The emergence of circadian timekeeping in the intestine

The circadian clock is a molecular timekeeper, present from cyanobacteria to mammals, that coordinates internal physiology with the external environment. The clock has a 24-h period however development proceeds with its own timing, raising the question of how these interact. Using the intestine of Drosophila melanogaster as a model for organ development, we track how and when the circadian clock emerges in specific cell types. We find that the circadian clock begins abruptly in the adult intestine and gradually synchronizes to the environment after intestinal development is complete. This delayed start occurs because individual cells at earlier stages lack the complete circadian clock gene network. As the intestine develops, the circadian clock is first consolidated in intestinal stem cells with changes in Ecdysone and Hnf4 signalling influencing the transcriptional activity of Clk/cyc to drive the expression of tim, Pdp1, and vri. In the mature intestine, stem cell lineage commitment transiently disrupts clock activity in differentiating progeny, mirroring early developmental clock-less transitions. Our data show that clock function and differentiation are incompatible and provide a paradigm for studying circadian clocks in development and stem cell lineages.

In this manuscript Parasram and colleagues examine the fimecourse of appearance of clock funcfion in the Drosophila intesfine.Using a variety of different approaches they show that clock genes are not expressed in the intesfine unfil the emergence of the adult fly, and that a robust circadian rhythm of clock gene expression does not appear unfil day 4 of adult life.Furthermore, they show that a mature clock first appears in the intesfinal stem cells.Interesfingly, they find that there is a transient loss of clock gene expression as cells transifion between discrete fates.Finally, they show that the combinafion of daily photoperiod and feeding cycles together synchronize the fiming of intesfinal circadian transcripfion cycles during clock maturafion.The work reported here provides a careful descripfion of the fimecourse of gene expression in different cell types as the intesfine develops from the larval to the adult stage.And, because the key clock genes are not expressed unfil adult emergence, it is clear from their analyses that there is no funcfional clock in the intesfine unfil adulthood.Beyond this, however, this reviewer is not convinced about the exact fimecourse of emergence of clock funcfion in this fissue.Specifically: 1-The results shown in Fig. 2A do not preclude the possibility that there is a funcfional clock by day 1, but that the clocks of different cells are not synchronized, leading to a lower overall rhythmicity.To address this quesfion the authors would need to follow individual cells, and determine when a daily rhythm emerges.For this the fissue could be cultured and the intensity of their clock reporters in individual cells followed over fime.(If I understood the legend correctly, in the fimecourses shown in Figs.1B and 1C each intesfine contributed a single measurement, which I assume is a measure of average fluorescence intensity over an area of the intesfine) 2-The statement (legend to Fig. 2) that "Clk/cyc transcripfional acfivity is inifially stochasfic and shows mature rhythms only by day 4 for ClockPER with a peak around ZT0 and trough around ZT12." is not convincingly supported by the data.By eye, it looks like Fig. 1C shows a daily rhythm starfing on day 1 with a daily minima at around ZT12. Regardless of what is seen by eye, it is really not possible to assert that rhythmicity does not emerge unfil day 4 absent a quanfitafive frequency analysis.Such a statement would also require having at least an extra day's worth of data, so as to have 2 days of robust rhythmicity (days 4-5) to contrast with the situafion on days 1-2, for instance.I was also confused by the stafisfics provided (in Supplemental informafion), which for Figure 2B and 2C indicate the values of an ANOVA analysis.An ANOVA analysis only tells us that there are stafisfically significant differences among the values for different fimes, but this does not inform us as to whether there are daily rhythmicifies and when such rhythmicity becomes significant.3-Finally, the emergence of circadian funcfion would also require records to be obtained under dark:dark (DD) condifions.The only such record is shown in Fig. S3, for which, incidentally, it isn't clear when the measurements were taken relafive to the start of DD (ideally, measurements should be taken starfing 24-48h after the beginning of DD, in case there is some residual rhythmicity).(As an aside, the legend to this figure states that "….full stafisfics are shown in Supplemental Informafion.",yet this figure is part of Supplemental informafion and no stafisfics are shown in the "Stafisfics" table.)4-Many of the data have to do with changes associated with the maturafion of the intesfine rather than specifically that of circadian clock functon(e.g., Fig. 4 and parts of Fig. 5).In addifion, and as detailed above, the "circadian" part of the results is not very conclusive.For these reasons I would suggest changing the emphasis of the study and focusing on the maturafion of the Drosophila intesfine in general rather than more narrowly on the appearance of circadian clock funcfion in this fissue.5-The methods secfion needs addifional details, in parficular with respect to the measurements of fluorescence intensity.

Minor comments:
-"phosphatase" should be deleted from line 106 "…. the phosphatase dbt, a kinase that regulates…".-The differences in color are not evident in the symbols shown in Figure 3 (especially purple vs. blue).Please make more pronounced.

Reviewer #2 (Remarks to the Author):
In the manuscript fitled "The Emergence of Circadian Timekeeping in the Intesfine", Parasram and colleagues characterized in detail the emergence and development of the circadian clock in the intesfine of Drosophila melanogaster.In this work, researchers explored how different circadian genes are expressed during the developmental stages and in which intesfinal cells these are more relevant.Using specific clock reporters, researchers showed changes in the acfivity and rhythmicity of TIM and PER in ISCs and their progeny cells, EB and EC.Importantly they concluded circadian rhythmicity and cell differenfiafion are exclusive processes, and the clock needs to be repressed in early differenfiated cells (EB) to be restored in fully differenfiated EC.Moreover, they demonstrated that expression of several genes started early in development (early pupa), but rhythmicity is only established in the adult after 4 days, highlighfing the complexity of the circadian regulafion in the gut.Interesfingly feeding and photoperiod have to some extent compensatory effects, upon the lack of one or another, in influencing the establishment of circadian rhythmicity in the gut.In summary, the manuscript demonstrates a novel approach to understanding circadian regulafion during organ and fissue development and these findings contribute significantly to the exisfing body of knowledge in the field.This manuscript addresses a very important biological quesfion, it is well-wriften and organized, making it easy to follow, ideas are clear, and figures and interpretafions are well presented.Overall, data presented supports the main conclusions.Major comments -Clock reporter acfivity in ISCs is not clear from the images presented.For instance, in Figure S2F, while nuclear reporter acfivity is clearly observed in the ECs, it is not present in ISC/EB and therefore the claim of the authors regarding this is not supported by this evidence.
-There is a lack of informafion on the changes in the localizafion of PER at different fime points.Authors should include a fime course of cell specific staining of PER and/or TIM in the gut to substanfiate the reporter informafion across intesfinal development and cell types.For example, include a fime course showing changes in localizafion of PER and/or TIM anfibody comparing larvae, pupa, immature adult and mature adult gut 4 looking at the different cell types in the gut.
-The weakest point in this manuscript refers to the funcfional data on molecular regulators of clock gene expression based on the scRNAseq data (Figure 4).Is any of the data presented in Figure 4D stafisfically significant?What is the rafionale behind knocking down these various genes in ECs and ISCs/EB, when the focus, as per the authors' statement and the transcripfional informafion presented in Figure 4B refers to the lafter cell type?Furthermore, what is the evidence leading to Bursicon signaling (rk) as a possible player here?No previous evidence has been presented on expression or funcfional role of rk in ISCs or ECs and therefore the rafional for these experiments.Is unclear.The data in this part of the manuscript needs to be re-evaluated and improved/strengthened.I suggest choosing a couple of the strongest candidates and analyse their funcfion in reporter expression over fime in ISCs, coupled with immunostaining with core clock components (e.g.PER, TIM) -Is the microbiome is playing a role in establishing the rhythmicity of the intesfine during development and in the adult stages analysed?-Is there any role of the central clock in the establishment of intesfinal clock during development?-Are all clocks in ISC synchronized or do you see different phases among them?This informafion could be obtained from the analysis of the ISC molecular signature in the scRNAseq.For example, if there were sub clusters of ISCs that express different levels or components of circadian genes.
-Consistent with the data presented here, Figure S5D, pdp1, is not essenfial for central clock expression and oscillafions but for regulafing circadian outputs, such as locomofion (Benito et al., 2007).Have the authors idenfified any funcfion of pdp1 in gut development or rhythmic behaviours (e.g.regenerafion)?The menfioned prior evidence and associated publicafion should also be acknowledged by the authors as relevant to the results they show in the gut.

Minor comments
General to all figures.Please include stafisfical significance (*) or p-value in the cases where differences were stafisfically significant to make it easy to follow.If clock gene expression graphs are going to be compared, please use the same scale.
• Figure 1-In H scales are different and it's hard to compare the different levels.Also, in panel D please indicate if the dashed line is delimitafing the intesfine.In Figure 1 D -G I'm concerned that only one fime point was measured.Could be that the clocks in pupa and larvae are present but the phase is different?Did the authors analyse any other fime point?
• Figure 2-In Figure 2A scales are also different between graphs please use the same to help interpret the data, in the same panel, clk levels after 4 days look quite low, is hard to see the oscillafion, please discuss in more detail why this could be happening.Why do PER and TIM reporters show different peaks of acfivity?In figure 2B use the same scale.TIM reporter has a peak at ZT0 and a minimum at ZT12 but PER reporter is delayed.Can you elaborate on this?In Figure S2A is histone=DAPI?Why B is the only one at ZT23?
• Figure 3. Please relocate panel D close to the other panels and the scale at the right boftom to make comparisons easier.
• Figure S4.When the authors said "We therefore used the higher-expressed clock genes as a and cwo, in this analysis the ones that clearly change are Pdp1 and Tim.Please discuss why they decided to use the others too.
• Figure 5.In panel A GFP is not very clear, is there any other picture where the intensity is a bit higher?
• Figure S6A.When comparing ad libitum and starving flies, the size of the gut is affected by starvafion but also the acfivity of the reporter, showing more acfivity when flies are in starvafion mode, can authors elaborate on this.

Reviewer #3 (Remarks to the Author):
In this manuscript, Karpowicz and colleagues invesfigated when and how the circadian clock is establishment in the developing intesfine of Drosophila.They ufilized specific clock acfivity reporters, RT-qPCR analysis, and single cell RNA-sequencing analysis to study the emergence and regulafion of the circadian clock during intesfinal development.The authors observed that the circadian clock emerged in the adult intesfine and established a daily rhythm three days after birth.They found increased circadian clock acfivifies in ISCs and ECs but not EEs during intesfinal maturafion.The emergence of the clock after birth was regulated by ecdysone and Bursicon hormone signaling pathways.The authors also demonstrated that circadian clock gene expressions were repressed during ISC-to-EC differenfiafion.They further showed that photoperiod and feeding synchronized the maturafion of the circadian clock during intesfinal development.
While this study provides valuable insights into the emergence and regulafion of the circadian clock during Drosophila intesfine development, there are some areas that require further clarificafion and mechanisfic insights.Here are specific comments: Major: 1.In Figure 2B, the authors showed that the daily clock rhythm was not established unfil three days after eclosion.However, the changes in some genes were not as remarkable as described in the main text.It would be nice for the authors to provide alternafive methods or evidence to support the establishment of the clock, such as demonstrafing the establishment of rhythmic feeding (if it occurs) that is not observed unfil three days post eclosion.
3. If the clock genes are restored in EEs and differenfiafing ECs, what would happen?This would provide insights into whether the temporal loss (for differenfiafing ECs) or permanent loss (for EEs) of the clock has any funcfional benefits.
4. In the cell lineage tracing experiments shown in Figure 5 and Figure S5, ISCs, EBs, and EEs are all small diploid cells in the lineage, which may not be reliably disfinguished without co-staining with cell typespecific markers such as Dl, NRE-lacZ, and Pros, respecfively for ISCs, EBs and EEs.
5. The authors examined photoperiod and feeding as means to synchronize the circadian clock in the intesfine, and the results were excifing and informafive.However, it would be nice to include a negafive control, such as fasted cry01 mutant flies, to provide a comparison.

Minor:
Please see our point-by-point response to the reviewer's comments/questions below.
---Reviewer #1 (Remarks to the Author): The authors have made a valiant effort to revise their manuscript and to respond to the reviewers' comments and suggestions.I have no further comments.
Reviewer #2 (Remarks to the Author): The revised version of this manuscript significantly addresses most of my major comments.Before publication, I would however suggest further clarification on the following points: The expression of the Clock reporter in ISCs/EBs remains unclear.What does the data in Figure S2G mean?There are two sets of data, one showing no reporter activity in either pupal or adult ISCs/EBs and the other does show reporter activity.
Figure S2G (now Figure S3G) shows ISC/EB expression of the Clock TIM reporter using two different lines that show differences in the strength of the GFP signal.Clock TIM (on chromosome III) expression, which is quantified in the violin plots, has a weaker GFP signal, than Clock TIM (II).To demonstrate unambiguously the ISC/EB signal, we included images from both reporters.To clarify this point, we have adjusted the Figure legend as follows: "(G) Clk/cyc activity in ISC/EBs (marked by mCherry expression in esg+ cells, representative cells are outlined) is similar in late pupa and immature adults, Mann-Whitney test p-values are shown on the graph.Two reporters are shown, the quantification corresponds to Clock TIM (III) but due to its weaker expression in ISC/EBs, we have included an image of Clock TIM (II) which shows a similar pattern but stronger GFP expression in ISC/EBs."I don't understand the data provided in Figure S2I.If cyc is over expressed in ISCs/EBs in the whole mutant animal, would one not expect to restore the reporter activity in these cells?I supposed the same would be the case in EC if the gene is over expressed in those cells?
In Figure S2I (now Supplementary Figure 3I) we restore cyc expression only in the ISC/EBs in the whole mutant animal.The ISC/EBs are marked with mCherry, and we see the reporter restored in the ISC/EBs.We have adjusted the text and image panels to clarify this point and indicate cells with reporter expression."(I) Representative images of Clock TIM intestines showing that the Clk/cyc activity that is present in the controls, is absent in the cyc 01 mutant, and restored strongly in the ISC/EBs when cyc is overexpressed (esg>mCherry, cyc).Arrowheads indicate mCherry+ ISC/EBs.Scale bar 10µm.Full statistics are shown in Supplemental Information." Yes, although we have not done the EC overexpression experiment, we would expect this to be the same case in ECs if the gene is overexpressed in those cells.The ISC/EB overexpression of nuclear receptors does affect EC reporter signal.We expect that the earlier Clk/cyc activity in ISC/EBs (precursors of ECs) allows the EC clock to develop earlier as well, since the ISCs would give rise to these clock positive cells earlier.The text has been updated to include this observation.
"Of note, EC reporter expression is also increased when these genes are overexpressed in ISCs, possibly due to the higher clock activity in their precursors." Reviewer #3 (Remarks to the Author): The majority of my concerns have been addressed by the authors.However, there are still a few minor issues that need attention: The curve for day 1 is missing in Fig. 2B.
The curve for day 1 in Figure 2B is missing because the Clock PER reporter is arrhythmic at day 1, therefore it does not fit a cosinor curve which could not be added to the graph.The figure legend has been updated with this clarification.We apologize for these mismatches; have verified that are matching in this final

Figure 5B :
Figure 5B: Over-expression of Hnf4 and Hr78 in ISC/EBs leads to clock activity increase in ECs.Why is that?

"
Cosinor fit analysis (right graph) shows arrhythmic activity on Day 1 (no cosinor curve can be fitted to the data) and 24-hour rhythms on Day 4." There are several citation mismatches.For instance, in line 199, Fig. S4E should be Fig.S4F.