Glial Hedgehog and lipid metabolism regulate neural stem cell proliferation in Drosophila
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Abstract
The final size and function of the adult central nervous system (CNS) is 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, and non-autonomously restrained to prevent ectopic Hh signalling in the NBs. In the context of cortex glial overgrowth, induced by Fibroblast Growth Factor (FGF) activation, Hh and lipid storage regulators Lsd-2 and Fasn1 were upregulated, resulting in activation of Hh signalling in the NBs; which in turn disrupted NB cell cycle progression and reduced neuronal production. We show that the LD regulator Lsd-2 modulates Hh’s ability to signal to NBs, and de novo lipogenesis gene Fasn1 regulates Hh post-translational modification via palmitoylation. Together, our data suggest that the glial niche non-autonomously regulates NB proliferation and neural lineage size via Hh signaling that is modulated by lipid metabolism genes.
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Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** In this study, the authors investigate the role of hedgehog signaling and lipid metabolism in the neural stem cell niche of the Drosophila larvae. They demonstrate that Hedgehog localizes to lipid droplets in glial cells and show that Hh is necessary but not sufficient for elaboration of glial membranes and normal rates of glial proliferation during development. In addition, they provide an extensive set of results in support of a model that FGF signaling functions upstream of lipid metabolism and hh in glial cells as well as a parallel ROS mediated pathway in glial cells to …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** In this study, the authors investigate the role of hedgehog signaling and lipid metabolism in the neural stem cell niche of the Drosophila larvae. They demonstrate that Hedgehog localizes to lipid droplets in glial cells and show that Hh is necessary but not sufficient for elaboration of glial membranes and normal rates of glial proliferation during development. In addition, they provide an extensive set of results in support of a model that FGF signaling functions upstream of lipid metabolism and hh in glial cells as well as a parallel ROS mediated pathway in glial cells to promote neuroblast proliferation. In general, the results provide strong support for the conclusions. Specifically, the approaches are sound, the images clearly demonstrate the phenotypes described, and the effects are quantified and tested for statistical significance. **Major comments:** 1.Since Hh RNAi decreases the glial compartment (which slows NB proliferation) and increases the frequency of pH3+ NBs, it is unclear why it would decrease the number of EdU+ NBs (Fig. S3C). 2.If overexpression of htl[ACT] slows the NB cell cycle (as evidenced by reduced pH3 and EdU positive cells), it unclear why it does not reduce the number of NBs (Fig. 4L). 3.What is the justification for presenting the EdU quantifications as an EdU index in which the experimental values are normalized to the average number of positive cells in the control? In many cases, the comparison is to the same w[1118] line so it does not control for a specific genetic backgrounds and yet this method may be obscuring experimental variation present between datasets. Likewise, why is glial number presented as a fold-change but NB number is presented as raw counts (e.g. 2D vs S3E)? **Minor comments:** On the top of P.14, "Figure S7A-C" should probably be "Figure S6A-C" Reviewer #1 (Significance (Required)): The cell autonomous regulation of growth and proliferation of neuroblasts in the larval brain have been well-studied, but much less is known about the non-cell autonomous signals. This paper significantly moves forward knowledge in this area by describing multiple steps of a molecular mechanism for glial regulation of the neuroblast cell cycle. These findings would be of interest not only to the study of Drosophila neuroblasts, but also to the broader adult stem cell field. My expertise is in Drosophila stem cell biology and genetics. Reviewer #2 (Evidence, reproducibility and clarity (Required)): **Summary:** The study by Dong et al., investigates the role of Hedgehog in the glial niche during larval neurogenesis in Drosophila. The authors describe the expression of Hh in cortex glia and its association with lipid droplets. They show that Hh expression in cortex glia is required for cortex glial proliferation, cell autonomously, and for maintenance of the normal cell cycle in neuroblasts. They go on to use a well characterised Drosophila glioma model, activation of FGF signalling, to investigate the requirement for Hh during cortex glial overgrowth. They show that FGF-activated cortex glial overproliferation requires Hh for modulation of neuroblast cell cycle, although Hh does not regulate cortex glial proliferation in this context. Finally, they show that inhibition of lipid modification of Hh rescues the neuroblast proliferation cell cycle defect caused by FGF activation in cortex glia. **Major comments:** 1.From the data in presented in Fig. 2H-K and Fig. S3C, I am very confused about role of Hh in the non-cell autonomous regulation of neuroblast cell cycle. Both RNAi and overexpression of Hh with Repo-Gal4 cause a reduction in the neuroblast EdU index (Fig. 2H-K and S3C). The authors conclude this section on p.7 saying "Together, our data suggests that high levels of glial Hh expression restricts NB cell cycle progression." This statement is not consistent with data. What is the normal physiological role of Hh if both decreased and increased levels of cortex glial Hh expression reduce neuroblast cell cycle? The discussion of p.15 does not clarify this issue. The model in Fig.7J relates to the role of Hh in the context of cortex glial FGF activation and does not illustrate the normal physiological role of Hh in the regulation of neuroblast cell cycle. 2.P.8 "Analysis of the total glial cell number indicates overexpression of htlACT, but not InRwt or EgfrACT, led to an increase in the number of cortex glial cells (Figure 4E-G, I-K)." This statement is confusing as Repo staining was used to quantify total glial numbers (including perineural, sub-perineural and cortex glia) but these data are then taken to represent and increase specifically in cortex glia. This should be clarified. 3.It should be mentioned on p.8 that the data in Fig.4A-K reproduce the findings of Avet-Rochex et al., 2012 and Read et al., 2009. 4.Figure 6F. Presumably due to the increase in glia cell number and dramatic increase in glial cell volume, any gene that is specific to, or enriched in, cortex glia will have increased expression levels in RepoGal4>htlACT larval CNS. Can the authors provide evidence that the increase in the expression of these genes is specific to FGF transcriptional regulation and not just a relative increase in the levels of these genes due to an increase in cortex glia as proportion of total CNS volume? Is there any evidence that Hh, fasn1 and lsd2 are direct transcriptional targets of FGF signalling in glia? 5.FGF signalling has been shown to be necessary and sufficient for cortex glial proliferation. So does knockdown of Htl, or expression of dominant negative Htl, cause a reduction in Hh, fasn1 and lsd2 expression in cortex glia? If so, does how does reduction of cortex glial numbers independent of FGF signalling, using for example knockdown of String or expression of Decapo, affect the expression of Hh, fasn1 and lsd2 in cortex glia? 6.Can the authors speculate on why and how increased levels of Hh in cortex glia, in the context of FGF activation, inhibit neuroblast cell cycle? Is this a physiological mechanism to limit neuroblast proliferation in the face of increased gliogenesis, or is it simply an indirect result of 'spillover' of excess Hh from cortex glia onto neuroblasts (which are autonomously regulated by Hh and so sensitive to this ligand) by due to increased cortex glia cells? **Minor comments:** -Figure 1C' some lipid droplets are extremely large, is this consistent with previous literature? -Including a profile plot of relative fluorescence intensity in Figure 1C',F',H' to illustrate colocalization of lipidTOX and Hh, would be helpful. -Figure S3A,B quantify Hh protein level and CNS size phenotypes with Hh RNAi. -p.6 include data showing overexpression of Hh does not cause glial overgrowth. -Top of p.14 should be FigS6A-C. -Include quantification of glial overgrowth and lipid droplet phenotypes with HtlACT plus catalase and SOD1 overexpression (Fig. S6D-K). Reviewer #2 (Significance (Required)): The is a novel and very interesting study, well written and the data are very clearly presented. It builds on and adds to the emerging literature on the glial niche and its role in neural stem cell regulation. It will be of great interest to Drosophila neurobiologists but also to the broader field of neural stem cell biology. My expertise is Drosophila neurobiology.
Dear editor
Below is our response to the reviewer’s comments and our experimental plan in addressing these concerns.
__Reviewer #1 __
Major comments:
1.Since Hh RNAi decreases the glial compartment (which slows NB proliferation) and increases the frequency of pH3+ NBs, it is unclear why it would decrease the number of EdU+ NBs (Fig. S3C).
Our experimental data suggests that accompanying glial niche disruption and downregulation of glia-derived signals, NBs are stalled in M phase (we detected an increase in the percentage of pH3+ NBs). As a consequence, less NBs are in G1 and S phase. Therefore, when we conducted a 15-min EdU incorporation, we observed a reduction in EdU incorporation. This NB phenotype (increase in pH3 index and decrease in EdU index) was also observed by Speder and Brand, 2018, when they induced glial niche impairment by inhibiting the PI3K signaling pathway (discussed in P7 of this ms).
To address whether glial-Hh knockdown reduces the ability of NBs to produce progeny, we plan to carry out two experiments:
We will assess the total number of neurons in the CB by assessing Elav+ neurons.
We will conduct two EdU pulse-chase experiments. First, we will assess the total number of EdU+ neurons produced within a 4-hr time window (neurons marked with Elav); and the secondly, we will mark the NB lineage (with either nerfin-1-GFP or pros-GFP) and quantify the number of EdU+ neurons produced per lineage during a 4-hr time window.
Together, these experiments should allow us to assess the consequence of glial-Hh knockdown on NB proliferation.
If overexpression of htl[ACT] slows the NB cell cycle (as evidenced by reduced pH3 and EdU positive cells), it unclear why it does not reduce the number of NBs (Fig. 4L).
The number of NBs in the larval CNS is specified at the beginning of post-embryonic neurogenesis, when quiescent NBs re-enter the cell cycle (reviewed by Homem and Knoblich, 2012). Once NBs re-enter the cell cycle, the number of NBs remain constant. NBs undergo asymmetric division to produce one daughter NB and a GMC, which divides once to generate two neurons. With each round of NB-division, the number of NBs remain constant. Therefore, changes in NB cell cycle speed does not alter the overall NB number, only the number of neurons produced.
To clarify this, we will add a schematic depicting NB asymmetric division to Figure 1.
3.What is the justification for presenting the EdU quantifications as an EdU index in which the experimental values are normalized to the average number of positive cells in the control?
EdU index is calculated as number of EdU+ NBs normalised to control EdU+ NBs. The number of EdU+ NBs reflects the NBs that progress through S phase in a 15-min time relative to the control. A similar method was used in Kanai et al., 2018. This method would not be valid only if NB number varied between control and experimental data sets, however, the number of NBs in all our genetic manipulations are not significantly altered relative to their control. We present the quantification of some key manipulations in Reviewer_Figure 1A, B.
As regards to why we normalise to control in each of these experiments, this is because in-vitro EdU incorporation rely on Click-IT chemistry, which is inherently variable due to incubation conditions. To overcome this, we always incubate control and experimental brains in the same tube and imaged them with the same confocal setting, and each experiment is normalised to its control done in parallel. We have now included Table 1 which includes all the raw data from these experiments (Table 1)
In the revised manuscript, we will clarify our methodology in greater detail in the Methods section, and we are happy to include Table 1in the supplementary data.
In many cases, the comparison is to the same w [1118] line so it does not control for a specific genetic backgrounds and yet this method may be obscuring experimental variation present between datasets.
We have used three different controls in our experiments, namely GAL4 or lexA >w1118, or UAS-mcherryRNAi, or UAS-luc. We detect no significant difference in terms of raw EdU+ NB numbers between the controls used in our experiments, as demonstrated below (Reviewer_Figure 1C). In our revised manuscript, we will include a sentence “As UAS-mcherryRNAi or UAS-luc are indistinguishable from the > w1118 control, we have used GAL4 driver > w1118 as control in place of UAS-luc in our results”.
Reviewer_Figure 1. Total NB number and Edu+ NB number quantification
- A) Hh knockdown or overexpression in glia does not significantly alter NB number compared to control.
- B) htlACT overexpression in glia does not significantly alter NB number compared to control.
- C) EdU+ NB number is not significantly different within the controls GAL4 or lexA >* w1118*, or UAS-mcherryRNAi, or UAS-luc. P-value was obtained performing student t-test in A, B and One-way ANOVA in C.
Likewise, why is glial number presented as a fold-change but NB number is presented as raw counts (e.g. 2D vs S3E)?
Glial number quantification was carried out using Fiji 3D object counter and a plug-in called “DeadEasy Larval Glia” (Forero et al., 2012), where the threshold of detection is dependent on the brightness of Repo staining in each experiment, this data is presented as fold-change, as control and experiment stained in the same tube are compared to each other. We represented this data as fold-change to allow easy comparison between experiments. The raw data is presented in Table 2. NB number is counted manually and is therefore presented as raw counts.
**Minor comments:**
On the top of P.14, "Figure S7A-C" should probably be "Figure S6A-C"
We will correct this.
Reviewer #1 (Significance (Required)):
The cell autonomous regulation of growth and proliferation of neuroblasts in the larval brain have been well-studied, but much less is known about the non-cell autonomous signals. This paper significantly moves forward knowledge in this area by describing multiple steps of a molecular mechanism for glial regulation of the neuroblast cell cycle. These findings would be of interest not only to the study of Drosophila neuroblasts, but also to the broader adult stem cell field.
My expertise is in Drosophila stem cell biology and genetics.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
**Major comments:**
1.From the data in presented in Fig. 2H-K and Fig. S3C, I am very confused about role of Hh in the non-cell autonomous regulation of neuroblast cell cycle. Both RNAi and overexpression of Hh with Repo-Gal4 cause a reduction in the neuroblast EdU index (Fig. 2H-K and S3C). The authors conclude this section on p.7 saying "Together, our data suggests that high levels of glial Hh expression restricts NB cell cycle progression." This statement is not consistent with data. What is the normal physiological role of Hh if both decreased and increased levels of cortex glial Hh expression reduce neuroblast cell cycle? The discussion of p.15 does not clarify this issue. The model in Fig.7J relates to the role of Hh in the context of cortex glial FGF activation and does not illustrate the normal physiological role of Hh in the regulation of neuroblast cell cycle.
With repo-GAL4>hhRNAi, the cortex glial niche enwrapping NBs is dramatically disrupted, which indirectly alters NB cell cycle progression, indicated by an increase in pH3 index and a decrease in EdU index. From these two pieces of data, it is likely that NBs are stuck in M phase, thus resulting in less NBs in G1 and S phase that are capable to incorporate EdU within a 15-min incubation time window. We will firm up this data with experiments proposed to address concerns of Reviewer 1, Point 1.
Both RNAi and overexpression of Hh with repo-GAL4 causes a reduction in NB EdU index is seemingly contradictory. However, it is consistent with a previous report from Speder and Brand, 2018, where it was shown that that glial niche impairment induced by the PI3K pathway inhibition also causes a similar NB phenotype (an increase in pH3 index and a decrease in EdU incorporation). Furthermore, with repo-GAL4>htlDN, which caused a similar glia niche impairment (data not shown), we observed a similar phenotype (an increase in pH3 index and a slight decrease in EdU incorporation). Therefore, we concluded that the NB cell cycle progression defects is due to a general cortex glial niche disruption rather than a direct effect of Hh inhibition on NBs. We are happy to include the repo-GAL4>htlDN data in the supplementary data if required.
With regards to the physiological role of Hh, we can only conclude from the data at hand that Hh is required for the development of cortex glial niche, which is required to maintain NB activities. In terms of how glial niche impairment impedes NB cell cycle progression, we observed that without a proper niche chamber, NBs cluster together instead of residing in separate niches (Figure 2F-G). Therefore, it is possible that the localization of other cell types (i.e. GMCs and neurons) are also altered as a result of NB clustering, which can potentially affect the NB cell cycle. While these questions will be interesting to explore in the future, they are beyond the scope of this current study.
In contrast, we robustly showed Hh signals, when overexpressed in glial niche, were capable of making contact with NBs (Figure 7C-C’) and triggering a slow-down of NB S-phase progression. Therefore, it is fair to conclude that “high levels of glial Hh expression restricts NB cell cycle progression”.
In the revised manuscript, we will discuss these findings in greater detail.
2.P.8 "Analysis of the total glial cell number indicates overexpression of htlACT, but not InRwt or EgfrACT, led to an increase in the number of cortex glial cells (Figure 4E-G, I-K)." This statement is confusing as Repo staining was used to quantify total glial numbers (including perineural, sub-perineural and cortex glia) but these data are then taken to represent and increase specifically in cortex glia. This should be clarified.
We thank the reviewer for picking this up. Our intention was to quantify the number of cortex glia cells in glial-specific htlACT, InRwt and EgfrACT manipulations. However, two reported cortex glial antibodies (PntP2 from Avet-Rochex et al., 2012 and SoxN described in Read, 2018), showed unspecific labelling of other cell types (Reviewer_Figure 2, arrows, neurons and NBs). As an alternative, we quantified the total glial cell number (Repo+) in htlACT, InRwt or EgfrACT overexpressed using a cortex glial driver (NP2222-GAL4). We expect that the alterations in glial cell number would be primarily attributed to cortex glial-specific gene manipulation. We agree that we should say that “overexpression of htlACT, but not InRwt or EgfrACT, led to an increase in the number of glial cell”.
In the revised manuscript, we will clarify this in the results section.
Reviewer_Figure 2: PntP2 staining in the larval CNS.
A-B) Representative images showing that PntP2 antibody stains cortex glial cells (marked by NP2222-GAL4>mGFP, yellow arrows)*, *NBs (white arrows) and neurons (blue arrows). B) is the zoomed in image of A). Scale bar = 50 mm.
It should be mentioned on p.8 that the data in Fig.4A-K reproduce the findings of Avet-Rochex et al., 2012 and Read et al., 2009.
We will correct this.
4.Figure 6F. Presumably due to the increase in glia cell number and dramatic increase in glial cell volume, any gene that is specific to, or enriched in, cortex glia will have increased expression levels in RepoGal4>htlACT larval CNS. Can the authors provide evidence that the increase in the expression of these genes is specific to FGF transcriptional regulation and not just a relative increase in the levels of these genes due to an increase in cortex glia as proportion of total CNS volume? Is there any evidence that Hh, fasn1 and lsd2 are direct transcriptional targets of FGF signalling in glia?
We agree that FGF activation causes a dramatic increase in glial cell number, thus will cause a relative increase in the level of hh, fasn1 and lsd2s. However, with RT-qPCR, the same amounts of total RNA (1μg) were extracted from control vs repo-GAL4> htlACT and reverse transcribed into cDNA for qPCR. Therefore, the mRNA level described in Figure 6 F are already normalized to the total amount of genetic material.
In the literature, it is not reported that hh, fasn1 and lsd2 are direct transcriptional targets of FGF signalling. However, lipid metabolism rewiring is well known as a hallmark of glioblastoma. For example, high levels of FASN has been linked with high grade glioblastoma (Grube et al., 2014). Furthermore, FGF signalling has also been shown to modulate lipid metabolism and alter the transcription of the Lsd-2 homologue called Plin2 in a mouse model (Ye et al., 2016).
To figure out whether hh, fasn1 and *lsd2 *are direct transcriptional targets of FGF signalling. we will have to first find out which TFs are altered in the glia upon altered FGF signalling via cortex glia specific RNA-seq, and then conduct DamID to identify their target genes. This would be interesting to follow-up but is however beyond the scope this current study.
We will add a section on this in the discussion section of the revised ms.
FGF signalling has been shown to be necessary and sufficient for cortex glial proliferation. So does knockdown of Htl, or expression of dominant negative Htl, cause a reduction in Hh, fasn1 and lsd2 expression in cortex glia?
In response to glial htlDN overexpression, we observed a significant reduction in total glial number and overall Hh expression. However, RT-qPCR showed that mRNA levels of hh, fasn1 or lsd-2 were not altered upon htlDNoverexpression (Reviewer_Figure 3).
This data will be included in the supplementary data in the revised ms.
Reviewer_Figure 3. Glial htlDN overexpression doesn’t alter the expression of hh, fasn1 and lsd2. The mRNA levels of hh, fasn1 and lsd2 are normalized to the reference gene rpl32.
Continued: If so, how does reduction of cortex glial numbers independent of FGF signalling, using for example knockdown of String or expression of Decapo, affect the expression of Hh, fasn1 and lsd2 in cortex glia?
To address this question, we plan to assess the expression levels of hh, fasn1 and lsd-2 using glia specific expression of an inhibitor of the PI3K (delta p60), which has been shown by Speder and Brand, 2018 to cause a reduction in cortex glial number. We will also ascertain whether Decapo overexpression causes cortex glial niche impairment. If so, we will also assess the expression levels of hh, fasn1 and lsd-2 in this setting.
6.Can the authors speculate on why and how increased levels of Hh in cortex glia, in the context of FGF activation, inhibit neuroblast cell cycle? Is this a physiological mechanism to limit neuroblast proliferation in the face of increased gliogenesis, or is it simply an indirect result of 'spillover' of excess Hh from cortex glia onto neuroblasts (which are autonomously regulated by Hh and so sensitive to this ligand) by due to increased cortex glia cells?
We favour the model that excess Hh in the glia compartment “spills over” to reduce NB proliferation, which are autonomously regulated by Hh and therefore are sensitive to this ligand. We can add this to the discussion.
**Minor comments:**
-Figure 1C' some lipid droplets are extremely large, is this consistent with previous literature?
These large lipid droplets are caused by lipid droplet fusion due to the use of detergent in this experiment. When we perform antibody staining together with lipid droplet staining, PBST detergent is required for antibody staining to work. However, this created the artefact of large lipid droplets, due to lipid droplet fusion. This has previously been reported by Bailey et al., 2015, and we have explained this in P19 of the Method section.
-Including a profile plot of relative fluorescence intensity in Figure 1C',F',H' to illustrate colocalization of lipidTOX and Hh, would be helpful.
We will include this in the revised ms.
-Figure S3A,B quantify Hh protein level and CNS size phenotypes with Hh RNAi.
We will include this in the revised ms.
-p.6 include data showing overexpression of Hh does not cause glial overgrowth.
We will include this in the revised ms.
-Top of p.14 should be FigS6A-C.
We will correct this.
-Include quantification of glial overgrowth and lipid droplet phenotypes with HtlACT plus catalase and SOD1 overexpression (Fig. S6D-K).
We will include this in the revised ms.
Reviewer #2 (Significance (Required)):
The is a novel and very interesting study, well written and the data are very clearly presented. It builds on and adds to the emerging literature on the glial niche and its role in neural stem cell regulation. It will be of great interest to Drosophila neurobiologists but also to the broader field of neural stem cell biology.
My expertise is Drosophila neurobiology.
Table 1. EdU+ NB numbers for each genotype described in each Figure
Figure
Genotype
EdU incubation time
Average EdU+ NB number
SEM
Number of samples
Figure 2J
repo-GAL4>w1118
15 min
66.63
1.79
16
Figure 2J
repo-GAL4>UAS-hh
15 min
57.35
1.35
20
Figure 2K
NP2222-GAL4>w1118
15 min
67.91
1.44
11
Figure 2K
NP2222-GAL4>UAS-hh
15 min
60.79
0.79
14
Figure 2P
dnab-GAL4>w1118
15 min
70.5
1.44
12
Figure 2P
dnab-GAL4>ciACT
15 min
60.1
1.48
10
Figure S3C
repo-GAL4>dcr2; mcherryRi
10 min
57.42
0.63
12
Figure S3C
repo-GAL4>dcr2; hhRi43255
10 min
48.56
2.65
9
Figure 3K
NP2222-GAL4>w1118
The same dataset as Figure 2K
Figure 3K
NP2222-GAL4>UAS-hh
Figure 3K
NP2222-GAL4>UAS-hh; mcherryRi
15 min
57.44
1.41
16
Figure 3K
NP2222-GAL4>UAS-hh; lsdRi34617
15 min
63.36
1.34
14
Figure 3K
NP2222-GAL4>UAS-hh; mcherryRi
15 min
58.83
2.61
6
Figure 3K
NP2222-GAL4>UAS-hh; lsdRi32846
15 min
64.5
1.2
14
Figure 5E
repo-GAL4>w1118
15 min
71.6
1.28
15
Figure 5E
repo-GAL4>UAS-htlACT
15 min
56
1.59
14
Figure 5E
NP2222-GAL4>w1118
15 min
70.2
1.58
10
Figure 5E
NP2222-GAL4>UAS-htlACT
15 min
54.75
1.24
16
Figure 6G
NP2222-GAL4>w1118
The same dataset as Figure 5E
Figure 6G
NP2222-GAL4>UAS-htlACT
Figure 6G
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
60
1.24
7
Figure 6G
NP2222-GAL4>UAS-htlACT;hhRi43255
15 min
67.17
1.13
12
Figure 6G
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
59.29
1.79
14
Figure 6G
NP2222-GAL4>UAS-htlACT;hhRi25794
15 min
68.55
1.68
11
Figure 6H
dnab-GAL4>mcherryRi
10 min
49.13
1.6
8
Figure 6H
dnab-GAL4>ciRi2125-R2
10 min
56.54
1.27
13
Figure 6H
repo-lexA>w1118
15 min
68.5
1.1
10
Figure 6H
repo-lexA>lexAop-htlACT
15 min
55.7
2.15
10
Figure 6H
repo-lexA>lexAop-htlACT; GFPRi
15 min
52
1.58
30
Figure 6H
repo-lexA>lexAop-htlACT; ciRiHMJ23860
15 min
62.4
1.79
15
Figure 6H
repo-lexA>lexAop-htlACT; GFPRi
15 min
56.33
1.49
12
Figure 6H
repo-lexA>lexAop-htlACT; ciRi2125-R2
15 min
62.86
1.81
7
Figure 6J
NP2222-GAL4>w1118
The same dataset as Figure 5E
Figure 6J
NP2222-GAL4>UAS-htlACT
Figure 6J
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
58.64
0.99
14
Figure 6J
NP2222-GAL4>UAS-htlACT;fasn1Ri3523R2
15 min
65
2.41
9
Figure 6J
NP2222-GAL4>UAS-htlACT;mcherryRi
The same dataset as Figure 6G control of NP2222-GAL4>UAS-htlACT;hhRi25794
Figure 6J
NP2222-GAL4>UAS-htlACT;lsd2Rikk102269
15 min
68.13
1.08
8
Figure S5H
NP2222-GAL4>mcherryRi
15 min
66.4
1.71
10
Figure S5H
NP2222-GAL4>fasn1Ri3523R6
15 min
65.5
1.38
10
Figure S5H
NP2222-GAL4>mcherryRi
15 min
66.4
1.13
15
Figure S5H
NP2222-GAL4>lsd2Rikk102269
15 min
64.2
0.94
10
Figure S5H
NP2222-GAL4>UAS-luc
15 min
65
1.07
10
Figure S5H
NP2222-GAL4>UAS-lsd2
15 min
64.9
1.51
10
Figure S5I
NP2222-GAL4>w1118
The same dataset as Figure 5E
Figure S5I
NP2222-GAL4>UAS-htlACT
Figure S5I
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
57.93
0.9
14
Figure S5I
NP2222-GAL4>UAS-htlACT;fasn1Ri3523R6
15 min
63.79
1.25
14
Figure S5I
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
50.25
2.52
8
Figure S5I
NP2222-GAL4>UAS-htlACT;lsd2Ri32846
15 min
59.3
1.2
10
Figure 7B
NP2222-GAL4>mcherryRi
15 min
65
0.93
10
Figure 7B
NP2222-GAL4>raspRi11495R2
15 min
65.13
1.29
15
Figure 7B
NP2222-GAL4>w1118
The same dataset as Figure 5E
Figure 7B
NP2222-GAL4>UAS-htlACT
Figure 7B
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
58.33
1.06
18
Figure 7B
NP2222-GAL4>UAS-htlACT;raspRi11495R1
15 min
63.95
1.05
21
Figure 7B
NP2222-GAL4>UAS-htlACT;mcherryRi
15 min
59.04
1.019
26
Figure 7B
NP2222-GAL4>UAS-htlACT;raspRi11495R2
15 min
63.07
0.92
29
Figure 7D
NP2222-GAL4>w1118
15 min
69.46
1.02
13
Figure 7D
NP2222-GAL4>UAS-hh.N.EGFP
15 min
52.25
1.9
12
Figure 7F
repo-GAL4>UAS-hh.N.EGFP;mcherryRi
15 min
54.4
1.18
15
Figure 7D
repo-GAL4>UAS-hh.N.EGFP;fasn1Ri3523R2
15 min
65.69
1.43
13
Figure S6L
NP2222-GAL4>UAS-htlACT; UAS-LacZ
15 min
59.17
1.18
12
Figure S6L
NP2222-GAL4>UAS-htlACT; UAS-Cat.A
15 min
64
1.31
12
Figure S6L
NP2222-GAL4>UAS-htlACT; UAS-LacZ
15 min
53.6
2.32
10
Figure S6L
NP2222-GAL4>UAS-htlACT; UAS-Sod.1
15 min
62.7
1.76
10
Table 2. Raw data on glial number
Figure
Genotype
Average Repo+glial number
SEM
Number of samples
Figure 2D
repo-GAL4>dcr2; mcherryRi
843
44.29
7
Figure 2D
repo-GAL4>dcr2; hhRi43255
666.5
46.77
8
Figure 4K
NP2222-GAL4>w1118
1165
20.55
10
Figure 4K
NP2222-GAL4>htlACT
2325
107.5
10
Figure 4K
NP2222-GAL4>InRwt
1189
85.92
10
Figure 4K
wrapper-GAL4>w1118
1305
51.78
7
Figure 4K
wrapper-GAL4>EgfrACT
1192
38.16
12
Reference:
Avet-Rochex, A., Kaul, A.K., Gatt, A.P., McNeill, H., and Bateman, J.M. (2012). Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain. Development* 139*, 2763-2772.
Bailey, A.P., Koster, G., Guillermier, C., Hirst, E.M., MacRae, J.I., Lechene, C.P., Postle, A.D., and Gould, A.P. (2015). Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila. Cell* 163*, 340-353.
Forero, M.G., Kato, K., and Hidalgo, A. (2012). Automatic cell counting in vivo in the larval nervous system of Drosophila. J Microsc* 246*, 202-212.
Grube, S., Dunisch, P., Freitag, D., Klausnitzer, M., Sakr, Y., Walter, J., Kalff, R., and Ewald, C. (2014). Overexpression of fatty acid synthase in human gliomas correlates with the WHO tumor grade and inhibition with Orlistat reduces cell viability and triggers apoptosis. J Neurooncol* 118*, 277-287.
Homem, C.C., and Knoblich, J.A. (2012). Drosophila neuroblasts: a model for stem cell biology. Development* 139*, 4297-4310.
Kanai, M.I., Kim, M.J., Akiyama, T., Takemura, M., Wharton, K., O'Connor, M.B., and Nakato, H. (2018). Regulation of neuroblast proliferation by surface glia in the Drosophila larval brain. Sci Rep* 8*, 3730.
Read, R.D. (2018). Pvr receptor tyrosine kinase signaling promotes post-embryonic morphogenesis, and survival of glia and neural progenitor cells in Drosophila. Development* 145*.
Speder, P., and Brand, A.H. (2018). Systemic and local cues drive neural stem cell niche remodelling during neurogenesis in Drosophila. Elife* 7*.
Ye, M., Lu, W., Wang, X., Wang, C., Abbruzzese, J.L., Liang, G., Li, X., and Luo, Y. (2016). FGF21-FGFR1 Coordinates Phospholipid Homeostasis, Lipid Droplet Function, and ER Stress in Obesity. Endocrinology* 157*, 4754-4769.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The study by Dong et al., investigates the role of Hedgehog in the glial niche during larval neurogenesis in Drosophila. The authors describe the expression of Hh in cortex glia and its association with lipid droplets. They show that Hh expression in cortex glia is required for cortex glial proliferation, cell autonomously, and for maintenance of the normal cell cycle in neuroblasts. They go on to use a well characterised Drosophila glioma model, activation of FGF signalling, to investigate the requirement for Hh during cortex glial overgrowth. They show that FGF-activated cortex glial overproliferation requires Hh for …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
Summary:
The study by Dong et al., investigates the role of Hedgehog in the glial niche during larval neurogenesis in Drosophila. The authors describe the expression of Hh in cortex glia and its association with lipid droplets. They show that Hh expression in cortex glia is required for cortex glial proliferation, cell autonomously, and for maintenance of the normal cell cycle in neuroblasts. They go on to use a well characterised Drosophila glioma model, activation of FGF signalling, to investigate the requirement for Hh during cortex glial overgrowth. They show that FGF-activated cortex glial overproliferation requires Hh for modulation of neuroblast cell cycle, although Hh does not regulate cortex glial proliferation in this context. Finally, they show that inhibition of lipid modification of Hh rescues the neuroblast proliferation cell cycle defect caused by FGF activation in cortex glia.
Major comments:
1.From the data in presented in Fig. 2H-K and Fig. S3C, I am very confused about role of Hh in the non-cell autonomous regulation of neuroblast cell cycle. Both RNAi and overexpression of Hh with Repo-Gal4 cause a reduction in the neuroblast EdU index (Fig. 2H-K and S3C). The authors conclude this section on p.7 saying "Together, our data suggests that high levels of glial Hh expression restricts NB cell cycle progression." This statement is not consistent with data. What is the normal physiological role of Hh if both decreased and increased levels of cortex glial Hh expression reduce neuroblast cell cycle? The discussion of p.15 does not clarify this issue. The model in Fig.7J relates to the role of Hh in the context of cortex glial FGF activation and does not illustrate the normal physiological role of Hh in the regulation of neuroblast cell cycle.
2.P.8 "Analysis of the total glial cell number indicates overexpression of htlACT, but not InRwt or EgfrACT, led to an increase in the number of cortex glial cells (Figure 4E-G, I-K)." This statement is confusing as Repo staining was used to quantify total glial numbers (including perineural, sub-perineural and cortex glia) but these data are then taken to represent and increase specifically in cortex glia. This should be clarified.
3.It should be mentioned on p.8 that the data in Fig.4A-K reproduce the findings of Avet-Rochex et al., 2012 and Read et al., 2009.
4.Figure 6F. Presumably due to the increase in glia cell number and dramatic increase in glial cell volume, any gene that is specific to, or enriched in, cortex glia will have increased expression levels in RepoGal4>htlACT larval CNS. Can the authors provide evidence that the increase in the expression of these genes is specific to FGF transcriptional regulation and not just a relative increase in the levels of these genes due to an increase in cortex glia as proportion of total CNS volume? Is there any evidence that Hh, fasn1 and lsd2 are direct transcriptional targets of FGF signalling in glia?
5.FGF signalling has been shown to be necessary and sufficient for cortex glial proliferation. So does knockdown of Htl, or expression of dominant negative Htl, cause a reduction in Hh, fasn1 and lsd2 expression in cortex glia? If so, does how does reduction of cortex glial numbers independent of FGF signalling, using for example knockdown of String or expression of Decapo, affect the expression of Hh, fasn1 and lsd2 in cortex glia?
6.Can the authors speculate on why and how increased levels of Hh in cortex glia, in the context of FGF activation, inhibit neuroblast cell cycle? Is this a physiological mechanism to limit neuroblast proliferation in the face of increased gliogenesis, or is it simply an indirect result of 'spillover' of excess Hh from cortex glia onto neuroblasts (which are autonomously regulated by Hh and so sensitive to this ligand) by due to increased cortex glia cells?
Minor comments:
-Figure 1C' some lipid droplets are extremely large, is this consistent with previous literature?
-Including a profile plot of relative fluorescence intensity in Figure 1C',F',H' to illustrate colocalization of lipidTOX and Hh, would be helpful.
-Figure S3A,B quantify Hh protein level and CNS size phenotypes with Hh RNAi.
-p.6 include data showing overexpression of Hh does not cause glial overgrowth.
-Top of p.14 should be FigS6A-C.
-Include quantification of glial overgrowth and lipid droplet phenotypes with HtlACT plus catalase and SOD1 overexpression (Fig. S6D-K).
Significance
The is a novel and very interesting study, well written and the data are very clearly presented. It builds on and adds to the emerging literature on the glial niche and its role in neural stem cell regulation. It will be of great interest to Drosophila neurobiologists but also to the broader field of neural stem cell biology.
My expertise is Drosophila neurobiology.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
Summary:
In this study, the authors investigate the role of hedgehog signaling and lipid metabolism in the neural stem cell niche of the Drosophila larvae. They demonstrate that Hedgehog localizes to lipid droplets in glial cells and show that Hh is necessary but not sufficient for elaboration of glial membranes and normal rates of glial proliferation during development. In addition, they provide an extensive set of results in support of a model that FGF signaling functions upstream of lipid metabolism and hh in glial cells as well as a parallel ROS mediated pathway in glial cells to promote neuroblast proliferation. In general, …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
In this study, the authors investigate the role of hedgehog signaling and lipid metabolism in the neural stem cell niche of the Drosophila larvae. They demonstrate that Hedgehog localizes to lipid droplets in glial cells and show that Hh is necessary but not sufficient for elaboration of glial membranes and normal rates of glial proliferation during development. In addition, they provide an extensive set of results in support of a model that FGF signaling functions upstream of lipid metabolism and hh in glial cells as well as a parallel ROS mediated pathway in glial cells to promote neuroblast proliferation. In general, the results provide strong support for the conclusions. Specifically, the approaches are sound, the images clearly demonstrate the phenotypes described, and the effects are quantified and tested for statistical significance.
Major comments:
1.Since Hh RNAi decreases the glial compartment (which slows NB proliferation) and increases the frequency of pH3+ NBs, it is unclear why it would decrease the number of EdU+ NBs (Fig. S3C).
2.If overexpression of htl[ACT] slows the NB cell cycle (as evidenced by reduced pH3 and EdU positive cells), it unclear why it does not reduce the number of NBs (Fig. 4L).
3.What is the justification for presenting the EdU quantifications as an EdU index in which the experimental values are normalized to the average number of positive cells in the control? In many cases, the comparison is to the same w[1118] line so it does not control for a specific genetic backgrounds and yet this method may be obscuring experimental variation present between datasets. Likewise, why is glial number presented as a fold-change but NB number is presented as raw counts (e.g. 2D vs S3E)?
Minor comments:
On the top of P.14, "Figure S7A-C" should probably be "Figure S6A-C"
Significance
The cell autonomous regulation of growth and proliferation of neuroblasts in the larval brain have been well-studied, but much less is known about the non-cell autonomous signals. This paper significantly moves forward knowledge in this area by describing multiple steps of a molecular mechanism for glial regulation of the neuroblast cell cycle. These findings would be of interest not only to the study of Drosophila neuroblasts, but also to the broader adult stem cell field.
My expertise is in Drosophila stem cell biology and genetics.
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