Author Response

We appreciate the thoughtful and thorough critique provided by the two reviewers, and generally agree with their assessment. The revised submission will address the issues they raise. In particular, we agree that the framework of the paper should be broadened to include bacteria and the deep literature associated with coincidental selection.

Author Response

Evaluation Summary:

The work by Volante et al. studied a new plasmid partition system, in which the authors discovered that four or more contiguous ParS sequence repeats are required to assemble a stable partitioning ParAB complex and to activate the ParA ATPase. The work reveals a new plasmid partitioning mechanism in which the mechanic property of DNA and its interaction with the partition complex may drive the directional movement of the plasmid.

Thank you for the kind evaluation. But we wonder about the description of the pSM19035 partition system we studied here as “a new plasmid partition system”. This system itself is quite old. The editor might have meant “new” as a subject of a research, but plasmid partition systems involving RHH-ParB proteins have been studied by number of groups for some time, including the Alonso Lab, which has worked on the pSM19035 partition system number of years prior to our current collaboration for this paper. Therefore, we wonder if the term “new” is the most appropriate.

Reviewer #1 (Public Review):

This is a very thorough biochemical work that investigated the ParABS system in pSM19035 by Volante et al. Volante et al showed convincingly that a specific architecture of the centromere (parS) of pSM19035 is required to assemble a stable/functional partition complex. Minimally, four consecutive parS are required for the formation of partition complex, and to efficiently activate the ATPase activity of ParA. The work is very interesting, and the discovery will allow the community to compare and contrast to the more widespread/more investigated canonical chromosomal ParABS system (where ParB is a sliding CTPase protein clamp, and a single parS site is often sufficient to assemble a working partition complex). All the main conclusions in the abstract are justified and supported by biochemical data with appropriate controls. A proposed multistep mechanism of partition complex assembly and disassembly (summarized in Fig 6) is reasonable. Perhaps the only shortcoming of this work is that the team does not yet get to the bottom of why four consecutive parS are needed.

Thank you for the kind evaluation. The last point is an important one. We would like to continue to test our current model to either obtain stronger supporting evidence or come up with better alternative model.

*Reviewer #2 (Public Review):

ParBs come in two variations, RHH and HTH. In this study, the authors examine the in vitro behavior of the RHH system, which is less studied. Two activities were carefully monitored; ATPase activation and ParA removal from DNA. The system is quite complex, but the authors have done a good job of examining parameter space. One question concerns the physiological relevance. Can this be assessed by uncoupling ParA/ParB expression (making it inducible with IPTG from the chromosome, for example) and testing plasmids with the various constructs?

This is an excellent point; we agree this a shortcoming of the current study. As described in response to “Essential Revisions”, we very much wanted to include an experiment testing in vivo plasmid stability for different size parSpSM sites in this paper, and we put a significant effort. However, we encountered certain technical issues with the approach we tried, and we failed to obtain conclusive data in timely fashion before we run out of time. Although, we had preliminary data, which appeared to be consistent with the notion that shorter parS sites are non-functional and full-size parS sites are functional, the experiment had certain flaw, which we could not rectify immediately to our satisfaction. Therefore, we decided to postpone this part of the project and plan for broader physiological evaluation of the parSpSM sequence arrangements in near future. In the revision, we mentioned at the beginning of discussion that in vivo functional test of parSpSM site requirements still remains to be examined.

The authors appear to suggest that the requirement for at least 4 ParB binding sites is due to the inability of ParBs of this type to spread inferring that for the ParB-HTH multiple ParBs bound to ParS are required. Has this been tested in this system?

ParB spreading has been shown to be essential for the HTH-ParB to perform its role in partition function. We clarified the importance of HTH-ParB spreading for partition function on lines 44-45.

In any event, another major difference between the two systems is that a peptide corresponding to the N-ter of ParB is sufficient to bind DNA indicating this type of ParB does not have to be bound to DNA to stimulate ParA. It would have been useful if the authors had commented on this.

There seems to be a mistype here. “N-ter of ParB is sufficient to bind DNA indicating ……” is incorrect. Perhaps this was meant to be “N-ter of ParB is unable to bind DNA, indicating ……” This is not a qualitative difference between the HTH- and RHH-ParBs: the N-terminal ParA interacting peptides of HTH-ParBs also can activate their cognate ParA ATPase without parS DNA binding, and parS-dependency of ATPase activation for HTH-ParBs appears to be significantly less stringent compared to the case for RHH-ParB we report here. ParBpSM1-27 , which cannot bind parSpSM, could only stimulate ParApSM ATPase to at most 1/10 of the full size ParBpSM in the presence of active parSpSM. We clarified this on lines 156-157, and also added discussion about this contrast between the HTH- and RHH-ParBs and possible implications on lines 458-467.

Reviewer #3 (Public Review):

Drs. Volante, Alonso, and Mizuuchi presented a milestone experimental finding on how the distinct architecture of centromere (ParS) on bacterial plasmid drives the ParABS-mediated genome partition process. Rather than driven by cytoskeletal filament pushing or pulling as its eukaryotic counterpart, the genome partition in prokaryotes is demonstrated to operate as a burnt-bridge Brownian Ratchet, first put forward by the Mizuuchi group. To drive directed and persistent movement without linear motor proteins, this Brownian Ratchet requires two factors: 1) enough bonds (10s' to 100s') bridging the PC-bound ParB to the nucleoid-bound ParA to largely quench the diffusive motion of the PC, and 2) the PC-bound ParB 'kicks" off the nucleoid-bound ParA that can replenish the nucleoid only after a sufficient time delay, which rectifies the initial symmetry-breaking into a directed and persistent movement. Although the time delay in ParA replenishment is established as a common feature across different bacteria, the binding properties of PC-bound ParB vary greatly, which begs the question of how Brownian Ratcheting adapts to different cellular milieu to fulfill the functional fidelity.

The finding in this work presented a new but important twist in the Brownian Ratchet paradigm. The authors showed that in the pSM19035 plasmid partition system, only four contiguous ParB-binding repeats in ParS are required for the ParA-ParB interactions that drive the PC partition. In other words, only four chemical bonds are needed for the PC partition. Crucially, the authors further demonstrated that distinct orientation (configuration?) of the ParB-binding repeats is required for this fidelity by their state-of-art biochemistry and reconstitution experiments. The authors then elaborated on a possible mechanism of how the smaller number of PC-bound ParB can drive directed and persistent PC movement by interacting with nucleoid ParA. If I understand correctly, in their proposed scheme, due to their specific orientation (configuration?), when two of the ParS-bound ParB molecules bind to the two nucleoid-bound ParA molecules there arises a torsional/distortional stress. Consequently, the thermal fluctuations preload the forming bonds, triggering the dissociation of the two ParB molecules from the PC. And the remaining PC-bound ParBs may kick off the ParAs that have a time delay in DNA-rebinding, while ParB molecules will replenish the ParS to initiate the next round. In this proposal, the key conceptual leap is that not only the substrate but the cargo remodels to underlie the Brownian Ratcheting.

We thank the reviewer for kind evaluation of our work. The model proposed is highly speculative at this point. Despite it may appear rather detailed in order to account for the unexpected findings, we consider it only a working hypothesis to be revised or replaced by a better model in future. We thank for many useful suggestions, which we will follow in our revision.

Author Response

REVIEWER #1 (PUBLIC REVIEW):

The study by Monterisi et al. reports that loss-of-function mutations in metabolic pathways do not necessarily have a negative impact on cancer growth. The authors suggest that small solutes transferred via gap junction channels formed between wild-type cells and cells express mutants defective in metabolic pathways rescue the metabolic-deficient cancer cells. Through the examination of multiple human cell lines with several advanced means to determine gap junction coupling, Cx26 was identified as a major connexin molecule involved in medicating gap junction coupling between colorectal cancer (CRC) cells. The gene mutations of three metabolic gene mutations were investigated for major metabolic function of the cell, pH regulation, glycolysis and mitochondrial function.

Strengths: The paper tests a new hypothesis that the mutations that inactivate key metabolic pathways do not incur functional deficits in cancer cells expressing the mutants due to their gap junction coupling to wild type cells.

From microarray data they identified multiple connexins expressed in various CRC cells. Several advanced analyses were used to assess gap junction coupling in CRC cells including fluorescence recovery after photobleaching (FRAP). The extent of permeability at steady state was evaluated using CellTracker dyes and coupling coefficients were determined. They also used flow-cytometry to study dye transfer, which will provide a quantitative, dynamic means for study cell coupling. The data showed that knocking down Cx26 could greatly reduce diffusive exchange in most of the CRC cells tested.

The study focused on three metabolic genes, Na+/H+ exchanger NHE1, a regulator of intracellular pH, a glycolytic gene, ALDOA and mitochondrial respiration gene, NDUFS1. These genes were knocked out in the selected CRC cells highly expressing these genes. The co-culture studies were well executed with fluorescence-markers distinguishing the WT and knockout cells and well-defined readouts such as intracellular pH, media pH, glucose/lactate levels and mitochondrial O2 consumption and glycolytic acid.

The experiments in general were well designed and conducted, and the data supported the conclusions. The paper is also logically written and figures were well presented providing clear graphic illustrations.

Thank you for recognising the strengths and novelty of our findings.

Weaknesses: Although the hypothesis is innovative, no clear justification is provided that illustrates the scenario representing the clinical situation. The remaining questions include: What kind of somatic mutations in cancer cells has little impact on their growth and progression?

We have now added in vivo data (Fig 8) and revised the Introduction and Discussion to address this point. Briefly, the broader clinical relevance our findings relates to the notion of essential genes and their negative selection. We show that connexin-dependent coupling can rescue a genetic deficiency, provided the mutation-carrying cell can access wild-type neighbours for the missing function. This rescue effect is limited to processes that handle solutes that can pass via connexin channels, i.e. metabolic processes. As such, sporadic loss-of-function mutations in “essential genes” may not produce a functional deficit in human cancers. We demonstrate rescue extensively in vitro, and now in a xenograft model.

We argue that our work can explain why certain metabolic genes are essential in vitro, but not in vivo. In monolayers of mutated cells, diffusion across gap junctions cannot rescue the mutant phenotype, because there is no wild-type cell available to supply the missing function. In contrast, mutations in vivo will arise sporadically and wild-type cells are typically available to couple onto the mutation-bearing cell, providing it with functional rescue. Thus, only in the former case would the lethality of essential genes emerge.

Indeed, many notable studies have found genes of various metabolic pathways to be essential for growth in vitro. Such genes would be expected to undergo negative selection in vivo, but this is exceedingly rare according to multiple observations. By demonstrating metabolic rescue in co-cultures (i.e. a setting closer to the tumour) and (now) in xenografts, our work provides an explanation for this apparent paradox. Indeed, cells such as NDUFS1-negative SW1222 grow very, very slowly in culture compared to wild-type cells and require regular media changes to keep pH alkaline. However, coupling onto wild-type cells can rescue knock-out cells in vitro and in vivo. We argue that this finding explains why loss-of-function mutations in NDUFS1 (and similar genes) do not undergo negative selection in human tumours (despite in vitro predictions).

The three proteins selected for this study were chosen to represent very distinct types of solute-handling processes. We illustrate our point in a (new) summary figure in Fig 8.

What types of WT cells, within the tumour cells or with neighbouring normal cells? Whether the current experimental design closely recapitulates the scenario in vivo?

Indeed, we find that stromal fibroblasts may also support cancer cells via gap junctions, as this is essentially the same concept (i.e. coupling onto a cell with wild-type genes). However, we feel that expanding our present submission to fibroblasts would make the volume of data exceeding large. Also, the methods we use for fibroblasts are different, and require a full manuscript on its own. For example, there is the issue of how to control for the radically different growth rates of fibroblasts and cancer cells. We chose co-cultures of WT and genetically altered CRC cells so that the co-cultures are of the same background, with just one element changing (i.e. the metabolite-handling gene). This makes our data easy to interpret, and thus strengthens our case. Our in vitro experiments were performed on monolayers, where cells can make contacts in 2D. In vivo, these contacts will spread in all dimensions, thus connectivity is likely to be even more significant. If anything, monolayers probably under-estimate the importance of connectivity, but this preparation is more accessible for studying cell-to-cell communication.

We recognise the importance of adding in vivo data to firm our conclusions. To that end, we have analysed xenografts established from co-cultures of wild-type DLD1 and NDUFS1-KO SW1112 cells on one flank of a mouse, and Cx26-KO DLD1 and NDUFS1-KO SW1112 cells on the other flank. This experiment tested whether Cx26-dependent connections between mitochondrially-defective NDUFS1 KO SW1222 cells and respiring DLD1 cells (on left flank only) are able to stimulate growth of the former (GFP-tagged). Indeed, NDUFS1-deficient cells grew faster when rescued by Cx26-expressing DLD1 cells. In contrast, their growth decelerated when DLD1 cells were Cx26-negative. We include these experiments and their controls in Fig 8.

The readouts for co-culturing for glycolytic ALDOA and NDUFS1 knockout are only cell mass, without determining the more relevant markers, glucose/lactate and mitochondrial O2 consumption and glycolytic acid production.

Our readouts are two-fold: total biomass and the size of the genetically altered compartment of co-cultures (GFP). We can therefore follow the relative growth of KO cells, which is essential for describing their growth (dis)advantage. We appreciate other markers are informative. Indeed, we characterised KO and WT cells in terms of O2 consumption and acid production in Fig 7. However, it would not be possible to measure glucose consumption selectively in GFP-positive KO cells of a co-culture, as the assays available for this measure ensemble rates for the entire population of cells (e.g. in a single well). Nonetheless, we believe that biomass as a readout is highly relevant to cancer, and we hope the reviewer concurs with us.

The study needs to include cells without functional gap junctions like the characterized negative control RKO cells.

This is an excellent suggestion, and we have added data for RKO cells to several figures. As expected, these do not form a syncytium and cannot rescue genetic defects in co-cultured cells. New data are shown in Fig 3G-H, Fig 6-supp2 and Fig 7H, adding to existing RKO controls in Fig 2A/B. Briefly, RKO cells do not exchange CellTracker dyes in monolayers (Fig 3F/G), cannot rescue cells that are ALDOA-deficient (Fig 6-supp2), and cannot rescue NDUFS1-deficient cells (Fig 7H). We also added Cx26-KO DLD1 cells to the CellTracker experiments in Fig 3.

REVIEWER #2 (PUBLIC REVIEW):

This paper is a logical extension of the 50 year-old concept of the "bystander effect" in tumours, wherein the effects of anti-tumour chemotherapeutics extend beyond the cells that take them up due to spread through gap junctions to adjacent cells. In this case, however, the authors have creatively realized that the reverse might also occur, and that tumour cells with otherwise fatal mutations in essential metabolic pathways can be rescued by their neighbours through passage of the missing metabolites through gap junctions. This can explain why mutations in other critical pathways, such as protein synthesis and transporters, are selected against in rapidly growing tumours, but others in equally critical pathways of glycolysis, electron transport, etc. are not, despite these genes having been demonstrated to be essential in in vitro KO studies (where all cells in the plate have the critical gene knocked out). A series of elegant experiments are used to test this proposal in several colorectal cancer (CRC) cell lines using three examples - pH regulation (defective Na+/H+ exchanger NHE1), glycolysis (defective Aldolase A (ALDOA)) and oxidative phosphorylation (defective Complex 1 - NDUFS1).

Thank you for these positive comments. We have added key references to the bystander effect in the Introduction, and explain how our findings build on these milestones.

The authors first determine the levels of different Cx proteins expressed in each cell line, and determine that for most Cx26 and 31 are dominant, although come lines have a subset of cells with high Cx43 expression. They then use Cell Tracker Green to pre-label cells and use FRAP as a means to measure how well the cell population is coupled. This is a useful measurement but is significantly over-interpreted by the authors as a "permeability" in uM/min. This is not really a permeability, which requires knowledge of the concentration gradient of the permeant species, relative cell volumes, etc. Rather it is a rate of fluorescent recovery that is presumably correlated with, but not quantitatively related to, levels of coupling.

Thank you for this comment. We would like to explain why we believe our FRAP experiments are able to estimate permeability in units of um/s. The rate of recovery of a solute in a cell following its “destruction” (here, photobleaching) is given as follows:

dCcell/dt = p⋅P(Ccell-Csurround) … [1]

Where subscripts ‘cell’ and ‘surround’ refer to the cell and its neighbours. P is the permeability of the barrier between these two compartments, and p is the ratio of the surface area of the barrier (i.e. membrane) to volume of the bleached cell. Within a “bleached” cell, we measure fluorescence.

Since fluorescence (F) is proportional to concentration (C), we can substitute:

C = α⋅F

where α is a constant of proportionality. Thus, the rate of recovery (L.H.S. of equation [1]) becomes:

dC/dt = d(α⋅F)/dt = α⋅dF/dt … [2]

And the R.H.S. of equation [1] is re-written as: P⋅(Ccell-Csurround) = P⋅(α⋅Fcell-α⋅Fsurround) = α⋅P⋅ (Fcell-Fsurround) … [3]

Putting [2] and [3] together,

dFcell/dt = p⋅P⋅(Fcell-Fsurround)

Prior to photobleaching, there are no (net) gradients, thus initial Fcell and Fsurround are equal.

Thus, we can re-write the equation in terms of normalized fluorescence (f=F/F0):

dfcell/dt = p⋅P⋅(fcell-fsurround)

P can therefore be expressed as:

P = dfcell/dt / (p⋅ (fcell-fsurround))

Here, dfcell/dt is measured from the fluorescence recovery time course and fcell-fsurround is measured experimentally (in fact, bleaching in the cell is set to 50%, thus this takes the value of 0.5 by default). We can approximate the monolayer as a network of cuboidal cells. The cell’s volume is thus ‘area’ times ‘height’, and the cell’s surface (at which it contacts its neighbors) is the ‘cell’s perimeter’ times ‘height’. Thus, for the bleached cell,

p = perimeter × height / area × height = perimeter / area.

The perimeter and area can be measured from the acquired fluorescence images. Thus, we can describe permeability using data obtained from image stacks. We appreciate that this method makes certain geometrical approximations, but we believe these are not unreasonable. We explain the assumptions and calculations in Appendix 1. More information about the method is published by us in https://pubmed.ncbi.nlm.nih.gov/28368405/. Of course, we accept that these calculations are less accurate than, say, electrical conductance measurements, and to that end, we added a note of caution to the main text.

This fluorescent recovery is shown to be sensitive to siRNA KO of Cx expression, but strangely its reduction is only correlated with KD of Cx26 in the 5 cell lines examined. KD of Cx43 (in LOVO cells) and Cx31 in all 5 cell lines had no effect or in some cases seemed to increase the rate of recovery (DLD1 and SNU1235). This is a notable finding, yet the authors choose to completely ignore it and continue with Cx26 KDs in studies of specific metabolite transfers. Some discussion should be included as to why KD pf these Cxs has no effect or causes an apparent increase in coupling of the cells.

The effectiveness of GJB2 knockdown in ablating ensemble connectivity is most likely a reflection that Cx26 is likely the dominant conductance inherited from the parent epithelium. Other isoforms are expressed, but in most CRCs cells, these do not produce major coupling, as GJB2 knockdown was sufficient to uncouple many CRCs. These observations justify our choice of connexin for studying metabolic rescue functionally. These findings are also consistent with the good correlation between ensemble connectivity and GJB2 levels.

Our data show a trend that GJB3 (Cx31) KD in DLD1 and SNU1235 cells and of GJA1 (Cx43) KD in LOVO cells produce an increase in coupling. However, when analysed by hierarchical (nested) analysis, these effects are not statistically significant, and for that reason we did not elaborate on these trends in the original submission. The apparent increase in conductivity in cells treated with GJA1 or GJB3 siRNA could reflect a compensatory response to the ablation of a specific message, closer contacts between cells allowing Cx26 to strengthen its connections, or a shift away from heterotypic channels involving Cx26 and Cx31/Cx43, towards homotypic Cx26. We did not see any consistent change in the intimacy of cell-cell contacts. We now performed western blots for connexins to probe for compensatory changes (see Fig 2-supp1). In comparison to wild-type cells, expression of Cx31 was not changed by GJB2 (Cx26) or GJA1 (Cx43) knockdown in DLD1 cells. GJB2 KO DLD1 cells did not induce expression of the other major isoform, Cx43. Also, in DLD1 cells, KD of GJB3 or GJA1 did not substantially change Cx26 levels. Similarly, KD of GJB3 did not affect Cx43 levels. In GJA1-high C10 cells, KD of GJB3 did not alter Cx43 levels, although a small decrease was observed with GJB2 KD on Cx43. Also in C10 cells, KD of GJB2 and GJA1 did not induce an increase in Cx31 levels.

We agree that complex interactions between connexin genes are possible, but we feel that a molecular study of Cx gene regulation would fall outside the scope of the present manuscript. Our findings point to a prominent role of Cx26 in metabolic rescue, and to strengthen this point, we show that Cx26-negative cells that express other connexins (e.g. C10 cells or NCIH747 cells) cannot rescue ALDOA-deficient counterparts or NDUFS1-KO SW1222 cells (new data in Fig 6 and 7). We share the Reviewer’s enthusiasm about the interplay between connexins and will endeavour to study this further in the near future.

Rather than just focus on acute transfer of dye between cells, the authors develop a system using 50/50 mixes of cells labelled with two junctionally permeant dyes and measured the degree of mixing at equilibrium (48 hours). This is presented as a "coupling coefficient", but how it is calculated, and its significance is not well described, and does not correlate with the historical use of this term in the literature. Nonetheless, the studies do seem to demonstrate a good degree of equilibration, although it would have been informative to determine of the cells that do not exchange dyes express Connexins. To document that this equilibration requires gap junctions, the authors employ low density cultures, which significantly decrease dye exchange. However, in at least one cell line (SW1222) dye exchange is only reduced by <50%, indicating a very high background to this assay. This is not addressed.

Thank you for these comments. We agree that our description of the method was inadequate, and we have added the necessary information in Appendix 1. We have also added information about actual confluency and restructured the figure. We also added new data for RKO cells and DLD1-Cx26 KO cells, i.e. two negative controls (Fig 3H). We pondered about the best name for describing the numerical output of the method, and concluded that “coupling coefficient” is reasonable (provided we improve our description of it) because it is dimensionless, and like many coefficients has a finite range (here, zero to one). With further explanation, we hope this terminology is acceptable. The issue with SW1222 cells is that both low- and high-seeding densities produce clusters of cells. Even though overall cell numbers were different in high and low seeded cultures, actual connectivity within “islands” of cells remained high, hence their similar coupling coefficients (see Fig 3E). Indeed, this CRC line is unusual in this behaviour, so we only present data from the higher density.

The most compelling part of the study is the use of reporters to directly demonstrate a role of Cx26 coupling of cells to rescue cells with mutations of the three genes mentioned above when mixed with normal neighbours. This case was most convincing in the cases of ALDOA and NDUFS1, with the data for the pH regulation requiring more explanation for full understanding of the data shown (e.g. Figs 7 G and H).

Thank you for this comment. Studies of pHi regulation provide a unique opportunity to obtain single-cell resolution (unlike e.g. glycolytic assays). We took advantage of this, and therefore the figure on pHi presents a greater depth of analysis. Nonetheless, we agree the pH data need further explanation. We have expanded the text, and also added a bar plot of data on day 7, which now provides a clearer illustration of the rescue effect. This form of presentation was also adopted for ALDOA and NDUFS1 experiments in the subsequent figures.

Overall, the study does a credible job of demonstrating that Cx26 coupling of CRC cells serves to rescue cells with mutations in critically necessary metabolic pathways, presumably due to transfer of metabolites from surrounding wt cells. However, some of the results indicate this is not a simple process where all connexins behave similarly, and some effort should be made to investigate if Cx31 and 43, which do not seem to play the same roles in maintaining cell coupling as Cx26, also play any role in such metabolic rescue.

Thank you for this comment. We have addressed this by selecting three additional cell lines for study: RKO – a cell line with no major Cx expression; C10 – a cell line that expresses Cx43, but very low levels of Cx26; NCIH747 – a cell line that expresses Cx31, but low levels of Cx26. These additional experiments cover lines that are GJB2 (Cx26)-low/negative to test whether metabolic rescue is best achieved with Cx26. Our new data show that these cells are unable to rescue metabolic defects (new data provided in Fig 6H/I, Fig 7H, and Fig 6-supp2). These findings strengthen our case for a major role of Cx26, at least in CRC networks. Indeed, recent analyses by Robert Gatenby and colleagues have shown that mutations in GJB2 (Cx26) are exceedingly rare in cancer (a property not shown for other connexins genes). This is interpreted to mean that Cx26 plays a particularly prominent role, ostensibly for metabolic rescue.

REVIEWER #3 (PUBLIC REVIEW):

Strengths of the study include that it appears to be a careful and well thought out set of experiments. The analysis and treatment of multiplexed data is also sophisticated. For the most part, the work is clearly and logically described, as well as well illustrated. In general, the authors achieved their experimental goals, and the methods while not entirely new, do provide new twists and augmentations that should be useful to the field. A general weakness is that this is not entirely a new story. Instead, it is a variant of one of the oldest concepts in the field of gap junction biology i.e. "Metabolic cooperation". The term "Metabolic cooperation" (i.e., as mediated by gap junctions) was not mentioned by the authors, but it is a long-established and foundational concept in the field. Indeed, in a classic paper by Gilula and colleagues published in 1972, the experimental approach used was similar to that of the study in hand. These earlier authors showed how transformed cell lines with deficiencies in hypoxanthine metabolism can be "rescued" by "metabolic cooperation" in co-culture with metabolically competent cells via passing a gap junctional permeant molecule. This and other relevant papers were not cited. More importantly, the extant literature places the onus on the authors to explain and convince reviewers why this study is more than an incremental step.

We apologise for not quoting these important and classical references. We have now added these works to our reference list (quoted in Introduction). At the time of these seminal discoveries, Loewenstein and colleagues made a case that connexins are absent in cancer, and this belief persisted for many decades. More recently, the role of gap junctions in cancers has garnered attention. With new gene manipulations (e.g. CRISPR/Cas9) and imaging techniques and improved xenografting, it is now possible to precisely study the impact of GJ on cancer metabolism. Moreover, we have a wide panel of cancer cell lines to study, and identify the prominent role of Cx26. We highlight that our study is the first to offer a mechanistic explanation for the absence of negative selection in cancer, a phenomenon which was not known in the 1970s. To strengthen our novelty, we now add in vivo data to Fig 8 that confirm in vitro findings.

Author Response

Reviewer #1 (Public Review):

1. “The major weakness of the study is that with the interpretation of the results. The changes in tractography, behavior and TBM are what would be expected following lesions of the neostriatum”

We appreciate this comment and would like to offer clarification. We respectfully disagree that the pattern of results presented in this manuscript are akin to what would be expected following striatal lesions. In NHPs, striatal lesions typically cause more extreme phenotypes than what we observed in our 85Q-treated animals. In macaques, bilateral putamen lesions can result in phenotypes that include seizures, inappetence, hyper-aggression, and other severe features.  This strongly impacts clinical scores and can make it unfeasible to care for the animals for multiple years. For these reasons, recent NHP HD lesion models have used only unilateral putamen lesions coupled with bilateral caudate lesions to model HD (as in the recent paper by Lavisse et al, 2019). Of additional relevance is that even the cognitive effects of these striatal lesions are more severe than what we observed in our 85Q-treated animals: for example, Lavisse reported reduced performance on similar “prefrontal” cognitive tasks by ~50%, whereas our AAV-HTT model exhibited only ~10% reductions in working memory. This mild, but significant, change in cognitive performance and motor function seen in our 85Q animals is much more akin to that which is observed in the early stages of HD.

2. “The results have been interpreted as showing a progressive model, although evidence that there is progression is limited”...“begs the question as to whether or not the 85Q-lesioned monkeys would recover to a level similar to the 10Q animals if left for another 12 months”

At the request of Reviewer 1, we added an additional 30-month timepoint and re-ran all of the analyses to include these new data.  All of the behavioral and neuroimaging data were re-analyzed with this final timepoint included (see Lines 125-141, 146-163, 173-194, 228-255, 270-294, 314-345). Additionally, due to the unidirectional nature of our hypothesis and on the advice of our bio-statistician, we applied one-tailed tests to the planned comparisons in this revision. To address the Reviewer’s point directly: 85Q-treated animals showed minimal evidence of functional recovery between the 20- and 30-months timepoints on the behavior tasks. In particular, working memory deficits measured with SDR and fine motor skills measured with Lifesaver Retrieval did not improve between 20- and 30-months (Figure 1C and 1F). Additionally, neurological rating scores in group 85Q remained consistently elevated (in the 5-7 range) between the 20- and 30-month timepoint. Taken together, we feel confident that these results do not show evidence of any significant functional recovery, out to 2.5 years (30-months). In terms of the longitudinal trajectories of the behavioral measures, we appreciate the Reviewer’s feedback regarding the use of the term ‘progressive’ and have tempered our language appropriately. We removed all instances of the word progressive/progressed except in the context of the motor rating scores, which show a significant Group x Timepoint interaction and demonstrate a clear progression.

3. “The whole manuscript is written as though this is a genetically-relevant progressive model of HD. But the animals are normal, and so there is no genetic context relevant to HD”

We thank Reviewer 1 for this comment. We recognize that viral-based animal models of HD, including the model characterized here, are not as genetically similar to the human condition compared to some of the other modeling approaches currently under investigation (ex. knock-in and gene editing). Limitations of the AAV-based HTT85Q model include: 1) vector packaging restrictions that prohibit expression of full-length HTT, 2) the use of a CAG promoter vs. an endogenous promoter that leads to overexpression of the transgene, 3) the use of cDNA versus genomic DNA excludes introns and therefore lacks the ability to produce alternatively spliced variants (ex, Exon 1), 4) the use of a mixed CAG-CAA repeat may preclude the possibility of somatic instability and 5) expression of HTT that is restricted to specific brain regions and cell types. All of these important limitations have been added to the discussion section in this re-submission (Lines 503-517).

Despite these limitations, we feel that this AAV2:AAV2.retro-HTT85Q based model has some features that make it genetically-relevant to human HD including: 1) the expression of an N-terminal fragment of human HTT (N171), 2) the N-terminal fragment bears a pathological PolyQ expansion (85Q), 3) the expressed mHTT fragment forms neuronal aggregates that can be detected in the nucleus, 4) mHTT fragments are expressed in many of the same brain regions where aggregates are detected in human HD cases, with both regional and sub-regional specificity (ex. higher expression in anterior vs posterior cortical regions and expression primarily limited to deep cortical layers V/VI) and 5) expression of mHTT fragments in these regions leads to many of the same pathological and behavioral changes observed in HD patients.  Importantly, expression of the N-terminal portion of HTT allows for the evaluation of HTT lowering therapeutics that target first 3 exons (ASOs, miRNAs, zinc finger repressors, CRISPR-based therapies, etc), which cannot be evaluated in lesion-based models.

4. “The authors state in the Abstract that the injection resulted in "robust expression of mutant huntingtin in the caudate and putamen". These data are not in the manuscript.”

Evidence of mHTT expression in the caudate and putamen, as well as several other brain regions, via immunohistochemical and immunofluorescent staining is now included in the manuscript. Please see additions to the methods, results and discussion sections regarding these findings, as well as a new Figure 5, (see Lines 347-376, 756-788). Additionally, further details regarding an associated PET imaging study in this same cohort of animals using a mHTT aggregate-binding radioligand has been added to the discussion, (see Lines 437-443). Please also see response #13 (below).

5. “The authors chose to use a fragment of the HD gene, with a very long repeat that is seen only in juvenile patients”

Comments regarding the need to use a fragment of the HTT gene, versus the full-length gene, due to packaging constraints of the viral vector, were added to the discussion in the context of limitations (Lines 503-517), and also discussed above in response #3.  The choice to use a CAG repeat length of 85 (83 pure CAGs followed by a CAA/CAG cassette -see response #17 below for further details), was based off previous studies wherein similar CAG repeat lengths were used to create animal models of HD over the past several decades. Interestingly, while CAG repeat lengths in patients with adult-onset HD typically range from ~40-60, longer repeat lengths (>60) are typically required in animal models of HD to elicit pathological and behavioral manifestations of disease: transgenic, knock-in and viral vector-based rodent models (ranging from 72-150 CAGs), OVT73 transgenic sheep model (73 CAGs), transgenic and knock-in minipig models (ranging from 85-150 CAGs), transgenic and viral vector-based macaque models (ranging from 82-103 CAGs). See Ramaswamy et al, 2007 and Howland et al, 2021 for thorough reviews of these models.

6. “For their cognitive testing, the authors used a task (delayed non-match to sample) that measures object recognition and familiarity. Before surgery, only 11/17 of the animals were successfully trained to complete this task. It is not clear how useful the data are when only 64% of the animals can be included.”

We appreciate the Reviewer’s concerns and have decided to conservatively remove this data from the revised manuscript.

7. “It is not clear how this monkey model will be useful for developing either disease biomarkers or therapeutic strategies for HD (as stated in the abstract)”. “The authors state that they hope the model will become a widely used resource. This seems an unlikely scenario, given the limitations of the current study and the challenges associated with using monkeys. They say that a major advantage of their technique is being able to generate large numbers of monkeys. But this is not a relevant argument if the usefulness of the model to investigate HD is not proven.”

We thank the reviewer for requesting clarification on these important points. We believe that this model will be useful for developing therapeutic strategies because the HTT85Q-treated macaques express mutant HTT, along with HTT aggregates, in several key brain regions that are affected in human HD, along with undergoing regional gray matter atrophy and white matter microstructural alterations that correlate well with behavioral dysfunction. Studies currently under review elsewhere also show reduced dopamine neurotransmission and regional hypometabolism via PET imaging in this model. Together, or individually, these imaging and behavioral changes can serve as outcome measures when screening potential therapies. Possible therapeutic interventions that are amenable to screening in this model are included in the discussion.

Regarding biomarker development, we have already engaged in PET imaging biomarker development in this model in collaboration with the CHDI foundation and the Molecular Imaging Center at the University of Antwerp, evaluating a candidate radioligand that binds to aggregated mHTT. See #13 below for a more detailed description of this PET study, including recent data showing its ability to bind to aggregated species of mHTT in several brain regions in this same cohort of HTT85Q macaques that correspond to 2B4 and em48 IHC staining (a manuscript describing these results has been prepared for submission and the PDF is included for the reviewers to peruse).

The authors do envision this AAV-based macaque model becoming a resource for the HD research community. While this model does have certain limitations (now detailed in the Discussion), we respectfully assert that all of the HD animal models, both small and large, each have their own important limitations to consider when deciding on which to use to screen therapeutics. Selecting a specific animal model based on the individual scientific questions being asked will be required, and employing a combination of models may be an even more prudent strategy.

While NHP research presents unique challenges (cost, housing requirements and recent challenges in availability, among them), we believe that viral vector-based NHP models could be more accessible to the HD research community compared to some of the other established large animal models; in that they may able to be readily created at contract research organizations (CROs), in addition to various academic research institutions. There are now many CROs that exist in the US, and elsewhere around the world, that have developed specific expertise in MRI-guided, intracranial delivery of AAVs into the NHP brain (including the caudate and putamen), in the context of assessing therapeutic interventions for a variety of neurological disorders (HD, PD, and MSA, among others). Most of these same CROs also have expertise in NHP imaging (MRI/DTI) and behavioral assessments across multiple domains. It seems feasible that AAV-mediated HD macaques could be produced in sufficient numbers to appropriately power therapeutic studies, using the outcome measures established in the current study.

Reviewer #2 (Public Review):

1. “The major weaknesses are the manner in which the data is presented”

We replotted all of the figures with improved color palettes and larger font sizes to make them easier to read. We also added additional details throughout the results section to aid in clarity and improve readability.

2. “The authors would benefit from talking more about their model in the introduction and including references to some key points. For example, there has been critical new data in the field showing the importance of poly (CAG) in disease, not necessarily poly(Q), and the community will want to know (and not be required to look up), the nature of the transgene. Is it a pure CAG repeat? A mixed repeat? If it is pure, do they see or could they measure somatic expansion in the various brain regions impacted? How does that data match the phenotypes seen? Since this is a transgene, there is no possibility for the exon1/intron1 splicing variant to appear - how does this impact their interpretation”

Further details regarding the transgene have been added to the Viral Vector Section of the Methods (Lines 531-550). The repeat is not pure and contains a single CAA interruption. The glutamine encoded repeat for HTT85Q contained 83 pure CAG repeats, followed by a single CAA/CAG cassette, while the glutamine encoded sequence for HTT10Q contained 8 pure CAG repeats followed by a single CAA/CAG cassette. Both constructs contained a proline stretch distal to the glutamine repeat in the following allelic conformation where QT represents the total glutamine length:

HTT85Q: QT\=85, (CAG)83(CAACAG)1(CCGCCA)1(CCG)7(CCT)2

HTT10Q: QT\=10, (CAG)8(CAACAG)1(CCGCCA)1(CCG)7(CCT)2

There are plans to probe for somatic expansion in various brain regions, including the caudate and putamen, as well as several distal cortical regions. That analysis is ongoing and not in the scope of the present manuscript; however, these analyses are now mentioned in the discussion section (lines 540-560), as well as a discussion on the ability to either remove or duplicate the CAA/CAG cassette to potentially increase or decrease the rate of disease progression, respectively, based on the work of Ciosi et al. 2019. Additionally, Reviewer 2 is correct in that the lack of intronic sequences in the transgene precludes the formation of splicing variants, such as the exon1/intron1 variant, which we know is pathological based on the work of Bates et al. This drawback has been added to the discussion, along with other limitations of this viral vector-based model (Lines 503-517).

3. “What about RAN translations? Is RAN translation noted at all in this over-expression model? How does that contribute (or not) to the progressive phenotype they see in their NHPs?”

We are also curious regarding the assessment of toxic protein products from RAN translation of the expanded repeat sequence in this model. These studies are planned, and the results of these assays will be included in a future manuscript describing other ongoing post-mortem evaluations in this model.

Author Respose

Reviewer #1 (Public Review):

This manuscript reports a new genetically encoded neuronal silencer BoNT-C. They show that it fully blocks neurotransmission in two classes of Drosophila motor neurons (Is and 1b; tonic and phasic, respectively). They also update a GCaMP postsynaptic reporter SynapGCaMP to express GCaMP8f instead of 6f. They selectively silence 1b or 1s neurons to disambiguate the neurotransmission properties of each neuron. Finally, they show that silencing either 1b or 1s neurons does not induce heterosynaptic structural or functional plasticity (only neuron ablation triggers plasticity). The data are convincing. The new silencing tool will be widely used.

We thank this reviewer for his positive assessment of our study and for highlighting the utility of the new silencing tool presented in this study.

Reviewer #2 (Public Review):

The conclusions of this paper are properly supported by the provided data.

Overall this work opens a new window to examine novel aspects of heterosynaptic structural and functional plasticity.

We also thank this reviewer for his positive assessment of our study and for putting the importance of our findings in context.

Reviewer #3 (Public Review):

The strength of the manuscript by Han et al. is the comprehensive characterization of BoNT-C, showing that it truly abolishes all evoked and mini responses without structural alteration of the NMJ. Based on this, the authors then show that ablation of all neurotransmission in either Ib or Is does not cause any compensatory changes (neither functional nor structural) in the 'other' (i.e. looking at Is when silencing Ib or looking at Ib when silencing Is).

The weakness of the manuscript lies in the modest gain over the previous work. Specifically, Aponte-Santiago had already shown that many parameters are not changed (in Ib when Is is perturbed, or in Is when Ib is perturbed), including that 'the Is terminal failed to show functional or structural changes following loss of the coinnervating Ib input' (quote from 2020 paper). Hence, the only major difference is that Han et al now show that Ib also does not really change when Is is silenced. Aponte-Santiago also clearly showed a ~50% EJP reduction when Is or Ib are perturbed alone, and adding these two equals wild type. The highly emphasized finding of Han et al. that (quote) ' composite values of Is and Ib neurotransmission can be fully recapitulated by isolated physiology from each input' quite obviously follows from the one key finding that one does not affect the other, as mentioned above in the strengths. The wording is a bit odd, but really adding Is (with Ib perturbed) and Ib (with Is perturbed) inputs is really not adding much over either the main finding nor the previous work.

We thank this reviewer for his/her/their assessment of our study and for highlighting the strengths in characterizing the impact of BoNT-C expression at the NMJ. We also understand and appreciate the criticisms raised. It is important to note from the outset that the motivation and central goal of this study was not primarily to mechanistically dissect heterosynaptic plasticity between tonic and phasic motor inputs at the Drosophila NMJ. Rather, it was to develop an approach that would, for the first time, enable accurate isolation of complete neurotransmission from entire MN-Is or MN-Ib NMJs (both miniature and evoked transmission). By the reviewer’s own admission, we were entirely successful at achieving this central goal in our comprehensive characterization of BoNT-C.

Next, the reviewer raises the valid question about whether this achievement is a significant advance over previous work, and discusses recent experimental findings regarding heterosynaptic plasticity at the fly NMJ. We want to emphasize here that having a tool that is capable, for the first time, of accurately discriminating complete transmission from Is vs Ib alone is a major advance, one that it is not clear the reviewer sufficiently appreciates. As summarized in Fig. 1, no previous attempts have been successful in accurately isolating synaptic transmission between Is vs Ib synapses. In particular, no previous approach was capable of isolating miniature activity from Is vs Ib, and as we show in our manuscript, miniature events exhibit major differences between the two inputs. Thus, without isolating miniature transmission, one cannot know baseline synaptic function in Is vs Ib nor whether heterosynaptic functional plasticity has been induced. Further, we detail major confounds with some of the previous approaches the reviewer alludes to in prior studies, including selective optogenetic stimulation.

Finally, the reviewer discusses at length recent findings regarding heterosynaptic plasticity and questions whether the new insights revealed by BoNT-C provides a sufficient advance. In particular, the reviewer refers to previous work published in 2020 and 2021, where important initial insights into Is vs Ib structure and transmission after differential manipulations to either input was reported. The reviewer appears to believe that it was settled in these studies that no heterosynaptic functional plasticity was induced.

However, a critical point that the reviewer appears not to appreciate is that while the two previous studies on heterosynaptic plasticity at the Drosophila NMJ were able to assess structural plasticity (AponteSantiago et al., 2020; Wang et al., 2021), no accurate or quantitative conclusions can be made about heterosynaptic functional plasticity from these studies. This is due to the authors not knowing what baseline synaptic function is at Is vs Ib (miniature frequency, miniature amplitude, and evoked transmission), so that in their manipulations they cannot accurately determine whether any functional changes are observed after their manipulations. Further complicating the interpretation of the previous studies is that at the muscle 1 NMJ (2020 study), like the muscle 4 NMJ (2021 study), ~30% of these NMJs fail to be innervated by a Is input in wild-type larvae. This major confound makes it difficult to know how or whether adaptive plasticity is induced in wild-type NMJs with or without Is innervation (since, interestingly, evoked transmission does not appear to change in wild-type m1 or m5 NMJs with or without a Is input), and then to determine whether any heterosynaptic plasticity is induced. Indeed, we have also struggled with how to accurately determine whether synaptic function changes compared to baseline throughout our studies at earlier stages, despite the fact that the muscle 6/7 NMJ we use in our study does not suffer from the variable Is innervation confounds observed at muscle 4 (Wang et al., 2021) and muscle 1 (Aponte-Santiago et al., 2020).

Respectfully, we contend that the only way one can accurately and quantitatively determine baseline synaptic transmission (miniature amplitude, frequency, evoked, quantal content), and whether any changes are observed following manipulations to Is or Ib, is to fully and accurately recapitulate wild type (blended Is+Ib) neurotransmission from isolated Is vs Ib transmission. This is why we believe the data shown in Fig. 7 (and also Fig. S7 in the revised manuscript) is so important. It is true that numerous previous studies established relative and qualitative differences between Is vs Ib (miniature events are larger at Is relative to Ib, Is drives larger depolarization in response to single synaptic stimulation over Ib, etc). However, in no case did previous studies accurately assess baseline Is vs Ib synaptic function from entire inputs, and therefore could not conclude with certainty whether heterosynaptic functional plasticity was induced.

On a different but somewhat similar topic, UAS-BoNT-C is not a new tool. I am a bit put off by the wording ' We have developed a botulinum neurotoxin, BoNT-C...'. More on this and the way the previous BoNT-C paper (Backhaus et al., 2016) is cited in the detail comments below in the recommendations for the authors.

We understand these points raised by the reviewer. Our BoNT-C transgenic line is indeed a new tool, the only one in which synaptic transmission has ever been electrophysiologically characterized and shown to completely silence synaptic transmission in Drosophila. That being said, in retrospect, we can appreciate that the term “developed” might imply a level of innovation that reasonable people can disagree about. We have therefore elected to change the apparently offensive wording to “We have employed a botulinum neurotoxin, BoNT-C…” in the abstract of the revised manuscript.

Additionally, the manuscript does not really dive into an analysis of phasic versus tonic functions (that's just a correlation with the Is and Ib dominant modes of function).

We absolutely agree that selective silencing by BoNT-C now enables a rigorous study of tonic vs phasic neurotransmission at MN-Is vs MN-Ib NMJs, but that in the current manuscript we have not focused on this interesting question. We have adopted the convention the field has used to classify MN-Is and MN-Ib subtypes based on their apparent firing modes as “phasic” vs “tonic”, but like previous studies, we have not analyzed these functional distinctions on a deeper level. Although the focus of the current manuscript was to establish the properties of BoNT-C and highlight its utility as a tool for the field, we are now in the process of preparing an entirely new manuscript focused on just this reviewer’s question about the differences in tonic vs phasic synaptic physiology. This eight-figure manuscript will be entitled “Electrophysiological properties and nanoscale distinctions that define tonic vs phasic glutamatergic synapses” and is focused on the central question raised by the reviewer - how and why synaptic transmission differs between tonic vs phasic inputs. While this interesting question is outside the scope of the current manuscript, we will submit this new manuscript within the next few months, which is based on new experimental insights now enabled by selective BoNT-C silencing established in the current manuscript.

Finally, since the authors show that loss of Is or Ib function does not cause any change in the other, we are left to wonder what actually DOES cause heterosynaptic plasticity. TNT or rpr DO cause some heterosynaptic plasticity and they also DO cause some structural changes - but whether the structural changes themselves are important here remains unclear. Substantial progress would have been to take the starting point that BoNT-C does not cause heterosynaptic plasticity, and then identify the signal that does (is it morphology? or signaling between Is and Ib? Or with the muscle?).

We certainly agree with the reviewer that understanding how heterosynaptic plasticity is induced is an important question and worthy of additional investigation. As stated above, the focus of our current study was to establish the tool, BoNT-C, that will now enable a variety of fascinating and important future studies, both at understanding how and why synaptic strength differs between tonic vs phasic synapses and also how heterosynaptic plasticity signaling occurs at the NMJ. It required substantial time and experimental effort to establish that BoNT-C works to cleanly silence transmission without inducing structural and functional plasticity in the current manuscript (Figures 1-7 and several supplemental figures). Respectfully, we believe it is unreasonable to expect all of this data to be relegated to a “starting point” to then go on and probe heterosynaptic plasticity in more detail, all compressed into a single paper.

It appears this reviewer is particularly interested in heterosynaptic plasticity, which we agree is a fascinating topic. First, we should clarify that in our experiments, TNT expression does NOT induce any heterosynaptic structural or functional plasticity (see Figures 6 and Table S2), at least in our studies at m6/7, m12/13, and m4 NMJs. Rather, TNT expression alters synaptic structure in the neuron in which it is expressed (“intrinsic structural plasticity”, Fig. 6), but does not induce any changes to the convergent input. Hence, the only evidence for actual heterosynaptic plasticity is the rather minor adaptations in synaptic structure and function observed following ablation of Is motor inputs (Fig. 6 and 8).

In addition to the important insights revealed by BoNT-C in accurately distinguishing tonic vs phasic transmission outlined above, it appears that the reviewer does not fully appreciate the mechanistic constraints that the new BoNT-C tool reveals about heterosynaptic signaling. We would therefore like to highlight the key insights our study has revealed specifically about heterosynaptic plasticity. First, we show that at the muscle 6/7 NMJ, loss of MN-Ib completely eliminates Is innervation – this was not the finding reported in the 2020 study (Ib ablation was not reported in the 2021 study). Rather, AponteSantiago et al. 2020 reported that elimination of Ib did not trigger compensatory changes in active zone or bouton numbers of the Is input, no were compensatory increases in the Is EPSP reported. This may be due to the confounding variable Is innervation at the muscle 1 and muscle 4 NMJs used in the previous studies. Second, to what extent miniature transmission changes after manipulating activity from Is vs Ib could not be accurately assessed in previous studies because spontaneous activity persists following TNT expression as does innervation following rpr.hid expression. Third, and perhaps most important, our study is the only one that can demonstrate no heterosynaptic functional plasticity is induced by the physical presence but functional silencing of neurotransmitter release between tonic vs phasic inputs at NMJs with consistent innervation by both Is and Ib inputs.

It is clear to us now that we did not do a sufficient job of emphasizing these advances our study has now revealed about the baseline and heterosynaptic relationships between Is vs Ib. We have added additional details throughout the revised manuscript to ensure these insights are highlighted in an effort for the reader to better appreciate the importance of this study.

Overall, while an initial reading of the manuscript sounded rather exciting, a deeper analysis of the work in context of the literature of the last few years diminishes my enthusiasm for the novelty and progress provided.

We have responded to the major criticisms raised by this reviewer above and hope that he/she/they can more fully appreciate the importance of the new tool we developed, the impact it will have on the field in opening new studies on tonic vs. phasic transmission, and establishing the rules of heterosynaptic plasticity between convergent tonic and phasic inputs on common targets.

Author Response

Reviewer #1 (Public Review):

It should also be noted that their immunohistochemical studies of human fetal tissue for TBX5 and PTK7 are not convincing. There appears to be widespread staining of multiple cell types, suggesting either very broad expression of both genes or poor specificity of the primary antibodies.

We appreciate the reviewer’s comment that the immunohistochemistry staining does not provide definitive evidence for the functional importance of TBX5 and PTK7 in PUV, however these images do confirm that the proteins are ‘in the right place at the right time’ during normal human urinary tract development. We have updated the discussion on page 19, line 441-445 to emphasise this. To further support a putative role for these proteins in urinary tract development we have added additional images from a second human embryo at the same gestation which confirms these distinct patterns of staining (Figure 8 – figure supplement 1 on page 14, lines 313-317). Even if these proteins can also be detected in other tissues or cell types, this does not detract from this idea, as in other locations the proteins may have redundant or different roles. 

PUVs have not been described as a clinical manifestation of disease associated with mutations of either gene in humans.

The reviewer is correct that rare variants affecting TBX5 and PTK7 have not previously been associated with PUV. They have however been associated with other developmental anomalies (as stated in the discussion on page 18, line 408-411 and page 19, line 434-437) confirming a clear role in embryonic development for both these genes.

The fact that rare variant association testing did not identify an increased burden of rare, likely deleterious variants in these two genes (although with limited power in this cohort) suggests that PUV is not driven by ultra-rare, highly penetrant alleles in these genes. However, the identification of common and low-frequency variants using GWAS suggests a complex mode of inheritance for PUV likely in combination with maternal_/in utero_ factors. As with other complex traits, these signals provide potential insights into the underlying biology of this disease as opposed to the diagnostic implications of conventional monogenic gene discovery associated with purely Mendelian conditions. A paragraph on the Mendelian/complex trait implications of the findings of the study has been incorporated into the discussion (page 21-22, line 594-502).       

Discuss how variants in either gene or in the patterns of structural variants that they found associated with PUV intersect with sex to result in this exclusively male condition.

The fact that PUV is a uniquely male disease is most likely the result of differences in urethra and bladder development and length differences in urethra between males and females. Sex hormones may also potentially result in tissue-specific differences in gene expression (Ober, Loisel, and Gilad 2008). We have added a paragraph into the discussion to clarify this (page 20, line 454-463) as well as clarified the results of the chromosome X and sex-specific analyses (page 7, lines 149-155; see also Reviewer 2, point 5 below) as suggested. 

Reviewer #2 (Public Review):

Major:

1. The replication study is problematic given that different genotyping methods are used for cases (targeted KASP) versus controls (WGS). This may introduce differential bias. Moreover, the ancestry of the control cohort (UK-based) does not seem to be well matched to the cases (predominantly German and Polish), and the lack of genome-wide data for the cases precludes proper adjustment for population stratification. The case-control design is also imbalanced in the replication study. The authors should reconsider their replication strategy to include a more balanced cohort with ancestry-matched controls and uniform genotyping. As an alternative, genome-wide genotyping of the replication case cohort would significantly enhance the study and should be considered.

Many thanks to the reviewer for their valuable comments regarding the replication study case-control cohort. While different sequencing technologies were used to compare allele counts at the lead variants in the replication study (KASP genotyping for cases vs WGS for controls), both techniques exhibit > 99.5% accuracy and are subjected to variant level quality control metrics. Only individuals with reliably called genotypes were included in the replication analysis. This has been clarified in the methods section (page 30, line 693).

We were able to obtain genome-wide genotyping data for 204 of the 395 European cases in the replication cohort. While (despite sustained effort on our part) we were unable to analyze this data jointly with the control cohort in the 100KGP due to enforced limitations on data sharing, we were able to demonstrate similar ancestry of the replication study cases and controls:  we performed PCA on a set of ~80,000 overlapping autosomal, high-quality, LD-pruned variants with MAF > 10% and projected the cases and controls separately onto (the same) data from the 1000 Genomes Project (Phase 3) labelled by ‘population’ (Figure 5). This clearly demonstrates that both cohorts have homogeneous European ancestry, as stated now in the results (page 8, lines 166-168).

We note with thanks the reviewer’s comments regarding the case-control imbalance in the replication study which can sometimes result in a type 1 error. To address this, the case control ratio was reduced from 1:27 to 1:10.5 by including only the 4,151 male controls from the cancer cohort of the 100KGP. The results remained significant for both lead variants and have been updated in the manuscript (page 8, line 162-176; Table 2).

When the number of controls was reduced to 500 males (a case:control ratio of 1:1.3), rs10774740 (TBX5 locus) remained significant demonstrating that case-control imbalance was not driving the observed signal (P\=9.9x10-3; OR 0.77; 95% CI 0.63-0.94). rs144171242 (PTK7 locus) however did not reach significance due to insufficient power (P\=0.06; OR 2.24; 95% CI 0.93-5.36). For a rare variant such as rs144171242 (MAF ~ 1%), a replication study with 500 controls is only powered to detect association with large effect size (OR > 3.5). A case:control ratio of ~1:10 is therefore optimal to maximize power to detect association, while minimizing unnecessary noise from excess controls. This has been added to the results section of the manuscript (page 8-9, lines 178-184).

2. I am reassured that the TBX5 signal remains genome-wide significant in European-only analysis. However, the signal at PTK7 appears much less robust - it has borderline statistical significance (especially given that the authors test for all rare and common variants across the genome) and is represented by a single variant with a relatively rare risk allele that is differentially distributed by ancestry. Therefore, I would like to see more information for this specific signal:

Information on the depth of coverage and the quality of the top variant

This has been incorporated into the manuscript for both lead variants (Page 7, lines 142-145). For rs144171242 at the PTK7 locus, the meanDP was 29.34 and the meanGQ was 75.59.

Information if the top PTK7 variant remain genome-wide significant after application of genomic control. Of note, the calculation of genomic inflation is dependent on sample size - lambda of 1.05 may represent an underestimate given low power of the cohort, and this point deserves at least a comment. Some methods correcting lambda for sample size have been proposed, and the authors should consider applying these methods.

We appreciate the reviewer’s comments that the value of lambda may be affected by sample size and have added a comment to this in the manuscript (Page 7, line 136-137). Despite extensive searching, we were unable to find a recent published example of how to correct lambda for sample size and would be grateful if the reviewer could suggest a reference for this.

To answer the reviewer’s specific question, application of genomic control to the lead variant at PTK7 results in P\=4.37x10-8 which remains below the threshold for conventional genome-wide significance. However, while the genomic inflation factor provides a reasonable indication of possible confounding by population structure, there are recognized limitations to applying it as a corrective factor as it assumes that all variants are confounded i.e., the same correction is applied irrespective of differences in population allele frequency which can be insufficient for some variants and lead to a loss of power in others. Furthermore, in addition to sample size, lambda can vary with heritability and disease prevalence (Yang et al. 2011) and its use for correction can therefore be too conservative and reduce power to detect significant associations. In this manuscript we therefore chose to use the mixed model approach (as part of SAIGE – detailed in the methods on page 28, lines 647-648), which has largely superseded older methods such as genomic control, to robustly correct for both population structure and cryptic relatedness and minimize false positive associations (Shin and Lee 2015).

This locus requires more robust replication as discussed above. If more robust replication study is not possible, additional functional studies could provide more evidence in support of this locus.

Please refer to point 1 regarding the revised and more robust evidence of replication. 

3. There is no validation of sensitivity and specificity of SV detection by variant size or type (e.g. inversions, deletions, duplications). Also, since burden differences are not replicated independently, the authors should stress the exploratory nature of these analyses.

We appreciate the reviewer’s comment that there is no independent validation of SV detection (e.g., by microarray or long-read sequencing) and this was reported as a limitation of our study in the discussion (page 22-23, line 520-524). However, one of the main strengths of this study is the use of clinical-grade WGS data where all samples have been sequenced on the same platform and undergone variant calling using the same bioinformatics pipeline. This essentially eliminates confounding due to differences in data generation and processing and the sensitivity and specificity of SV detection will therefore be the same for both cases and controls.

We agree with the reviewer that the SV analyses have not yet been replicated independently and, as they suggest, have stressed the exploratory nature of the findings in the discussion (page 21, line 491-493).

In the discussion (especially second paragraph, but also throughout), the authors overemphasize multi-ancestry nature of their study. The reality is that the included non-Europeans are very small in numbers (18 SAS cases, 11 AFR cases, and 14 admixed cases). I would suggest for the authors to specifically state these case counts and make it clear that expanded efforts to recruit non-Europeans are still needed given these very low numbers.

We appreciate the reviewer’s comment about the overemphasis on the multi-ancestry nature of the study and the small absolute numbers of individuals included, however as a proportion of the cohort, a third of the cases are non-European: 14% are of South Asian ancestry, 8% are of African ancestry and 11% are admixed. This breakdown comprises a greater proportion of non-white European ancestry individuals than the UK as a whole (DOI: 10.5257/census/aggregate-2001-2), where the discovery cohort was based. This provides evidence that our study eliminates at least some of the Euro-centric bias present in existing genetic and genomic literature, at least as far as the UK population is concerned. Clearly, global studies fairly representing all populations would be needed to address this issue perfectly. The case counts were reported in Table 1 but we have now referenced the low absolute numbers and included the reviewer’s suggestion about expanding efforts to recruit non-European populations in the main text (page 22, line 518-520). We have also edited paragraph two of the discussion in response to the reviewer’s comments (page 17, line 387-398).   

Supplemental figure 2 -provide case-control counts in each ancestral group (Y axis).

These have been added to the figure legend of Figure 6 – supplemental figure 4 (previously Figure 5 - supplemental figure 2).

Supplemental figure 3 is misleading since allelic frequencies in the cases are pooled and are not available individually for all depicted populations.

Figure 5 - supplemental figure 3 has been removed and replaced by Figure 6 – supplemental figure 3 to show only the individual case, control and gnomAD AF by ancestry for AFR, SAS and EUR population groups instead of using the pooled allele frequencies.

5. I did not see details of chr. X analysis. This is important given that the case group involves only Males and control group involves both Males and Females. Also, please explain how sex was used as a fixed effect (as stated in the methods) given that the case cohort is 100% male.

We thank the reviewer for their insightful comments. Sex was used as a covariate (or fixed effect) to control for the anatomical differences in development of the urethra (and in utero hormonal changes) between the sexes in the control cohort (clarified in the methods, page 28, lines 651-653). Given the PheWAS findings (page 13, line 292-297) reveal an association between the lead variant near TBX5 and female genital prolapse and urinary incontinence, this suggests that while women do not develop PUV (due to differences in urethral development) they may manifest other lower urinary tract phenotypes. In theory, removing the female individuals from the control cohort should therefore strengthen the association as the signal would not be diluted by ‘affected’ women (i.e., those with potentially unknown lower urinary tract phenotypes). We tested this by performing a sex-specific male-only GWAS and found that the strength of association at both lead variants increased. The results of this have been added to the manuscript (page 7, line 149-155).

The results of the chromosome X rare variant analysis are shown on the Manhattan plot (Figure 9), with no significant genes identified. We have added chromosome X to the mixed-ancestry and European GWAS as suggested (with no significant results) and the Manhattan and Q-Q plots have been updated in Figure 2 and Figure 6. The number of analyzed variants in each analysis has also been updated accordingly.

Author Response

Reviewer #2 (Public Review):

Feeding behaviour in C. elegans has been extensively studied over decades. Several methods  of measuring feeding exist, but none can directly measure both pumping and locomotion  behaviour in freely-moving worm populations. The authors have developed a new  imaging-based method for automated detection of pharyngeal pumping events in freely moving

C. elegans populations, and can thus simultaneously measure pumping and locomotion  behaviour in tens of worms, at a single-worm, single-pump resolution that is not possible with  previous methods. This user-friendly method can be applied to several research directions, such  as large-scale foraging, behavioural coordination, and high throughput screening.

The authors designed their new method to be broadly applicable and user-friendly, for easy  adaptation in other research labs. However, adding direct evidence to show that "the method is  relatively insensitive to the optical instrument used" will better support this claim of wider  application.

We appreciate the reviewer’s suggestion to show evidence that our method will also work on  data acquired on different microscopes. We now present data obtained on a second  epi-fluorescent microscope, which was downscaled and analyzed in Fig. 1H-J.

The authors carefully benchmarked their new method against expert annotations and existing  results from previous methods, to both validate their method and reveal additional advantages.  They also assessed potential pitfalls of the method such as by examining the effect of  fluorescence imaging on the behavioural outcome, albeit only at the timescale of minutes. The  effect of longer-term fluorescence imaging should be further explored, which is relevant for  large-scale foraging experiments that the authors discussed. It could be helpful to determine the  maximum total exposure for the method to still be valid, both in terms of pump detection (which  could be sensitive to photobleaching) and behavioural modulation (which could be sensitive to  higher phototoxicity).

We thank the reviewer for this comment. In response to their comment and related comments  by the other reviewers, we have provided bleaching curves and evidence of long-term imaging  to show the potential of the methods for longer scale assays. We found that with our illumination  intensity (see methods), bleaching was significant at a time scale of ~1h. We then added  triggered illumination and could extend the recording time to ~5 h (Methods). Additionally, we  perform a supplementary control for viability of worms exposed to continuous light (not  triggered) for 5 hrs. We do not observe any apparent phototoxic effect.

Overall, the manuscript is well-written and the results are clearly presented both in terms of  statistics and interpretation. Methodological details are well-documented and openly accessible.

We thank the reviewer for their positive view of our work and their appreciation for our efforts to  document both data and software.

Reviewer #3 (Public Review):

In this manuscript, the authors present a method for simultaneous assessment of pharyngeal  pumping (feeding) and locomotion in many C. elegans simultaneously. In this technique,  imaging of the fluorescent labeled pharynx provides a measure of velocity and pumping rate,  through analysis of the spatial variations in fluorescence.

The technique is clearly described, well-validated, and yields some novel results. It has the  advantage that it can be performed using microscopes found in many C. elegans laboratories.

We appreciate that the reviewer recognizes the wide applicability of the method across many C.  elegans  laboratories.

Some limitations of the method include its reliance on fluorescence imaging, which is a  hindrance to genetic analysis, computational intensiveness, and phototoxic effects of  fluorescence excitation that are not fully explored in the manuscript.

The authors show the utility of their method by assessing pharyngeal pumping and motor  behavior (1) during development, (2) in the presence or absence of food, and (3) in the  presence of two mutations affecting feeding.

Although I understand these are proof-of-principle demonstrations, I still came away feeling  underwhelmed by these examples. I did not see any results here that could not have been  obtained fairly easily with conventional techniques.

We appreciate the constructive criticism of the reviewer and highlight in the revised version the  fact that using conventional techniques such studies would require tens of hours of experiment  time. We would like to emphasize the comparisons in Table 1 where we show other methods  and their current limitations. Obtaining a dataset such as in Figure 3 which comprises a total of  34 worm-hours of pumping observation from unrestrained animals is to our knowledge currently  impractical with competing methods. We would like to remind the reviewer that, using our  method we were able to reveal bimodal distributions within a population as illustrated, for  instance, in Fig. 3F, 4B, and 4F. These observations are not possible when the single worm  resolution is not accessible or when large statistics are not feasible as it happens with previous  methods.

Given these limitations, I feel the method's eventual impact in the field will be relatively small.

In this study, we present a method allowing performing behavioral studies on worm populations  at high throughput and reduced costs. Such a technique opens the door to many laboratories  that can not do EPG recordings or microfluidics due to the technical difficulties, or that want to  study animals in their normal plate context. We also would like to emphasize that there are already more than 1500 strains containing myo-2  promoter transgene available on CGC, which  would be amenable to our imaging approach. These transgenic strains form broad classes of  interest, such as thermotolerance, ER stress resistance, aging and neural-circuit specific genes.

Pharyngeal pumping has also been used as a read-out for pharmacological screens, for  example, bacteria pre-loaded with pharmacological agents are tested for their effect on  pharyngeal pumping rate. Pharaglow offers a high-throughput and sensitive method to measure  the pumping rate. This will benefit the field who use C. elegans  pumping for pharmacological  screens, and pave the way for the researchers who plan to use but are hindered by existing  techniques.

Author Response

Reviewer #1 (Public Review):

7) Can the primary cells in Figure 2E and AML#1 and AML#2 be studied for mTORC1 activity by Western, as in 2D?

For reasons that we do not understand, we have been unable to effectively culture primary FLT3-ITD AMLs, despite being able to culture most other AMLs for weeks. This issue has prevented us from being able to perform biochemical analyses of FLT3-ITD AMLs in response to FLT3 inhibition.

8) Additional genetic information should be provided if possible for the primary AML cells - what other mutations in addition to FLT3 were present? Were there any mTOR pathway alterations?

We provided the other mutations of AML#1 sample (NPM1 mutation) in the section METHODS-Therapeutic modeling in mice, as well as Figure legends 2E and 3D. There were no evident alterations in the mTOR pathway (beyond the FLT3-ITD mutation).

Author Response:

We thank the reviewers for their thoughtful critiques and helpful suggestions for how to improve our manuscript. Described below is our response clarifying a number of issues raised by the reviewers.

We agree with the reviewer that we cannot definitively conclude that the first division chromosome segregation defects and the later mid-blastula transition CI-induced defects are the result of distinct mechanisms. In fact, we raise this possibility in the discussion. However, our finding that the CI phenotype induces a temporally and developmentally deferred chromosome segregation defects in the late blastoderm divisions (in addition to the well-studied first division defect) alters the established view of the CI phenotype and must be taken into account when considering mechanisms of CI. Our current view is that the distinct early and late defects could be caused by either 1) a common mechanism (possibly a chromosome mark/defect inherited through the early blastoderm divisions causing segregation defects in the late blastoderm divisions) or 2) distinct early and late mechanisms that do not strictly “depend” upon one another. We have clarified this point in the revised manuscript.

We disagree with the reviewer that this result is to be expected given previous studies. In D. simulans, a small percentage of embryos derived from the CI cross hatch. These embryos are thought to have bypassed the first division defect. It is not obvious why there must be late defects in these embryos that “escape” early CI-induced defects and subsequently hatch. Previous studies interpreted embryos that exhibit late division errors as those that have lost their entire paternal complement of chromosomes as a result of strong CI-induced defects during the first mitotic division and develop as maternal haploids. These studies, including transgene- induced CI, have focused primarily on embryos that have undergone the first mitotic division embryonic defects. To the best of our knowledge, no group has thoroughly examined embryos that progress normally through the pre-cortical cycles 2-9 as performed in this manuscript. Thus, it was entirely unexpected that these embryos would exhibit the mitotic defects during the late blastoderm divisions and the MBT. We discuss how this finding requires modified current models for the mechanisms of CI.

Regarding the comment that “the primary claim of the paper that later-stage embryos die for different reasons than early-stage embryos,” we make no such claim. In fact, we provide evidence that the failure to hatch (late embryonic lethality) is, at least in part, due to haploid development—a direct result of the first division CI defect. The focus of our studies are those CI-derived embryos that progress normally, maintain the normal complement of chromosomes through the first division, and exhibit chromosome segregation errors during the late blastoderm divisions. We do not know the fate of these embryos, and previous studies have demonstrated that embryos suffering extensive late blastoderm segregation errors are able to hatch (Sullivan, 1990, Development 110:311-323). We have clarified these points in the discussion.

While we agree that transgenic tools have proven invaluable in the study of CI, they are not appropriate for these studies. The purpose of our study was to undertake an unbiased re-examination of the CI phenotype. Of necessity, the transgenic studies rely on exogenous host promoters rather than the natural endogenous Wolbachia/Prophage promoters. Thus, while informative, it is unlikely the that the transgenic alleles would capture all of the complexities and nuance of the CI phenotype. In addition, the transgenic studies, of which we are aware, have only interrogated a single pair of the CI-inducing genes, while the Wolbachia genome contains both Cid and Cin CI-associated gene pairs and possibly other yet-to-be-identified CI/Rescue genes.

Our unbiased re-examination of the CI phenotype induced by W. riverside in D. simulans identified a previously unsuspected temporally and developmentally distinct set of CI-induced defects that occur during and after the mid-blastula transition. This finding must be taken into account when considering the mechanisms that cause CI. In our revisions, we clarify the above points and qualify our statements to appropriately interpret our results in context of the nuances and uncertainties of CI and early Drosophila embryogenesis.

Author Response:

Reviewer #3 (Public Review):

The authors revealed the novel role of the DLL-4-Notch1-NICD signaling axis played in platelet activation, aggregation, and thrombus formation. They firstly confirmed the expression of Notch1 and DLL-4 in human platelets and demonstrated both Notch1 and DLL-4 were upregulated in response to thrombin stimulation. Further, they confirmed the exposure of human platelets with DLL-4 would lead to γ-secretase mediated NICD (a calpain substrate) release. Stimulating platelets with DLL-4 alone triggered platelet activation measured by integrin αIIbβ3 activation, P-selectin translocation, granule release, enhanced platelet-neutrophil and platelet-monocyte interactions, intracellular calcium mobilization, PEVs release, phosphorylation of cytosolic proteins, and PI3K and PKC activation. In addition, Susheel N. Chaurasia et al. showed that when platelets were stimulated with DLL-4 and low-dose thrombin, the Notch1 signaling can operate in a juxtacrine manner to potentiate low dose thrombin mediate platelet activation. When the DLL-4-Notch1-NICD signaling axis was blocked by γ-secretase inhibitors, the platelets responding to stimulation were attenuated, and the arterial thrombosis in mice was impaired.

This study by Susheel N. Chaurasia et al. was carefully designed and used multiple approaches to test their hypothesis. Their research raised the potential of targeting the DLL-4-Notch1-NICD signaling axis for anti-platelet and anti-thrombotic therapies. Interestingly, compared to thrombin, a potent physiological platelet agonist, the signaling cascade triggered by DLL-4 was relatively weak. Given that the upregulation of DLL-4 and Notch1 happened in response to thrombin stimulation, how much DLL-4 mediated signaling could contribute to in vivo platelet activation in the presence of thrombin is questionable. This could potentially limit the application of targeting Notch1 as an anti-thrombotic therapy. Further, the authors showed that Notch1 signaling could operate in a juxtacrine manner to potentiate low dose thrombin mediate platelet activation, which means the DLL-4 mediated platelet signaling can act as an accelerator of early-stage hemostasis. Again, inhibition of Notch1 may slow down the hemostasis process. But given the fact that there are other platelet agonists (ADP, collagen...) existing simultaneously, blocking Notch1 signaling may not have a strong anti-platelet effect.

We concur with the Public Reviewer that, further study is needed to delineate extent of contribution of DLL-4 signaling in thrombin-activated platelets. However, it is now amply clear that Notch signaling plays a central role in development of thrombinactivated phenotype of platelets. Further, DLL-4-Notch1 interaction on surfaces of adjacent platelets within the thrombus reinforces platelet-platelet aggregate formation. This is further reflected from significant inhibition of thrombus formation in vivo in presence of DAPT in a mouse model of intravital thrombosis. Given that there is a lot of redundancy in stimulation of platelets employing different physiological agonists (ADP, collagen, thrombin etc.), none of the present-day drugs is fully capable of effective platelet inhibition due to parallel signaling pathways. Thus, discovery of Notch signaling and its seminal role in platelet activation could explain redundancy associated with anti-platelet drugs, and Notch inhibition could complement with existing anti-platelet regimen in evoking effective and complete platelet inhibition.