of biologists who employ Drosophila to understand glial cell biology.

  6. Version published to 10.1101/2022.08.01.502305 on bioRxiv