Comparative developmental transcriptomics of Drosophila mushroom body neurons highlights the mevalonate pathway as a regulator of axon growth

Reviewer #1 (Public review):

Summary:

Fahdan et al. present a study investigating the molecular programs underlying axon initial growth and regrowth in Drosophila mushroom body (MB) neurons. The authors leverage the fact that different Kenyon cell (KC) subtypes undergo distinct axonal events on the same developmental timeline: γ KCs prune and then regrow their axons during early pupation, whereas α/β KCs extend their axons for the first time during the same pupal period. Using bulk Smart-seq2 RNA sequencing across six developmental time points, the authors identify genes enriched during γ KC regrowth and α/β KC initial outgrowth, and subsequently perform an RNAi screen to determine which candidates are functionally required for these processes.

Among these, they focus on Pmvk, a key enzyme in the mevalonate pathway. Both RNAi knockdown and a CRISPR-generated mutant produce strong γ KC regrowth defects. Knockdown of other mevalonate pathway components (Hmgcr, Mvk) partially recapitulates this phenotype. The authors propose that Pmvk promotes axonal regrowth through effects on the TOR pathway.

Overall, this work identifies new molecular players in developmental axon remodeling and provides intriguing evidence connecting Pmvk to γ KC regrowth.

While the Pmvk knockdown and loss-of-function data are compelling, the evidence that the mevalonate pathway broadly regulates γ KC axon regrowth is less clear. RNAi knockdown of enzymes upstream of Pmvk (Hmgcr, Mvk) produces only mild phenotypes, and knockdown of several downstream enzymes produces no phenotype. The authors attribute this discrepancy to the possibility of weak RNAi constructs, which is plausible but not fully demonstrated. It would be helpful for the authors to discuss alternative explanations, including non-canonical roles for Pmvk that may not require the full pathway, and clarify the extent to which the current data support the conclusion that the mevalonate pathway, rather than Pmvk specifically, is a core regulator of regrowth.

It is not clear from the Methods whether γ KCs and α/β KCs were sorted from the same brains using orthogonal binary expression systems (e.g., Gal4 > reporter 1 and LexA > reporter 2), or isolated separately from different fly lines. If the latter, differences in genetic background, staging, or batch effects could influence transcriptional comparisons. This should be explicitly clarified in the Methods, and any associated limitations discussed in the manuscript.

The authors have made important findings that contribute to our understanding of axon growth and regrowth. As written, some major claims are only partially supported, but these issues can be addressed through reframing and clarification. In particular, the manuscript would benefit from (1) a more cautious interpretation of the mevalonate pathway's role, potentially considering Pmvk non-canonical functions, and (2) addressing methodological ambiguities in the transcriptomic analysis.

Reviewer #2 (Public review):

Fahdan et al. set out to build upon their previous work outlining the genes involved in axon growth, targeting two axon growth states: initial growth and regrowth. They outline a debate in the field that axon regrowth (For instance, after injury or in the peripheral nervous system) is different from initial axon growth, for which the authors have previously demonstrated distinct mechanisms. The authors set out to directly compare the transcriptomes of initial axon growth and regrowth, specifically within the same neuronal environment and developmental time point. To this end, the authors used the well-characterized genetic tools available in Drosophila melanogaster (the fruit fly) to build a valuable dataset of genes involved at different time points in axon growth (alpha/beta Mushroom Body Kenyon cells) and regrowth (gamma Mushroom Body Kenyon cells). The authors then focus on genes that are upregulated during both initial axon growth and axon regrowth. Then, using this subset of genes, they screen for axonal growth and regrowth deficits by knocking down 300 of these genes. 12 genes are found to be phenotypically involved in both axon growth and regrowth based on RNAi gene-targeted knockdown in the Mushroom Body. Of these 12 genes, the authors focus on one gene, Pmvk, which is part of the mevalonate pathway. They then highlight other genes in this pathway. But these genes primarily affect axon regrowth, not initial axon growth, implicating metabolic pathways in axon regrowth. This comprehensive RNA-seq dataset will be a valuable resource for the field of axon growth and regrowth, as well as for other researchers studying the Mushroom Body.

Strengths:

This paper contains many strengths, including the in-depth sequencing of overlapping developmental time points during the alpha/beta KCs' initial axon growth and gamma KCs' regrowth. This produces a rich dataset of differentially expressed genes across different time points in either cell population during development. In addition, the authors characterized expression patterns at developmental time points for 30 Gal4 lines previously identified as alpha/beta KC-expressing. This is very helpful for Drosophila

Mushroom Body researchers because the authors not only characterized alpha/beta expression but also alpha'/beta' expression, gamma expression, and non-MB expression. The authors comprehensively walked through identifying differentially expressed genes during alpha/beta axon growth, identifying a subset of overlapping upregulated genes between cell types, then systematically characterized whether knockdown of a subset of these genes produced an axonal growth defect, and finally selected 1 of 3 cell-autonomous genes important for gamma KCs regrowth to further study.

The authors utilized the developing Mushroom Body in Drosophila melanogaster, which happens to have new neurons developing axons and neurons that have undergone pruning and are regrowing neurons at the same developmental time. They are also in the same part of the brain (the Mushroom Body) and, in theory, since the authors implicate a metabolic pathway, they will have similar metabolic growth conditions.

Identifying Pmvk and two other components of the mevalonate pathway in axon regrowth opens up novel avenues for future studies on the role this metabolic pathway may have in axon growth. The authors of this paper are also very upfront about their negative results, allowing researchers to avoid running redundant experiments and truly build on this work.

Weaknesses:

While the dataset produced in this study is a strength, certain aspects make it more challenging to interpret. For instance, the authors state that roughly equal numbers of males and females are used for sequencing, and this vagueness, coupled with only taking a subset of the GFP-labeled neurons during FACs sorting, can introduce confounds into the dataset. This may hold true in imaging studies as well, in which males and females were used interchangeably.

Additionally, a rationale is needed to explain why random numbers of 1-7 were assigned to zero-expressing genes in the DESeq analysis. This does not seem to conform to the usual way this analysis is normally performed. This can alter how genes across the dataset are normalized and requires further explanation.

The display and discussion of the data set do not always align with the authors' stated goal of having a comprehensive description of the genes that dynamically change during axon
growth and regrowth. Displaying more information about genes differentially expressed in the alpha/beta KCs, or any information about the genes diƯerentially expressed in the gamma KCs when using the same criteria as the alpha/beta KCs, or the 676 overlapping upregulated genes, would significantly add to this paper. The authors previously performed a similar study across developmental time points for gamma KCs, and it is not clear whether any overlapping genes were identified. Also, more information on the genes consisting of PC1 and PC3 when showing the PCA analysis would be helpful. Within the text, there is a discussion of why certain genes or gene groups were omitted or selected, such as clusters 1 and 2, and then some of their subgroups based on expected genes. There is also some discussion of omitted gene groups, but this is not complete across the different clusters, nor is there a discussion of why PC2 was not selected or of which genes might exhibit greater variability than cell type. The authors would make a stronger case for the genes they pursued if they showed that groups of genes already known to be involved in axon growth clustered within the selected groups. Since we do not see the gene lists, this is unclear and adds to the sometimes arbitrary nature of the author's choices about what to pursue in this paper. A larger set of descriptors, such as gene lists and Gene Ontology analysis beyond what is shown, would be very helpful in putting the results in context and determining whether this is a resource beneficial to others.

While the Pmvk story is interesting, the authors appear to make some arbitrary decisions in what is shown or pursued in this paper. Visually, CadN and Twr appear to be more severe axon regrowth phenotypes, where the peduncle appears intact, and axons are not regrowing in Figures 3 N and O. In contrast, Pmvk visually appears to lose neurons in Figure 3 M. With a change of the Gal4 driver (Figure 4), Pmvk now produces a gamma axon regrowth phenotype similar to CadN and Twr in Figure 3. This diƯerence in the use of Gal4 for characterizing axonal phenotypes is not discussed, making some interpretations more challenging due to diƯerences in Gal4 expression strength. For instance, the sequencing work was done with a diƯerent Gal4 MB expressing line than the characterization of gene knockdowns. Further characterization of the Pmvk was performed in the same Gal4 lines as the sequencing (Figure 4), suggesting a potential diƯerence in Gal4 strength that may play a role in their rescue experiments if they are using a slightly weaker Gal4 for gamma lobe expression. A broader discussion of this may make the selection of Pmvk less arbitrary if the phenotype is similar to those of CadN and Twr. Along the lines of the sometimes arbitrary nature of the genes chosen to pursue further, the authors state that they selected genes that showed differential expression at any time point. As they refine their list of genes to pursue further, they seem to prioritize genes that change at 18-21 APF. This appears to be the early period for axon growth in alpha/beta KCs and gamma KCs, based on Figure 1. A stronger case might be made at longer time points when the axon is growing or regrowing.

The paper would benefit from scaling back the claim that the mevalonate pathway is involved. The authors identified only a subset of genes from the mevalonate pathway, all immediately upstream of Pmvk, with no effect on downstream genes. Along these lines, the paper would benefit from a discussion of non-canonical PmvK signaling.

While the ability to take neurons at the same developmental time and from the same brain region is a strength, they are still 2 different types of neurons. Although gamma neuron axon growth occurs very early in development, it would be interesting to know whether the same genes are involved in their initial growth. A caveat to the author's conclusion is that these are 2 different cell types, and they might use different genetic programs or use overlapping ones at other times. The authors did not show that gamma KCs use these genes in their initial axon growth.