Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila
2021; Springer Nature; Volume: 22; Issue: 5 Linguagem: Inglês
10.15252/embr.202052130
ISSN1469-3178
AutoresQian Dong, Michael Zavortink, Francesca Froldi, Sofya Golenkina, Tammy Lam, Louise Cheng,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoArticle10 March 2021free access Source DataTransparent process Glial Hedgehog signalling and lipid metabolism regulate neural stem cell proliferation in Drosophila Qian Dong Qian Dong orcid.org/0000-0002-0829-8724 Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Michael Zavortink Michael Zavortink Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Francesca Froldi Francesca Froldi Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Sofya Golenkina Sofya Golenkina Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Tammy Lam Tammy Lam Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Louise Y Cheng Corresponding Author Louise Y Cheng [email protected] orcid.org/0000-0001-9712-4082 Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia The Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Qian Dong Qian Dong orcid.org/0000-0002-0829-8724 Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Michael Zavortink Michael Zavortink Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Francesca Froldi Francesca Froldi Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Sofya Golenkina Sofya Golenkina Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Tammy Lam Tammy Lam Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Louise Y Cheng Corresponding Author Louise Y Cheng [email protected] orcid.org/0000-0001-9712-4082 Peter MacCallum Cancer Centre, Parkville, Vic., Australia Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia The Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Vic., Australia Search for more papers by this author Author Information Qian Dong1,2, Michael Zavortink1,2, Francesca Froldi1,2, Sofya Golenkina1,2, Tammy Lam1,2 and Louise Y Cheng *,1,2,3 1Peter MacCallum Cancer Centre, Parkville, Vic., Australia 2Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic., Australia 3The Department of Anatomy and Neuroscience, University of Melbourne, Parkville, Vic., Australia *Corresponding author. Tel: +61 450053363; E-mail: [email protected] EMBO Reports (2021)22:e52130https://doi.org/10.15252/embr.202052130 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The final size and function of the adult central nervous system (CNS) are determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. Here, we explore non-autonomous glial regulation of NB proliferation. We show that lipid droplets (LDs) which reside within the glial niche are closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation. Upon FGF-mediated cortex glial overgrowth, glial Hh non-autonomously activates Hh signalling in the NBs, which in turn disrupts NB cell cycle progression and its ability to produce neurons. Glial Hh’s ability to signal to NB is further modulated by lipid storage regulator lipid storage droplet-2 (Lsd-2) and de novo lipogenesis gene fatty acid synthase 1 (Fasn1). Together, our data suggest that glial-derived Hh modified by lipid metabolism mechanisms can affect the neighbouring NB’s ability to proliferate and produce neurons. SYNOPSIS The glial niche regulates the proliferation of neural stem cells in the Drosophila larval CNS. Glial Hh signaling autonomously facilitates cortex glial niche formation, and non-autonomously regulates neuroblast proliferation dependent on the lipid regulators Fasn1 and Lsd2. Hh is associated with lipid droplets (LDs) in the cytoplasm of glial niche cells surrounding neuroblasts (NBs). Hh is required for the growth of cortex glial niche cells, which maintain NB activity during development. FGF-induced cortex glial overgrowth increases the expression of Hh as well as lipid regulators such as Fasn1 and Lsd2 in the niche. Fasn1 and Lsd2 modulate Hh activity to activate the Hh signaling pathway in NBs, which slows down their cell cycle. Introduction Most stem cells reside within specialised groups of cells, collectively referred to as a niche, that provide the trophic, structural and nutritional microenvironment to sustain and protect the stem cells during development (Scadden, 2014). The niche relays developmental and physiological states of the animal to the stem cells and influences the stem cells’ ability to divide in accordance with the environmental state of the organism. Asymmetrically dividing and multipotent neural stem cells in both mammals and invertebrates are responsible for generating the adult nervous system (Homem & Knoblich, 2012). In Drosophila, the vast majority of NBs are specified during embryogenesis, proliferate throughout larval development and terminate divisions during pupal stages. Type I NBs located within the ventral nerve cord (VNC) and the central brain (CB) are the predominant type of NBs, whilst type II NBs are eight NB lineages located on the dorsal surface of the CB (Homem & Knoblich, 2012). During each type I NB cell division, NB self-renews and produces a smaller ganglion mother cell (GMC) that creates a limited number of neurons or glia (Fig 1A). The ability of NBs to divide and generate appropriate progeny number and cell diversity is determined by their ability to maintain asymmetric division, regulate the speed of their cell cycles and timely enter/exit the cell cycle at the beginning and end of neurogenesis (Homem & Knoblich, 2012). Cell intrinsic mechanisms such as the temporal regulation of NB identity via transcription factors that are expressed throughout the life time of the NBs impact on both the numbers and the types of neurons generated by the NB (Doe, 2017). However, more recently, attention has shifted towards understanding how cell extrinsic signals are interpreted by the NBs to alter their behaviour (Ramon-Canellas et al, 2019). Figure 1. Hh is localised to the LDs within cortex glial cells Images in this and following figures are of larval central brains (CBs) at 96ALH. A. Schematic showing NBs that undergo asymmetric division to self-renew and produce GMCs, which terminally differentiate to generate post-mitotic neurons or glial cells (left). Each NB is surrounded by a microenvironment, composed of glial cells (right). B–D”. Representative images showing that Hh accumulates on the surface of LDs in glial cells of the CB (yellow arrows), quantified in (I) (n = 20 LDs). Glial cells are marked by repo-GAL4 > GFP and CB is circled in (B). E–G”. In the posterior compartment of the developing wing disc (WD) pouch region where Hh is expressed, LDs and Hh are not tightly associated (white arrows), quantified in (J) (n = 17 LDs). H–J. Hh-LD association is quantified by plotting the pixel intensities of both Hh (cyan) and LDs (red) along a line across LDs. Y-axis represents grey intensity values, and X-axis represents relative LD position. K, L”. Hh-LD associations are observed in the cortex glia (yellow arrows, NP2222-GAL4 > mGFP). Data information: Hh is detected with a Hh antibody and LDs are visualised with LipidTOX unless otherwise stated. (D-D’’), (G-G’’), (L-L’’) are zoomed in images of (C, F, K), respectively. Scale bar = 50 μm in (C and F), scale bar = 20 μm in (D-D’’, K-L’’), scale bar = 10 μm in (G-G’’). Error bar represents SEM. In (I): Welch’s t-test, (****) P < 0.0001. In (J): unpaired t-test, (ns) P = 0.7113. Source data are available online for this figure. Source Data for Figure 1 [embr202052130-sup-0005-SDataFig1.xlsx] Download figure Download PowerPoint Larval NBs and their progeny are surrounded by a scaffold of glial cell processes, which form the stem cell niche in the CNS (Fig 1A). Glial cells fall into three classes: (i) surface (perineural and subperineural) glia that enwrap the CNS to form the blood brain barrier (BBB); (ii) cortex glia that encapsulate neuronal soma and NBs; and (iii) neuropil glia that are located at the cortex–neuropil interface and form a sheath around the neuropil compartments (Freeman, 2015). The intimate relationship between glial cells and NBs has been extensively studied in the context of NB entry into the cell cycle at the beginning of post-embryonic neurogenesis shortly after larval hatch (Ding et al, 2020). Feeding has been shown to trigger insulin production by surface glial cells, which in turn activates the insulin/insulin-like growth factor pathway in neighbouring NBs and stimulates their growth and proliferation via activation of the phosphoinositide 3-kinase (PI3K) signalling pathway (Chell & Brand, 2010; Sousa-Nunes et al, 2011). Once NBs enter into the cell cycle, glial cells continue to play active roles in promoting NB proliferation. These reactivated NBs are found in close association with cortex glia (Hoyle, 1986; Hoyle et al, 1986; Pereanu et al, 2005), and this contact is maintained through adhesion via E-cadherin. Disruption of NB-cortex glia contact affects the NB’s ability to undergo mitosis (Dumstrei et al, 2003; Doyle et al, 2017), and the failure to expand the glial membrane also affects both neuronal survival as well as NB cell cycle progression (Speder & Brand, 2018; Yuan et al, 2020). Diffusible molecules that pass from glial cells to influence NB behaviour include Dally-like (Dlp) in the perineural glia, (Kanai et al, 2018) and Jellybelly (Jeb) in the cortex glia (Cheng et al, 2011). Furthermore, organelles such as lipid droplets (LDs) in the glial niche have been shown to buffer NBs proliferation from peroxidation chain reactions induced by oxidative stress (Bailey et al, 2015), suggesting that glial niche and the signalling molecules produced by these cells are important mediators of non-autonomous regulation of NB behaviour during developmental and environmental stress. In this study, we investigate how the stem cell niche and its dysfunction influence stem cell behaviour and the consequences on the brain as a whole. We found that the signalling molecule Hedgehog (Hh), involved in numerous developmental processes, resides within the cortex glial membrane that surrounds NBs. Hh is autonomously required to promote glial niche growth as well as acts non-cell autonomously to activate the Hh signalling in the NB, triggering its delay in S phase progression. Maintaining cortex glial size is important, as overgrowth induced by FGF activation, a mutation implicated in glioblastoma (Morrison et al, 1994; Yamada et al, 1999; Dienstmann et al, 2014; Jimenez-Pascual & Siebzehnrubl, 2019), phenocopied the effects of glial Hh activation on NBs. Indeed, inhibiting Hh rescued NB proliferation defects. Furthermore, we demonstrated that downstream of glial FGF signalling, Hh activity and its ability to signal to NBs are modulated by two lipid storage regulators Lsd-2 and Fasn1. Together, our data show that a dysfunctional niche can non-autonomously affect NB’s ability to produce the correct number of neurons that make up the adult CNS. This process mechanistically involves the Hh signalling pathway and its modulation by lipid metabolism. Results Hh is localised to the LDs within cortex glial cells To identify potential morphogens that facilitate glia-NB communication in the CB, we assayed for secreted molecules which are known to act in a paracrine fashion. We found that Hh is expressed at high levels at 96 h After Larval Hatching (96ALH) in glial cells labelled using Repo-GAL4 > GFP (Fig 1B–D’). Hh is a morphogen that was first identified to regulate embryo segmentation and wing imaginal disc development (Nusslein-Volhard & Wieschaus, 1980; Heemskerk & DiNardo, 1994). In the wing disc, it is expressed in the posterior compartment and is distributed in a gradient to regulate target gene expression in the anterior compartment (Fig 1E and F) (Chen et al, 2017). In the glial niche, however, we found Hh staining was mostly distributed in ring-like structures in the glial cytoplasm (yellow arrows, Fig 1C and D’). We then assessed whether Hh is associated with specific organelles. LDs are round-shaped organelles, consisting of a hydrophobic core for the storage of neutral lipids and a phospholipid monolayer containing LD surface proteins. As LDs have previously been reported to be enriched in the glial niche (Bailey et al, 2015; Kis et al, 2015), we therefore tested whether Hh ligands are associated with LDs. Using a neutral lipid stain, lipidTOX to visualise LDs and either an antibody, or a BAC encoded Hh:GFP to detect Hh (Chen et al, 2017), we found that Hh is localised to the surface of the LDs in the glial niche (yellow arrows, Figs 1C and D’’, I, and EV1A and B’’), but not in the wing disc (white arrows, Figs 1F and G’’, J, and Fig EV1-EV5’’) nor at earlier stages of development in the CB (48 ALH, Fig EV2A and B’’). Together, our data suggest that Hh is localised to the surface of LDs in the glial niche. Click here to expand this figure. Figure EV1. Hh forms complexes with LDs in the CB but not the wing discs (related Fig 1) A–B’’. HhGFP and LDs are associated in the CB glial cells (outlined with yellow dashed lines). C–D’’. HhGFP is not associated with LDs in the posterior wing disc (white arrows, the posterior compartment is separated from the anterior with yellow dashed lines). Data information: (B-B’’, D-D’’) are zoomed in images of (A and C), respectively. Scale bar = 50 μm in (A and C). Scale bar = 10 μm in (B-B’’ and D-D’’). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Hh-LD associations are specifically observed in cortex glial cells in the CB during late larval stages (related to Fig 1) A–B’’. Hh and LDs are present at low levels at 48 ALH, and do not form specific association (white arrows, brain lobes are circled with purple dashed lines). C–D’’’. Hh and LDs are not associated in the optic lobe glial cells (white arrows). E–F’’’. Hh and LDs associate only in the cortex glial cells (yellow arrows) but not the surface glial cells (white arrows, surface glia are separated from cortex with magenta dashed lines). G. Left and middle panel, representative image showing a surface glial clone (circled with white dashed lines, repo-MARCM, glial nucleus marked by Stinger in pink). Hh is localised to the cortex glial cells (yellow arrows) underneath the clone marked in green. Right panel, a schematic depicting XZ cross-section of CB glial cells and their relative position. Data information: Glial cells are visualised with repo-GAL4 > GFP in (C-F’’’). (B-B’’, D-D’’’, F-F’’’) are zoomed in images of (A, C, E), respectively. (C) is the same image as Fig 1C, with the optic lobe region highlighted in a yellow dashed square. Scale bar = 50 μm in (C, E) and XY section in (G). Scale bar = 20 μm in (A-B’’, D-D’’’, F-F’’’), Scale bar = 10 μm for XZ section in (G). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Effects of Hh overexpression and knockdown on Hh level, NB number and EdU index (related to Fig 2) A–D. Representative images showing pan-glial Hh knockdown (repo-GAL4 > GFP with UAS-dcr2) efficiently reduces Hh staining in the CB (outlined with yellow dashed lines) and brain lobe size, quantified in (C) (n = 6, 6 brain lobes) and (D) (n = 15, 16 brain lobes), respectively. E. Representative image from EdU incorporation assays used throughout the manuscript. During a 15 min EdU pulse, type I NB (yellow arrow) and its GMC (white arrow) both incorporate EdU. EdU index quantifications include only EdU+ type I NBs. F. Hh knockdown (repo-GAL4 > GFP with UAS-dcr2) significantly reduces NB EdU index (n = 12, 9 brain lobes). G. Hh knockdown or overexpression in glial cells (repo-Gal4>) does not significantly alter the number of CB NBs (n = 12, 10; 12, 12 brain lobes). H. Schematic depicting EdU pulse-chase experiment. Larvae are fed with EdU-containing food for 3 h and then chased with EdU-free food for 3 h before CNS dissection at wandering stages. NBs and newly generated GMCs are marked with Mira; GMCs and newly generated neurons are marked with ProsGFP. I–K. Representative images showing that Hh knockdown in cortex glial cells (NP2222-GAL4) significantly reduced the number of EdU+ cells that are marked by ProsGFP+, quantified in (K) (Box plot, the boxes extend from the 25th to 75th percentiles; the median is marked by a central band inside the box; and the whiskers go down to the minimum value and up to the maximum value. n = 94, 104 NB lineages imaged from 8, 8 brain lobes, respectively). L–O. Representative images showing that Hh overexpression in cortex glial cells (NP2222-GAL4 > mGFP) does not alter cortex glial membrane size and total Repo+ glial cell numbers, quantified in (N) (n = 15, 14 brain lobes) and (O) (n = 15, 14 brain lobes), respectively. P–Q’’. Representative images showing Ptc:mcherry is expressed in NBs (yellow arrows). (Q-Q’’) are zoomed in images of (P). Data information: Scale bar = 50 μm in (A, B, E, L, M). Scale bar = 20 μm in (I, J, P, Q-Q’’). Error bar represents SEM. In (C): unpaired t-test, (****) P < 0.0001. In (D): Mann–Whitney test, (**) P = 0.0017. In (F): Welch’s t-test, (*) P = 0.0101. In (G): unpaired t-test, (ns) P = 0.3645; Mann–Whitney test, (ns) P = 0.7621. In (K): Welch’s t-test, (****) P < 0.0001. In (N): unpaired t-test, (ns) P = 0.5151. In (O): unpaired test, (ns) P = 0.9690. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Glial htlACT overexpression affects NB asymmetric division, size and cell cycle exit (related to Fig 5) A. Schematic depicting the distribution of polarity proteins in M phase NBs. Apical polarity proteins include the Par complex (aPKC/Par3/Par6), the PINS/MUD/Gαi complex and the adaptor protein, inscuteable (magenta); basal polarity complex comprises the cell fate determinants Brat/ Pros/ Numb and their adaptor proteins Mira (red) and PON. B–G. Representative images showing that in pH3+ NBs, Insc and Mira mislocalise to the cytoplasm or cortex upon FGF activation in cortex glia (NP2222-GAL4 > htlACT). H. Schematic depicting a NB undergoing telophase. I–K. Representative images showing that NBs in telophase (Mira+; pH3+) give rise to more size-symmetric daughter cells upon cortex glial (NP2222-GAL4>) htlACT overexpression, quantified in (K) (n = 43, 63 NBs from 10, 9 brain lobes, respectively). L. Glial (repo-GAL4>) htlACT overexpression causes an increase in M phase NB diameter (n = 70, 53 NBs from 12, 7 brain lobes, respectively). M–O. Representative images showing that NB nucleoli are significantly enlarged upon glial (repo-GAL4>) htlACT overexpression, quantified in (O) (n = 33, 23 NBs from 9, 7 brain lobes). NBs are marked by Histone (red), surrounded by glial cells (grey, repo-GAL4 > GFP), nucleoli are marked by Fib (Cyan). P–S. Representative images showing that the timing of NB cell cycle entry (visualised by EdU incorporation at 26ALH) is not significantly altered by pan-glial (repo-GAL4>) htlACT overexpression, quantified in (S), where EdU voxels are normalised to control (n = 19, 25 brains). The region of interest is outlined in yellow. T–W. Representative images showing that the number of CB NBs (Mira+) at 24APF is significantly increased with pan-glial (repo-GAL4>) htlACT overexpression, quantified in (W) (n = 8, 8 brains). The region of interest is outlined by yellow dashed lines and NBs are marked with yellow arrows. Data information: NBs are outlined with yellow dashed lines in (B-G, I and J). Scale bar = 50 μm in (Q, R, U, V). Scale bar = 10 μm in (B-G); Scale bar = 20 μm in (I, J, M and N). Error bar represents SEM. In (K): Mann–Whitney test, (***) P = 0.0002. In (L): Welch’s t-test, (****) P < 0.0001. In (O): unpaired t-test, (****) P < 0.0001. In (S): Welch’s t-test, (ns) P = 0.8152. In (W): unpaired t-test, (*) P = 0.0134. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Characterisation of the effects of glial hh, fasn1 and lsd2 RNAis on glial size, LDs and NB proliferation (related to Fig 6) A–D. Representative images showing that induction of hh RNAi in cortex glial cells with htlACT overexpression do not alter the size of cortex glial membrane (NP2222-GAL4 > mGFP) nor the number of LDs in CB (outlined by yellow dashed lines). E–G. Representative images showing that glial (repo-GAL4>) induction of RNAis against fasn1 and lsd2 efficiently reduce the number of LDs in CB (outlined by yellow dashed lines). H. Knockdown of lipogenesis genes fasn1 and lsd2 or overexpression of lsd2 using a cortex glial driver (NP2222-GAL4>) do not significantly affect NB EdU index (n = 10, 10; 15, 10; 10, 10 brain lobes). I. The NB EdU incorporation defects due to cortex glial (NP2222-GAL4) overexpression of htlACT is rescued by overexpression of additional RNAis lines against fasn1 and lsd2 (related to Fig 6J; n = 10, 16; 14, 14; 8, 10 brain lobes). The NP2222-GAL4 > w1118 versus htlACT columns depict the same data as those in Fig 5E. Data information: Scale bar = 50 μm in (A–G). Error bar represents SEM. In (H): Mann–Whitney test, (ns) P = 0.9555; unpaired t-test, (ns) P = 0.1799; unpaired t-test, (ns) P = 0.9574. In (I): unpaired t-test, (****) P < 0.0001; unpaired t-test, (***) P = 0.0008; unpaired t-test, (**) P = 0.0032. Download figure Download PowerPoint We next explored whether Hh-LD associations are specifically localised to a glial subtype. Hh-LD associations were largely absent from both surface glial cells that forms the blood brain barrier (BBB) of the CNS (white arrows, Fig EV2E and F’’’), as well as optic lobe glial cells (white arrows, Fig EV2C and D’’’). In fact, Hh-LD associations were enriched in the cortex glial cells, underneath the sheath-like surface glial clone generated via repo-MARCM (yellow arrows, Fig EV2G). Using a cortex-specific driver, NP2222-GAL4 (Hayashi et al, 2002; Awasaki et al, 2008), we confirmed that the Hh-LD associations were localised to the cortex glia (Fig 1K and L’’). Hh autonomously regulates cortex gliogenesis and non-autonomously regulates NB proliferation In the mouse brain, Hh has been shown to promote astrocyte proliferation (Takezaki et al, 2011; Chandra et al, 2015; Ugbode et al, 2017). In glioblastoma (GBM), the Hh/Gli1 signalling pathway acts to accelerate cell proliferation (Chandra et al, 2015). To investigate the role of Hh in Drosophila cortex glial cells, where Hh is most abundant, we used pan-glial driver repo-GAL4 to express hh RNAi together with UAS-Dcr2 (Dicer-2) which sufficiently depleted Hh expression and reduced overall CNS size (Fig EV3-EV5). The reduction in CNS volume was accounted for by a significant decrease in Repo+ glial cell number and membrane size (labelled using NP2222-GAL4 > GFP, Fig 2A–D). Using a pan-glial driver repo-GAL4, we found cortex glial chambers were significantly disrupted upon Hh knockdown (Speder & Brand, 2018; Yuan et al, 2020), leading to clustering of NBs (compare Fig 2G to F). However, overexpression of Hh did not affect glial cell number nor membrane size (Fig EV3-EV5). This suggests Hh is necessary but not sufficient for glial expansion during CNS development. Figure 2. Hh autonomously regulates cortex gliogenesis and non-autonomously regulates NB proliferation A–D. Representative images showing that upon knockdown of Hh in cortex glial cells (NP2222-GAL4 > mGFP with UAS-dcr2), cortex glial membrane and overall Repo + glial cell number are significantly reduced, quantified in (C) (n = 7, 8 brain lobes) and (D) (n = 7, 8 brain lobes), respectively. E–G. Hh knockdown in glia (repo-GAL4 > GFP) results in niche disruption and clustering of NBs (circled with yellow dashed line), as well as an increase in the percentage of NBs in M phase (pH3+), quantified in (E) (n = 12, 10 brain lobes). H–K. Representative images showing that Hh overexpression using pan-glial (repo-GAL4) and cortex glial (NP2222-GAL4) drivers both result in a decrease in NB EdU index, quantified in (J) (n = 16, 20 brain lobes) and (K) (n = 11, 14 brain lobes), respectively. L–M”. Representative images showing ci-lacZ is expressed in NBs (yellow arrows). (M-M’’) are zoomed in images of (L). N–P. Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n = 12, 10 brain lobes). Data information: NBs are marked with Mira and EdU+ NBs are circled by yellow dashed line. Scale bar = 50 μm. Error bar represents SEM. In (C): Mann–Whitney test, (**) P = 0.0059. In (D): unpaired t-test, (*) P = 0.0176. In (E): unpaired t-test, (*) P = 0.0215. In (J): unpaired t-test, (***) P = 0.0002. In (K): unpaired t-test, (***) P = 0.0001. In (P): unpaired t-test, (****) P < 0.0001. Source data are available online for this figure. Source Data for Figure 2 [embr202052130-sup-0006-SDataFig2.xlsx] Download figure Download PowerPoint Given the role of Hh as a secreted ligand that can act over short range within the NB lineage (Chai et al, 2013) and that it is highly expressed in the cortex glial niche surrounding NBs, it is plausible that glial Hh non-autonomously affects NB proliferation. We next explored the potential impact of glial Hh on the activities of type I NBs. As Hh is required to maintain the glial niche (Fig 2A–D, F and G) and niche impairment has been shown to induce NB elimination (Read, 2018), we first assessed for alterations in NB number. We found that pan-glial Hh knockdown (repo-GAL4) did not significantly alter NB number (Fig EV3G), suggesting that NB survival is unaffected. We then investigated the effects of glial Hh knockdown on NB proliferation. Glial Hh knockdown using repo-GAL4 induced a small increase in the percentage of NBs in M phase (pH3 index; Fig 2E). To assess NB S phase progression, we examined EdU (5-ethynyl-2′-deoxyuridine) incorporation during a 15-min time window (EdU index, Fig EV3E, yellow arrows). Here, we found EdU incorporation was significantly reduced upon glial Hh knockdown, suggesting that fewer NBs entered into the S phase of the cell cycle (Fig EV3F). Interestingly, a similar alteration of NB pH3 and EdU index was observed upon glial niche impairment caused by PI3K signalling inhibition (Speder & Brand, 2018). Therefore, it is likely that glial Hh depletion indirectly causes NBs to stall at M phase via inhibition of cortex glial chamber formation. To investigate the effect of glial Hh depletion on NB progeny production, we conducted EdU pulse-chase assay, where larvae were fed food supplemented with EdU for 3 h and chased for 3 h in EdU-free food (Fig EV3H). Using pros:GFP which marks individual NB lineages, we found the number of GFP+ and EdU+ cells per lineage was significantly reduced upon cortex glial Hh knockdown (Fig EV3-EV5K). Together, these results suggest that NB proliferation is inhibited upon cortex glial niche impairment caused by Hh knockdown. The subperineural glial Dlp and the cortex glial Jeb promote NB proliferation during development; however, overexpression of these signalling molecules in the glial niche was not sufficient to increase NB cell cycle rate (Cheng et al, 2011; Kanai et al, 2018). We next assessed the effect of glial Hh overexpression on NB behaviour. Pan-glial induction of Hh did not significantly alter NB number (Fig EV3G). However, pan-glial and cortex glial-specific Hh overexpression caused a reduction in NB EdU incorporation (Fig 2H–K). Using reporter lines of Hh activity ci -lacZ (Schwartz et al, 1995) and Ptc:mCherry (Varjosalo & Taipale, 2008; Chen et al, 2017), we found that Hh signalling is highly active in the NBs (Figs 2L and M’’ and EV3P and Q’’), consistent with a previous report by (Chai et al, 2013). Furthermore, activation of Hh transcriptional activator cubitus interruptus (ciNc5m5m or ciACT) with a NB-specific driv
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