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Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system

['Sergio B. Velarde', 'Centro De Biología Molecular Severo Ochoa', 'Consejo Superior De Investigaciones Cientificas', 'Csic', 'Universidad Autonoma De Madrid', 'Uam', 'Madrid', 'Alvaro Quevedo', 'Carlos Estella', 'Antonio Baonza']

Date: 2021-08

Damage in the nervous system induces a stereotypical response that is mediated by glial cells. Here, we use the eye disc of Drosophila melanogaster as a model to explore the mechanisms involved in promoting glial cell response after neuronal cell death induction. We demonstrate that these cells rapidly respond to neuronal apoptosis by increasing in number and undergoing morphological changes, which will ultimately grant them phagocytic abilities. We found that this glial response is controlled by the activity of Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated after cell death induction, and their functions are necessary to induce glial cell proliferation and migration to the eye discs. The latter of these 2 processes depend on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. We also present evidence that a similar mechanism controls glial response upon apoptosis induction in the leg discs, suggesting that our results uncover a mechanism that might be involved in controlling glial cells response to neuronal cell death in different regions of the peripheral nervous system (PNS).

Funding: This study was supported by grants from: Fundación Ramón Areces (to AB); Programa Estatal de Generación de conocimiento y fortalecimiento científico y tecnológico del sistema de I+D+I (Ministerio de Ciencia, Innovación y Universidades) grants PGC2018-095144-B-I00 (to CE) and BFU2014-54153-P (to AB); The National Council of Science and Technology from México (CONACyT) (to SBV); Fellowship from the Ignacio Larramendi foundation (to SBV); Consejeria de Ciencia. Universidades e innovacion, Comunidad Autonoma de Madrid. “Ayudas para la contratacion de Investigadores Predoctorales” (to AQ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we use the eye disc to explore the mechanisms involved in promoting glial cell response to the induction of neural apoptosis during development. We demonstrate that eye glial cells respond by increasing in number and undergoing morphological changes that confer them phagocytic activity. We found that this glial response is controlled by the activity of the Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated in glial cells upon apoptosis induction in the retina region, and their function is necessary for stimulating the proliferation and migration of glial cells to the eye discs. This latter process depends on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. Remarkably, we present evidence indicating that a similar mechanism controls glial response upon apoptosis induction in the leg discs. As in the eye discs, most leg glial cells are born in the CNS/PNS transition zone during larval stages and migrate into the forming leg [ 16 ]. We observed that after apoptosis induction in leg discs, glial cells accumulate in this region. The function of Dpp and JNK, but not Hh signalling, is required for this glial response; hence, our results uncover a mechanism that might be involved in controlling glial cell response to neural cell death in different regions of the PNS.

Many studies using eye discs as a model system have contributed to uncovering the basic mechanisms and signalling pathways involved in the coupling of neuronal and glial development and have shown that Drosophila glial cells can serve as an experimental model to gain insights into mammalian glial biology [ 6 , 7 , 10 , 18 , 19 ]. However, despite the widespread use of this model, little is known about the response of glial cells upon apoptosis induction of neural tissue and the signalling pathways that might be mediating this function. The unique developmental features of the fly eye disc make this structure an excellent model to analyse the signals emitted by dying neural tissue and the mechanisms involved in regulating glial cell response. Furthermore, considering the similarities between non-myelinating Schwann cells and WG, the eye discs might provide new insights into the signalling pathway network involved in regulating glial cells behaviour in response to apoptosis induction in the PNS.

The eye discs contain distinct glial cell types [ 10 ]. Subperineurial cells, the so-called carpet cells, are 2 large cells that cover the entire differentiated part of the eye disc epithelium. Sitting basally to these 2 cells are the perineurial (PN) glial cells that have a distinct morphology in the optic stalk and in the eye disc [ 10 , 16 ]. These cells define a reserve pool, which can generate glial cells when necessary (for plasticity and development); accordingly, these cells maintain the ability to divide during eye disc development [ 10 ]. In addition, carpet cells separate the PN glia from the underlying wrapping glia (WG), a group of cells that derive from the PN glial and enwrap all axons produced by the photoreceptors. The WG cells perform functions that resemble the non-myelinating Schwann cells forming Remak fibers in the mammalian peripheral nervous system (PNS) [ 7 ]. Likewise, Schwann cells play a key role in promoting regeneration and provide the high ability of the peripheral nerves to regenerate [ 17 ].

Drosophila melanogaster is an excellent model system to discover evolutionarily conserved gene functions and gene networks. Previously, the eye disc of Drosophila has been used to analyse basic mechanisms regulating the migration of glial cells along their neuronal partners [ 2 , 5 – 11 ]. The eye disc develops from a group of ectodermal cells with embryonic origin, distinct from the neuroectodermal cells that form the central nervous system (CNS). Thus, unlike its mammalian counterpart, Drosophila eyes are not part of the CNS. Nevertheless, similarly to mammalian systems, they do contain neurons and glial cells. The eye primordia develops progressively, from posterior to anterior, over the course of approximately 2 days. A morphogenetic furrow (MF) sweeps across the disc during this period, leaving in its wake, developing clusters of photoreceptor cells that will become the individual units of the compound eye, known as ommatidia [ 12 ]. Therefore, while the region anterior to the furrow is mainly composed of proliferating, undifferentiated cells, the region posterior to the furrow consists predominantly of cells that have exited the cell cycle and have begun to differentiate into photoreceptors [ 12 – 14 ]. Unlike photoreceptors, the progenitors of all subretinal glia are not generated from the eye disc cells. During early embryonic stages, the anlage of the eye disc is established, and a few glial cells are born in the initial segment of the Bolwing nerve, which will later become the optic stalk and serve to connect the developing imaginal disc to the brain [ 9 , 10 , 15 ]. These new glial cells are the precursors of the eye disc glial cells. During larval stages, these precursor cells proliferate, forming new glial cells that accumulate in the optic stalk. As the eye imaginal disc grows and neurogenesis is initiated behind the MF, glial cells leave the optic stalk and migrate onto the eye disc [ 9 , 10 , 11 , 15 ].

A complex nervous system is comprised of neurons and glial cells whose development and function are mutually interdependent. The intricate interaction between these 2 cell types is essential for the generation and maintenance of a functional nervous system. During development or after neuronal damage, cells within the nervous system undergo changes in order to preserve structural integrity and function. Glial cells actively participate in all aspects of nervous system development, including mechanisms involved in maintaining structural robustness and functional plasticity. In response to neuronal damage, glial cells proliferate, change their morphology, and alter their behaviour [ 1 – 4 ]. This glial cell response is associated with their regenerative function and is found across different species. The signalling pathways underlying glial response and how they are coordinated remain poorly understood. We can have a better understanding of the mechanisms involved in regulating this process by exploring how glial cells respond to the induction of neuronal apoptosis.

Results

Glial cell proliferation increases in response to apoptotic induction in the retinal cells The increased number of glial cells observed in damaged eye discs may be due to over-migration of these cells from the optic stalk and/or an excess of glial proliferation. To distinguish between these possibilities, we next examined the proliferation pattern of the glial cells in GMR-Gal4 tub-Gal80ts UAS-rpr eye discs after inducing cell death during 72 hours. We observed that upon cell death induction, the proportion of glial cells in S phase was higher than in age-matched control animals, as assayed by 5-Ethynyl-20-deoxyuridine (EdU) incorporation (S2A–S2B” and S2E Fig). In addition, we observed an overall increase in the number of dividing glia upon damage (S2F Fig). Only glial cells located in the basal layer of the eye disc undergo mitosis, as we do not find cells expressing PH3 outside this level. Next, we analysed the proliferation dynamics of glial cells during recovery time. To this end, we induced cell death in the retina region during a 24-hour period and then determined the number of positive PH3 glial cells at various time points post damage induction. We found that at T0 glial cell proliferation was already elevated compared to control discs. After 24 hours of recovering (T1), glial proliferation was still higher than in undamaged discs, but similar to that observed at T0. However, at T2 (after 48 hours of recovering), the ratio of glial proliferation was similar to that of control undamaged discs (S3E Fig), suggesting that the signals that promote glial division cease after 48 hours of apoptotic induction. To evaluate the contribution of cell proliferation on the increased number of glial cells observed in damaged discs, we blocked glial proliferation after inducing apoptosis in the retinal region. To this end, we used the QF/QUAS system [21,22] in combination with the Gal4 system. To induce genetic ablation, we expressed QUAS-rpr [23] in the retinal cells under the control of GMR-QF, whereas glial proliferation was simultaneously blocked by expressing a constitutively activated form of the Retinoblastoma Factor (UAS-rbfCA280) under the control of repo-Gal4 [24]. GMR-QF>rpr tub-Gal80ts repo>rbf280 larvae were raised at 17°C and then shifted to the restrictive temperature to block cell division for 24 hours. After this time, we found that glial cell proliferation was strongly reduced in both damaged and undamaged discs (Fig 3F). Accordingly, we detected that the number of glial cells was also sharply reduced in damaged discs (0.0178 ± 0.0004 glial cells/area μ2 in control damaged discs, n = 24 versus 0.0049 ± 0.001 glial cells/area μ2 in GMRQF>rpr repo>rbfCA280 discs, n = 11, p = <0.0001; Fig 3C–3E). However, in these discs, we observed more glial cells than in control undamaged discs in which proliferation was blocked (0.0049 ± 0.001 glial cells/area μ2 in GMRQF>rpr repo-Gal4>rbf280 versus 0.001 ± 0.00035 glial cells/area μ2 in repo>rbf280, p = 0.02; Fig 3E), suggesting that glial over-migration also contributes to the elevated number of glial cells found after inducing apoptosis. The overexpression of UAS-rbfCA280 for a prolonged period of time (72 hours) totally abolished glial cell proliferation, yet more glial cells were found when compared to undamaged discs in which proliferation was blocked (Fig 3E and 3F). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 3. Overexpression of rbfCA280 in glial cells reduces the number of glial cells observed after cell death induction. (A) The schematic illustration represents a transverse section of an eye disc where the region marked in red (expression domain of GMR-QF) corresponds to the area of the discs that has been damaged using QUAS-rpr GMR-QF. Glial cells are indicated in light blue. repo-Gal4 drives the expression of UAS-rbfCA280 under the control of UAS specifically in glial cells. (B–D) Third instar eye discs stained with anti-Repo (white) and anti-Elav (blue). Control undamaged GMR-QF; tub-Gal80ts repo-Gal4 eye disc (B), tub-Gal80ts/+; repo-Gal4/UAS-rbfCA280 (C) and GMR-QF; tub-Gal80ts/+; repo-Gal4/UAS-rbfCA280 disc (D). The overexpression of UAS-rbfCA280under the control of repo-Gal4 reduces the number of glial cells in undamaged (C) and damaged eye discs (D). (E) The graph represents the glial density of discs shown in B–D (Control, repo Gal4 -rbf 24hrs , GMR QF -rpr, GMR QF -rpr repo Gal4 -rbf 24hrs , and GMR QF -rpr repo Gal4 -rbf 72hrs ). (F) The graph shows the percentage of glial cells in mitosis (PH3 positive). Statistical analysis is shown in Table C in S1 Text. Error bars represent SEM. Scale bars, 50 μm. The numerical data used in this figure are included in S1 Data. GMR, glass multiple reporter; rpr, reaper; SEM, standard error of the mean. https://doi.org/10.1371/journal.pbio.3001367.g003 Taken together, our data suggest that after cell death induction in the retinal region, signals were generated that ultimately promote nonautonomously glial proliferation and migration, resulting in an increase in the number of these cells in the eye disc.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001367

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