The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila CNS

Reviewer #2 (Public Review):

This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.

Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.

Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.

Reviewer #3 (Public Review):

Summary:

The receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) in humans is nervous system expressed and plays an important role as an oncogene. A number of groups have been studying ALK signalling in flies to gain mechanistic insight into its various roles. In flies, ALK plays a critical role in development, particularly embryonic development and axon targeting. In addition, ALK was also shown to regulate adult functions including sleep and memory. In this manuscript, Sukumar et al., used a suite of molecular techniques to identify downstream targets of ALK signalling. They first used targeted DamID, a technique that involves a DNA methylase to RNA polymerase II, so that GATC sites in close proximity to PolII binding sites are marked. They performed these experiments in wild type and ALK loss of function mutants (using an Alk dominant negative ALkDN), to identify Alk responsive loci. Comparing these loci with a larval single cell RNAseq dataset identified neuroendocrine cells as an important site of Alk action. They further combined these TaDa hits with data from RNA seq in Alk Loss and Gain of Function manipulations to identify a single novel target of Alk signalling - a neuropeptide precursor they named Sparkly (Spar) for its expression pattern. They generated a mutant allele of Spar, raised an antibody against Spar, and characterised its expression pattern and mutant behavioural phenotypes including defects in sleep and circadian function.

Strengths:

The molecular biology experiments using TaDa and RNAseq were elegant and very convincing. The authors identified a novel gene they named Spar. They also generated a mutant allele of Spar (using CrisprCas technology) and raised an antibody against Spar. These experiments are lovely, and the reagents will be useful to the community. The paper is also well written, and the figures are very nicely laid out making the manuscript a pleasure to read.

Weaknesses:

The manuscript has improved very substantially in revision. The authors have clearly taken the comments on board in good faith. Yet, some small concerns remain around the behavioural analysis.

In Fig. 8H and H' average sleep/day is ~100. Is this minutes of sleep? 100 min/day is far too low, is it a typo?

The numbers for sleep bouts are also too low to me e.g. in Fig 9 number of sleep bouts avg around 4.

In their response to reviewers the authors say these errors were fixed, yet the figures appear not to have been changed. Perhaps the old figures were left in inadvertently?

The circadian anticipatory activity analyses could also be improved. The standard in the field is to perform eduction analyses and quantify anticipatory activity e.g. using the method of Harrisingh et al. (PMID: 18003827). This typically computed as the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition.

In their response to reviewers, the authors have revised their anticipation analyses by quantifying the mean activity in the 6 hrs preceding light transition. However, in the method of Harrisingh et al., anticipation is the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition. Simply computing the activity in the 6hrs preceding light transition does not give a measure of anticipation, determining the ratio is key.

Author response:

The following is the authors’ response to the previous reviews.

Reviewer #3:

Summary:

The receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) in humans is nervous system expressed and plays an important role as an oncogene. A number of groups have been studying ALK signalling in flies to gain mechanistic insight into its various roles. In flies, ALK plays a critical role in development, particularly embryonic development and axon targeting. In addition, ALK also was also shown to regulate adult functions including sleep and memory. In this manuscript, Sukumar et al., used a suite of molecular techniques to identify downstream targets of ALK signalling. They first used targeted DamID, a technique that involves a DNA methylase to RNA polymerase II, so that GATC sites in close proximity to PolII binding sites are marked. They performed these experiments in wild type and ALK loss of function mutants (using an Alk dominant negative ALkDN), to identify Alk responsive loci. Comparing these loci with a larval single cell RNAseq dataset identified neuroendocrine cells as an important site of Alk action. They further combined these TaDa hits with data from RNA seq in Alk Loss and Gain of Function manipulations to identify a single novel target of Alk signalling - a neuropeptide precursor they named Sparkly (Spar) for its expression pattern. They generated a mutant allele of Spar, raised an antibody against Spar, and characterised its expression pattern and mutant behavioural phenotypes including defects in sleep and circadian function.

Strengths:

The molecular biology experiments using TaDa and RNAseq were elegant and very convincing. The authors identified a novel gene they named Spar. They also generated a mutant allele of Spar (using CrisprCas technology) and raised an antibody against Spar. These experiments are lovely, and the reagents will be useful to the community. The paper is also well written, and the figures are very nicely laid out making the manuscript a pleasure to read.

We thank the reviewer for this analysis.

Weaknesses:

The manuscript has improved substantially in the revision. Yet, some concerns remain around the genetics and behavioural analysis which is incomplete and confusing. The authors generated a novel allele of Spar - Spar ΔExon1 and examined sleep and circadian phenotypes of this allele and of RNAi knockdown of Spar. The RNAi knockdown is a welcome addition. However, the authors only show one parental control the GAL4 / +, but leave out the other parental control i.e. the UAS RNAi / + e.g. in Fig. 9. It is important to show both parental controls.

We would like to express our gratitude for your insightful comments and feedback on our manuscript. We acknowledge the concerns raised regarding the genetics and behavioural analysis, and we appreciate the opportunity to address these issues. We have added the reciprocal UAS Spar-RNAi control in addition to the GAL4/+ control and we have incorporated both controls in the revised Figure 9, Figure 9 Supplementary Figure 1 and Figure 9 Supplementary Figure 2. Figure legends have been modified accordingly.

Further, the sleep and circadian characterisation could be substantially improved. It is unclear how sleep was calculated - what program was used or what the criteria to define a sleep bout was.

The data underwent analysis utilizing an Excel macro, as outlined in the study by Berlandi et al. (2017) (PMID: 28912696). As previously indicated in the methodology, sleep is characterized as 5 minutes of inactivity. The raw data acquired from the Trikenetics DAM system was input into an Excel spreadsheet, and the parameters, encompassing sleep and activity, were computed for each day of the trial as an average derived from the data of all living animals at that time. Subsequently, these parameters were exhibited over the course of the experiment. We have further detailed this part in the methods section to avoid confusion (Page 32 of revised MS).

In the legend for Fig 8c, it says sleep was shown as "percentage of time flies spend sleeping measured every 5min across a 24h time span". Sleep in flies is (usually) defined as at least 5 min of inactivity. With this definition, I'm not sure how one can calculate the % time asleep in a 5 min bin! Typically people use 30min or 60min bins.

We thank the reviewer for bringing this to our attention. As previously stated, in our experiments, sleep is defined as 5 minutes of inactivity. We have now modified the wording in the figure legend (Figure 8, Page 41), which was previously misleading.

The sleep numbers for controls also seem off to me e.g. in Fig. 8H and H' average sleep / day is ~100. Is this minutes of sleep? 100 min / day is far too low, is it a typo? The same applies to Figure 8, figure supplement 2. Other places e.g. Fig 8 figure supplement 1, avg sleep is around 1000 min / day.

The numbers for sleep bouts are also too low to me e.g. in Fig 9 number of sleep bouts avg around 4, and in Fig. 8 figure supplement 2 they average 1 sleep bout. There are several free software packages to analyse sleep data (e.g. Sleep Mat, PMID 35998317, or SCAMP). I would recommend that the authors reanalyse their data using one of these standard packages that are used routinely in the field. That should help resolve many issues.

We thank the reviewer for pointing this out. There was indeed a typo “missing 0”, resulting in 0 values as only 3 days of raw data were chosen for the analysis of the average sleep in the mentioned figures. We have corrected this mistake in all figures.

The circadian anticipatory activity analyses could also be improved. The standard in the field is to perform eduction analyses and quantify anticipatory activity e.g. using the method of Harrisingh et al. (PMID: 18003827). This typically computed as the ratio of activity in the 3hrs preceding light transition to activity in the 6hrs preceding light transition. The programs referenced above should help with this.

For consistency purposes we used the same macro excel (Berlandi et al, 2017) (PMID: 28912696) and followed the methodology of Harrisingh et al. (PMID: 18003827) to assess the anticipatory activity. We selected the activity in the 6 h period before lights on and defined it as a.m. anticipation, and the activity in the 6h period preceding the lights off and defined as p.m. anticipation (Figure 8 f-g).

Finally, in many cases I'm not sure that the appropriate statistical tests have been used e.g. in Fig 8c, 8e, 8h t-tests have been used when are three groups in the figure. The appropriate test here would an ANOVA, followed by post-hoc comparisons.

We agree with the reviewer’s comments. We have re-evaluated the data in Figure 8 b, c, e, h and h’ and Figure 8 Supplement 2 and 4 using a One-Way ANOVA followed by Tukey post-hoc test and we have indicated this in all legends.