Muscles owe their ability to contract to structural units called sarcomeres, and a single muscle fiber can contain many thousands of these structures, aligned one next to the other. Each mature sarcomere is made up of precisely arranged and intertwined thin filaments of actin and thicker bundles of motor proteins, surrounded by other proteins. Sliding the motors along the filaments provides the force needed to contract the muscle. However, it was far from clear how sarcomeres, especially the arrays of thin-filaments, are assembled from scratch in developing muscles.

When the fruit fly Drosophila transforms from a larva into an adult, it needs to build muscles to move its newly forming wings. While smaller in size, these flight muscles closely resemble the skeletal muscles of animals with backbones, and therefore serve as a good model for muscle formation in general. New muscles require new sarcomeres too, and now Shwartz et al. have observed and monitored sarcomeres assembling in developing flight muscles of fruit flies, a process that takes about three days.

The analysis made use of genetically engineered flies in which the gene for a fluorescently labeled version of actin, the building block of the thin filaments, could be switched on at specific points in time. Looking at how these green-glowing proteins become incorporated into the growing sarcomere revealed that the assembly process involves four different phases. First, a large store of unorganized and newly-made thin filaments is generated for future use. These filaments are then assembled into rudimentary structures in which the filaments are roughly aligned. Once these core structures are formed, the existing filaments are elongated, while additional filaments are brought in to expand the structure further. Finally, actin proteins are continuously added and removed at the part of the sarcomere where the thin filaments are anchored.

Shwartz et al. went on to identify a protein termed Fhos as the chief player in the process. Fhos is a member of a family of proteins that are known to elongate and organize actin filaments in many different settings. Without Fhos, the thin-filament arrays cannot properly begin to assemble, and the subsequent steps of growth and expansion are blocked as well.

The next challenges will be to understand what guides the initial stages in the assembly of the thin-filament array, and how the coordination between assembly of actin filament arrays and motor proteins is executed. It will also be important to determine how sarcomeres are maintained throughout the life of the organism when defective actin filaments are replaced, and which proteins are responsible for carrying out this process.

Sarcomeres constitute the basic functional units of muscle fibers, endowing these large and specialized cells with their contractile capacity. Central to sarcomere function is the lattice-like organization of two filament systems: an actin-based thin-filament array, which provides a stiff backbone along which thick filaments, composed of myosin motor proteins, 'slide' in order to produce force and contractile motion (Squire, 1997). The spatial organization and efficient operation of this remarkable cellular machinery relies on a host of dedicated proteins and protein complexes, which act to regulate sarcomere size and streamline its activity, and to coordinate between the multiple sarcomeric units that comprise individual myofibrils (Clark et al., 2002; Ehler and Gautel, 2008; Gautel and Djinovic-Carugo, 2016).

Despite their fundamental significance, elucidation of the molecular mechanisms underlying assembly, maturation and maintenance of thin-filament arrays remains one of the major open issues in the study of sarcomere structure and function. While mechanisms relating to size definition and stability of the arrays have been extensively investigated (Fernandes and Schock, 2014; Meyer and Wright, 2013), other key aspects of microfilament array formation and dynamics, including determination of distinct phases of array maturation, the identity and regulation of elements mediating filament nucleation/elongation, and the processes governing incorporation of additional filaments into nascent arrays are not resolved (Ono, 2010).

Here we address these matters in the context of formation and development of the Drosophila indirect flight muscles (IFMs). These are the largest muscles of the adult fly, which power flight by regulated contraction of the thorax (Dickinson, 2006). A major subset of the IFMs, the dorso-longitudinal muscles (DLMs), closely resemble vertebrate skeletal muscles in both their developmental program and in their mature myofibrillar structure (Dutta and VijayRaghavan, 2006; Roy and VijayRaghavan, 1999), making them a particularly attractive model system, in which the powerful molecular genetic approaches available to study Drosophila development can be harnessed to investigate and elucidate general principles of myogenesis.

DLM formation initiates by fusion of hundreds of individual myoblasts to a set of larval muscles during the first 24-30 hours of pupal development (Fernandes et al., 1991). The subsequent ~80 hrs of myogenesis leading up to eclosion of the adult fly include formation and maturation of a parallel arrangement of myofibrils and assembly and growth of sarcomeric units within them (Reedy and Beall, 1993; Weitkunat et al., 2014). This sequence of events takes place over a wide time window, providing an opportunity to temporally dissected and manipulate the coordinated processes giving rise to thin-filament array assembly and maturation.

IFM sarcomeres initiate as small, nascent structures, that grow considerably over the course of pupal development (Sparrow and Schock, 2009), reaching a final, uniform size of 3.4 µm in length and 1.5 µm in diameter. Spatial organization of the mature, evenly-spaced IFM sarcomeres closely mirrors that of striated vertebrate skeletal muscle (Reedy and Beall, 1993). Individual sarcomeric units are defined by Z-disc borders, which serve as anchoring sites for the barbed ends of the thin-filament arrays, and by a central, microfilament-free H-zone, bordered by the pointed-ends of neighboring thin-filament arrays. We utilized temporally-controlled expression of GFP-tagged actin monomers (Roper et al., 2005) to follow thin-filament array dynamics, and recognize key phases and distinct transitions of the arrays throughout sarcomerogenesis. In parallel we identified Fhos, the single Drosophila member of the conserved FHOD family of formin proteins (Schonichen and Geyer, 2010), as a major contributor to thin-filament array assembly and growth. An important aspect of Fhos involvement is its critical role in radial growth of the arrays, by mediating incorporation of new peripheral filaments to the nascent core structure. The elongation of thin filament arrays, shown to occur primarily from their pointed ends (Mardahl-Dumesnil and Fowler, 2001), is mediated by the WH2-domain actin regulator Sals protein. While the combined activities of Fhos and Sals can account for most aspects of thin-filament array growth and maturation during pupal stages, other elements are likely involved in additional processes that shape and maintain the arrays, such as continuous monomer exchange at the barbed-ends.

Formation of the adult Drosophila IFMs during pupariation provides an established model system to study formation of skeletal muscles, and in particular the generation of the repeated sarcomere structure, the core functional unit underlying muscle contractility. The IFM system possesses several key features that make it amenable for detailed analysis. These include an extended developmental time window of ~60 hr, the availability of genetic methods for investigation (e.g., RNAi-based knockdown) and the highly ordered and repetitive organization of sarcomeric units within IFM myofibrils, which allows for the detection and analysis of both major and subtle alterations and defects. Our study focused on the actin based thin-filament array component of sarcomeres. Towards this end, we utilized an additional, highly useful feature of the IFM system: the capacity to monitor the incorporation pattern of new actin monomers at discrete stages of the process, using transgenic GFP-actin constructs whose expression can be readily manipulated. The analyses performed using the various approaches and tools available for the study of the IFMs allow us to chart the principles, timeline and molecular basis for assembly, organization and maturation of thin-filament arrays within this model of sarcomerogenesis.

While it is likely that regulators of linear actin belonging to the formin family play a central role in the formation and maintenance of actin filament arrays in the sarcomere, redundancy among them appears to be commonplace in this context (Mi-Mi et al., 2012; Rosado et al., 2014), complicating the elucidation of distinct functional roles. Our detection of severe sarcomeric phenotypes following disruption of Fhos activity on its own, identifies Fhos as a key contributor to the processes governing IFM thin-filament array assembly, and sets the stage for studying the involvement of formins in this major aspect of microfilament organization, via utilization of genetic approaches. FHOD-family formins have been previously associated with sarcomere organization in cardiac muscle (Iskratsch et al., 2010; Kan et al., 2012; Taniguchi et al., 2009; Wooten et al., 2013), but the roles these proteins play during skeletal myogenesis have been difficult to ascertain. The ability to dissect the program of IFM sarcomere formation and to disrupt the activity of Fhos to different extents and at distinct phases, provides a comprehensive view of the role of this formin-family protein in the process, and of skeletal muscle thin-filament array assembly at large (Figure 7).

Figure 7

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1. The onset of pupal development is accompanied by a prominent burst of actin polymerization, generating a store of microfilaments for use during subsequent stages, when polymerization activity declines considerably. The identity of actin nucleators and in particular formins involved in the extensive initial polymerization is not known, and it is certainly possible that several formins act redundantly, given our failure to disrupt this process by single formin knockdowns, including that of Fhos. An initial sign of internal myofiber organization is seen in the segregation of the abundant microfilaments produced during the early phase of pupal development, into elongated myofibrils that align in parallel to the fiber. We do not know the nature of the signal governing this alignment, but it is noteworthy that previous studies of the dynamic organization of sarcomeres in cultured muscle cells have indicated that microtubules which are aligned along the fiber may provide an initial cue for the recruitment and orientation of myosin heavy chain and possibly microfilaments as well (Pizon et al., 2005).

2. The initial, widespread polymerization gives way to a more limited mode of incorporation, characterized by 'patches' of actin monomers that are added to a nascent microfilament array, suggesting that this period is devoted to proper structuring of the arrays within individual, uniformly-sized and regularly separated sarcomeric units. It is during this interim period that disruption of Fhos activity first leads to alteration of the monomer incorporation pattern, in what we interpret to be a telling fashion, as it retains an underlying, highly regular pattern of monomer incorporation at the pointed-ends of the thin filament arrays (Figure 4D–G). This observation constitutes a direct demonstration that IFM arrays elongate from their pointed-ends, contrary to the conventional barbed end-biased growth of microfilaments, and in keeping with previous findings based on studies of the pointed-end capping protein Tropomodulin in both IFMs and vertebrate cardiomyocytes (Littlefield et al., 2001; Mardahl-Dumesnil and Fowler, 2001). Furthermore, our investigation identifies the WH2-domain protein Sals as a major mediator of the pointed-end growth of IFM thin-filament arrays, similar to its function in Drosophila larval muscle sarcomeres (Bai et al., 2007). Interestingly, Leiomodin acts to mediate pointed-end elongation in vertebrate cardiomyocytes (Chereau et al., 2008; Tsukada et al., 2010), implying a conserved function for WH2-domain actin regulators in sarcomerogenesis.

Interfering with Fhos activity results in severe impairment of myofibril and sarcomere organization (Figure 2), raising the question of how to reconcile the strong mutant phenotypes with the limited degree of Fhos-dependent actin incorporation and array growth during the early and interim periods of pupal development. We suggest that, rather than participating directly in the process of actin polymerization, Fhos plays a key organizational role during this period, which leads to the initial structuring of thin-filament arrays, using microfilaments generated by other formins and actin regulators. FHOD-family formins are considered to be poor microfilament nucleators (Schonichen et al., 2013; Taniguchi et al., 2009), and are thought to act via alternative modes- such as microfilament bundling (Kutscheidt et al., 2014; Schonichen et al., 2013)- to provide shape and structure to microfilament arrays. Our analysis is consistent with this notion, as we have demonstrated that FhosI966A, a Fhos variant presumably lacking barbed end associated activities, supports formation of small but properly organized sarcomeres, similar to the sarcomeres normally formed during the early and interim stages of pupal development. We suggest therefore, that Fhos plays a critical early role, coupling organization of pre-existing filaments into ordered arrays, with a secondary but important capacity to coordinate array size and structure through 'patchy' monomer incorporation.

3. Once the rudimentary sarcomere 'core' is assembled and defined Z-discs with fixed spacing are established, further extension and addition of actin filaments is dictated by the initial organization of the sarcomere. It is at this stage that Fhos assumes a striated localization pattern corresponding to thin-filament array ends, and where localization to the barbed end region becomes critical for Fhos function. We identified three different modes of actin incorporation which contribute to the nascent sarcomere maturation during the later stages of pupal development:

1. Elongation. Thin-filament arrays continue to extend laterally. Growth occurs from the pointed-ends and is mediated primarily by Sals. On the other hand, no evidence for extension at the barbed ends is evident from actin-GFP incorporation. We suggest that the reduced lateral size of arrays observed following late-stage Fhos knockdown may arise from the compromised protection of the barbed ends immediately adjacent to the Z-disc.

2. Radial 'thickening'. The sarcomeres grow radially due to the circumferential addition of actin filaments at their periphery. The added filaments are predominantly synthesized at this later stage, since they are readily labeled by monomeric actin produced only at that time (Figure 1E,E’). Radial growth requires Fhos, and therefore constitutes a second major contribution of this formin to thin-filament array assembly. Fhos may contribute to radial thickening by bundling and recruiting complete filaments to the periphery of the array core. The reliance on localization to the vicinity of the Z-discs and the arrest in growth of FhosI966A sarcomeres prior to radial thickening strongly imply that this activity depends on association of Fhos with the barbed ends of the thin-filament arrays.

3. Barbed-end turnover. An unexpected finding was the identification of rapid actin turnover that is highly restricted to the barbed ends of the thin-filament arrays. The barbed-end turnover persists throughout the late phase of pupariation, and does not contribute to filament elongation (Figure 1G–J). Interestingly, barbed-end turnover could be detected with the GFP-tagged form of the ubiquitous actin isoform actin5C, but not with the IFM-specific isoform GFP-actin88F. This may reflect a structural constraint that underlies the use of distinct actin isoforms for different aspects of array growth and maintenance.

The contribution of barbed-end turnover to thin-filament array organization is not readily apparent. Turnover may be part of a mechanism that maintains the filament integrity by a continuous process of elongation and disassembly within the dynamic environment of the maturing Z-disc. Turnover does not require Fhos, and its molecular basis is currently unknown. It will be interesting to examine if a similar process is operating in adult muscles, to maintain their integrity in the face of extensive contraction activity during flight (Perkins and Tanentzapf, 2014).

In conclusion, our analysis of IFM sarcomeres implies that the assembly of the highly structured thin-filament array is an elaborate, stepwise process involving diverse aspects and machineries of microfilament nucleation, growth and organization. The formin protein Fhos plays a central role, contributing to array assembly at several stages of the process. Fhos acts initially to mediate the assembly of thin-filament arrays within discrete sarcomeric units. Fhos localization to the Z-disc region, an apparently conserved feature among FHOD-family formins (Iskratsch et al., 2010; Mi-Mi et al., 2012; Rosado et al., 2014), becomes essential for function during the later stages of IFM development, where Fhos plays an essential role in sarcomere radial growth. Interestingly, it has been suggested that localization of FHOD3 to the Z-lines of murine cardiomyocytes is a regulated process, relying on phosphorylation of a short domain encoded by an alternatively-spliced exon (Iskratsch and Ehler, 2011; Iskratsch et al., 2010), echoing the importance of Fhos Z-disc localization described here.

While this study has elucidated specific roles for a FHOD-family formin in a model system of skeletal muscle sarcomerogenesis, key issues remain open. The precise molecular nature of the microfilament-associated activities of Fhos, the mechanistic significance of its spatial localization patterns, regulation of its sarcomeric activities and their coordination with other functional elements of the actin-based cytoskeleton, all await further investigation.

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[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly" for consideration by eLife. Your article has been reviewed by three peer reviewers (with expertise in Drosophila cell biology, cytoskeleton, and muscle cell biology), and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Velia Fowler (Reviewer #2).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

In discussions, the referees agreed that the description of the stages involved in assembly of thin filaments was very thorough and well done. The referees agreed that the quality of data was very high as well.

However, several questions were raised (please see referee comments verbatim), in particular on the extent of mechanistic advance and the generality of the conclusions, considering previous work on other formins in muscle and the sals.

Reviewer #1:

The organization of actin-based filaments is central to function of sarcomere, the basic functional units of muscle fibres. In Drosophila, indirect flight musculature (IFM) is a well-established skeletal muscle model system for studying assembly and maturation of thin-filament arrays. This study (Arkadi Shwartz et al.) described the role of Fhos, the single Drosophila homolog of the FHOD sub-family of formins, as a primary mediator of indirect flight musculature (IFM) thin filament organization. The authors also identified several phases in the dynamic construction of thin-filament arrays: intensive microfilament synthesis -> assembly of nascent arrays -> elongation primarily from filament pointed-ends -> radial growth of the arrays via recruitment of peripheral filaments -> continuous barbed-end turnover. In addition, the authors showed that the WH2-domain protein Sals that is known to contribute to pointed-end filament elongation, was specifically responsible for pointed-end elongation.

Unfortunately, this manuscript lacks conceptual novelty and is not suitable for general readers of ELife. Other formin proteins such as Diaphanous has already been studied in skeletal muscle model and this manuscript on Fhos, another formin, does not provide significant mechanistic insight into actin-based thin filaments formation in sarcomere. Given that Sals was previously known to contribute to pointed-send filament elongation, the data on Sals in this manuscript also lacks novelty.

Reviewer #2:

This is an interesting study of the role of Drosophila Fhod subfamily formins (Fhos) in thin filament assembly into myofibrils in the Drosophila indirect flight muscle (IFM). The IFM is a great model for sarcomere and thin filament assembly, and the authors take advantage of the fly genetics as well as the ability to express 'pulses' of GFP-actin during IFM myofibril assembly to evaluate the role of the Fhos proteins in actin incorporation/assembly at the barbed vs pointed ends of the thin filaments, as well as assembly of new peripheral thin filaments during stages of myofibril growth. This is an important area in which relatively few careful studies combining genetic deletion or knockdown with tests of actin incorporation have been undertaken. The key findings are that the long isoform of Fhos (Fhos-PH) is required for actin assembly at barbed ends and peripheral thin filament addition during myofibril growth, and that the nucleation activity of Fhos is not essential for this function. In addition, data is presented suggesting that the SALS protein is important for pointed end actin addition and thin filament elongation. The data is of high quality and interesting and reinforce and extend substantially the earlier findings from Mardahl-Dumesnil and Fowler (2001) that thin filament elongation during myofibril assembly takes place at their pointed but not barbed ends, and from Bai et al. (2007) that SALS is important for thin filament elongation at pointed ends. There are a few areas that require clarification and extension to solidify the conclusions.

1) The image of the thin myofibrils in Figure 3D showing partial rescue of the Fhos MI01421 with the GFP-Fhos-PA (short Fhos) is described as having "only weak corrective influence on the disrupted microfilament organization and other sarcomeric defects in myofibrils (Figure 3C,D)". However, to my eye this image shows thin myofibrils but with clearly demarcated and short sarcomeres, quite similar to the image of the myofibrils for the point mutant Fhos I966A in Figure 6A-A', which is described as having "an organized structure of repeated sarcomeric units, with clear demarcation of the Z-discs (Figure 6A-A'), suggesting an arrest in sarcomere growth following proper initial assembly and organization." Thus, it appears to me that the rescue with the short GFP-Fhos-PA should be reinterpreted, and the text rewritten. See next point also.

2) The experiment in Figure 4 shows that the Fhos-i knockdown (which reduces long Fhos isoforms specifically) reduces GFP-actin incorporation at Z lines and at the myofibril periphery, but does not affect the GFP-actin incorporation at the M line. This is a very nice experiment. However, since the short Fhos-PA localizes to the M lines (Figure 3 H-H'), and the Fhos-PA does rescue the myofibril disruption phenotype to some extent (Figure 3D), this could mean that the short Fhos-PA functions at M lines to promote GFP-actin incorporation. However, this was not tested. Depending on the results, the model in Figure 7 may need to be modified.

3) The GFP-actin (act88F) incorporation in the sals RNAi experiment (Figure 5E-E') is not convincingly qualitatively different from the control in Figure 5D-D'. I can see both M and Z line incorporation, just that some M lines appear fainter than others, and overall the image appears to have reduced/fuzzier GFP-actin incorporation. (Note that the shorter sarcomeres due to reduction of sals are convincing (Figure 2—figure supplement 2) and repeat the previous study from Bai et al.) However, I wonder whether the sals is actually affecting pointed end actin elongation directly. Do the authors have stronger data for the GFP-actin incorporation locations? Can the relative M and Z line incorporation be quantified by line scans to compare the WT and the knockdown?

4) Along these same lines, the study of Mardahl-Dumesnil and Fowler implied that Tmod reduces actin pointed end incorporation and thus prevents elongation. However, these authors never tested a Tmod knockdown or deletion, or actin incorporation. Can the authors perform a knockdown of fly Tmod in the IFM to determine whether this affects GFP-actin incorporation at the pointed end? This would fill in the picture, and strengthen the unique role(s) of the Fhos isoforms.

5) In Figure 6, the overall thin/thick filament organization appears normal by TEM of cross sections in the small myofibrils that form in the Fhos I966A point mutant (Figure 6C). However, I noticed a few areas of discontinuity in the lattice (upper left of myofibril) in which there are missing or extra thin filament surrounding the thick filament. In Figure 6B, there is an impression that the M line is not perfectly straight and that H zone is not perfectly even, so that some thin filaments may be shorter and some longer. I wonder whether there may be some subtle thin filament length or organization defects. Can the authors comment on this?

6) In Figure 1, can higher magnification images of the GFP-actin88F for the 45 h incorporation be shown to help identify the location of the actin "patches"? It appears that they do not colocalize with the Z line marker, Zasp. Do they colocalize with the pointed end marker, Tmod? Similarly, in the FhosMI-GFP experiment in Figure 3 F-F', where are the FhosMI-GFP patches? The colocalization with α-actinin is not shown at high magnification. (The regular sarcomeres at later times make it easier to figure out what is going on).

Reviewer #3:

The study describes 3 stages of actin monomer incorporation during Drosophila indirect flight muscle differentiation. Initial bulk polymerisation is followed by ordering into units, which are then elongated and widened to match the uniform sarcomere size. The formin FHOD is implicated in organising and thickening the sarcomere, which requires its long isoforms with N-terminal Z-disk localisation domain. Sals is required for pointed end elongation (as had been shown previously). In both these areas actin88F is incorporated, while at the barbed end, a different actin isoform, actin5c is being built in in a FHOD independent manner.

FHOD seems not to require its barbed-end association (and any associated nucleation, elongation and capping function) until about 60h APF as a conserved mutation I966A can still form organised sarcomeres. Thus the authors argue that FHOD might use its bundling activity during the early stages. However, such a bundling activity is neither shown for FHOD in the present study, nor in the two cited papers.

It is slightly puzzling why FHOD needs to localise to the barbed end, but doesn't need to actually bind to the barbed end. Any mechanistic explanations provided are speculative as no data as to the activity of the mutant or mechanistic action of FHOD or Sals itself are contained in the paper. Thus interpretations rest on the assumption that these would function as other formins. Interestingly, FHOD3, the closest mammalian homologue of FHOD inhibits barbed end elongation, rather than accelerating it (Taniguchi 2009) – a fact that seems not to be discussed in this study.

While the N-terminal extension of the longer FHOD isoforms seem to be of functional importance to organise sarcomeres and the longer isoforms localise specifically to the Z-disk, a causal relationship cannot be drawn, i.e. it is not clear that the localisation itself is required or that the localisation is sufficient to enable the canonical elements of FHOD to fully function. This would need to be tested with more subtle mutations that affect localisation (rather than the removal of 120kD!) and/or using means to bypass the requirement for the N-terminal domain by a more direct recruitment of the shorter FHOD isoforms to Z-disks. In the absence of such mechanistic experiments, the observations are difficult to interpret and some of the statements would need to be altered or stated as being purely speculative.

To my understanding, no data show that radial size increase occurs through recruitment of peripheral filaments – instead incorporation of actin monomers suggest their new synthesis. Also, the barbed end-binding activity of FHOD seems to be required for this step, whether nucleating or inhibitory.

Overall a beautifully illustrated descriptive story identifying temporal and spatial patterns of actin incorporation and formin activity. However, the authors have over-interpreted their findings and make mechanistic claims that are not substantiated by data in the study (or elsewhere).

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "The Drosophila formin Fhos is a primary mediator of sarcomeric thin-filament array assembly" for consideration by eLife.

Your appeal has been reviewed by an additional peer reviewer, and the evaluation has been overseen by a Reviewing Editor and Vivek Malhotra as the Senior Editor. The reviewer has opted to remain anonymous.

While the additional referee sees the value of your description of actin polymerization and thin filament assembly in Drosophila IFM, they have raised a number of points, all of which seem very reasonable and geared towards tightening controls and improving imaging quality.

In summary, we would like you to revise the manuscript, taking into consideration the comments of all the four referees, and in particular on the substantive points raised by referees 2, 3, and 4. You already have the comments of reviewer# 1, 2 and 3, and below are the comments of reviewer #4

Reviewer #4:

Arkadi Shwartz et al. present a study on Drosophila indirect flight muscle actin assembly during the various stages of development and observe the incorporation of different actin isoforms (act88F, act5c) into specific locations of the sarcomere. The authors find that Fhos the single Drosophila homologue of the mammalian FHOD subfamily, which has a documented role in sarcomere assembly and/or maturation, is crucially required for IFM integrity. Knockdown and a previously described mutation lead to thin and randomly oriented myofibrils, with defects appearing after 50hs APF, suggesting an involvement at early stages of sarcomere formation. They further analyze the involvement of different isoforms as well as SALS in the regulation of actin assembly and find that the long isoform is targeted to the Z-disc, where it assembles and bundles the actin, while the short isoform is localized to the pointed ends and dispensable for myofibril formation. SALS, in contrast, as previously demonstrated (Bai et al., 2007) is needed to regulate the thin filament length at the pointed end. The article provides an unprecedented level of detail regarding the different stages of actin assembly during myofibrillogenesis and the involvement of FHOD proteins therein. There are however several points that need to be addressed before publication.

Major points:

1) The data presented regarding the unsuccessful rescue of the Fhos deletion phenotype by FHOS-PA is not entirely convincing. While there is an apparent lack of rescue in Figure 3—figure supplement 1A,A', the stronger overexpression of Fhos-PA results in clearly thicker and more organized sarcomeres in Figure 3D, suggesting that the short isoform can, at least at high expression levels take over the function and even localize to the Z-disc (Figure 3—figure supplement 1C,C'). The data from targeting the 5'coding exon provides further evidence for the involvement of the large isoforms (Figure 3 E,E'), however, the authors should perform rescue experiments with the PH isform as well, to show that it can fully restore the myofibril integrity.

2) Further, regarding the isoforms and the conclusions that the authors draw from their data:

a) The authors suggest that PA and PH are the main isoforms, but no data (qPCR or similar) is presented, or studies cited, to this regards.

b) Disregarding the expression levels, a rescue with the π isoform and localization of the π isoform should be tested as well to exclude major involvement of this isoform.

3) The authors state that the short isoform is inactive, due to its localization to the vicinity of the M-band (3H,H'). This is however not supported by the presented data since the authors find 1) addition of actin to M-Bands, and thickening of myofibrils at late stages of development (Figure 1E); 2) the authors argue, referencing the data from Schoenichen et al. for FHOD1, that Fhos has an important actin bundling activity, which requires filament side binding, however not necessary barbed end binding. 3) stage specific localization changes have been reported for FHOD3 as well, with early localization to the vicinity of the M-Band (Iskratsch et al., 2013). 4) the data from Figure 4D-G demonstrates a patchy incorporation of actin at 60hrs, not only at the Z-disc, which is lost after knockdown of Fhos. This demonstrates that localization to the Z-disc is not necessarily the single determinant of actin assembly activity actin filament turnover could be contributing to differing localization, especially since it was shown in living zebrafish that YFP-actin is incorporated all along the I-Band(Sanger et al., 2009). The authors could test this by performing FRAP experiments to investigate Actin turnover at the Z-disc, around the M-Band and the periphery of the sarcomeres in presence or absence of the various isoforms.

4) Several of the images comparing the control and Fhos deletion/knock downs are difficult to compare due to varying quality, with fuzzier staining also present in the other channels (especially F-actin and zasp in Figure 4 H vs I and J vs K, and F-actin vs Obscurin Figure 5 D vs E). The authors should provide better comparable images (or if not possible, discuss the reason for this difference). Also the authors should provide quantifications of the intensities (e.g. of the M and Z-band intensities normalized to the background).

5) The GFP-act88F localization around the M-bands is narrower in the sals-i, but still present. The authors should provide quantifications for the width (i.e. does this account for the difference in thin filament length) and test or at least discuss what other proteins could be responsible for the ongoing pointed end assembly in absence of sals.

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

The organization of actin-based filaments is central to function of sarcomere, the basic functional units of muscle fibres. In Drosophila, indirect flight musculature (IFM) is a well-established skeletal muscle model system for studying assembly and maturation of thin-filament arrays. This study (Arkadi Shwartz et al.) described the role of Fhos, the single Drosophila homolog of the FHOD sub-family of formins, as a primary mediator of indirect flight musculature (IFM) thin filament organization. The authors also identified several phases in the dynamic construction of thin-filament arrays: intensive microfilament synthesis -> assembly of nascent arrays -> elongation primarily from filament pointed-ends -> radial growth of the arrays via recruitment of peripheral filaments -> continuous barbed-end turnover. In addition, the authors showed that the WH2-domain protein Sals that is known to contribute to pointed-end filament elongation, was specifically responsible for pointed-end elongation.

Unfortunately, this manuscript lacks conceptual novelty and is not suitable for general readers of ELife. Other formin proteins such as Diaphanous has already been studied in skeletal muscle model and this manuscript on Fhos, another formin, does not provide significant mechanistic insight into actin-based thin filaments formation in sarcomere. Given that Sals was previously known to contribute to pointed-send filament elongation, the data on Sals in this manuscript also lacks novelty.

We take issue with the reviewer’s assessment of the novelty of our findings vis-a-vis published studies on Formin function during skeletal muscle sarcomerogenesis. Our study couples a detailed analysis of the dynamics of thin-filament array assembly, at high spatial and temporal resolution, with assignment of distinct functional roles to a single Formin sub-family (FHOD/Fhos). We are unaware of similar comprehensive studies, or of such clear demonstrations of sarcomeric Formin function, and believe that we have provided considerable novel insight, using state-of-the-art tools, to a fundamental and somewhat neglected aspect of skeletal muscle biology. Our analysis of Sals function complements the assignment of functional roles to Fhos, and generalizes the significance of this element to sarcomere formation.

Reviewer #2:

This is an interesting study of the role of Drosophila Fhod subfamily formins (Fhos) in thin filament assembly into myofibrils in the Drosophila indirect flight muscle (IFM). The IFM is a great model for sarcomere and thin filament assembly, and the authors take advantage of the fly genetics as well as the ability to express 'pulses' of GFP-actin during IFM myofibril assembly to evaluate the role of the Fhos proteins in actin incorporation/assembly at the barbed vs pointed ends of the thin filaments, as well as assembly of new peripheral thin filaments during stages of myofibril growth. This is an important area in which relatively few careful studies combining genetic deletion or knockdown with tests of actin incorporation have been undertaken. The key findings are that the long isoform of Fhos (Fhos-PH) is required for actin assembly at barbed ends and peripheral thin filament addition during myofibril growth, and that the nucleation activity of Fhos is not essential for this function. In addition, data is presented suggesting that the SALS protein is important for pointed end actin addition and thin filament elongation. The data is of high quality and interesting and reinforce and extend substantially the earlier findings from Mardahl-Dumesnil and Fowler (2001) that thin filament elongation during myofibril assembly takes place at their pointed but not barbed ends, and from Bai et al. (2007) that SALS is important for thin filament elongation at pointed ends. There are a few areas that require clarification and extension to solidify the conclusions.

1) The image of the thin myofibrils in Figure 3D showing partial rescue of the Fhos MI01421 with the GFP-Fhos-PA (short Fhos) is described as having "only weak corrective influence on the disrupted microfilament organization and other sarcomeric defects in myofibrils (Figure 3C,D)". However, to my eye this image shows thin myofibrils but with clearly demarcated and short sarcomeres, quite similar to the image of the myofibrils for the point mutant Fhos I966A in Figure 6A-A', which is described as having "an organized structure of repeated sarcomeric units, with clear demarcation of the Z-discs (Figure 6A-A'), suggesting an arrest in sarcomere growth following proper initial assembly and organization." Thus, it appears to me that the rescue with the short GFP-Fhos-PA should be reinterpreted, and the text rewritten. See next point also.

These points are well taken, and we have now added new observations and have substantially revised our presentation of rescue and localization data, and our discussion of their significance. A key new experiment is specific disruption of the short Fhos isoforms (via CRISPR-based indels- Figure 3 and Figure 3—figure supplement 1). Using this approach we can now state with confidence that the short isoforms are dispensable and the long isoforms are sufficient for full sarcomeric function. The inability of the short forms to provide significant function when expressed at physiological levels is demonstrated both by the severe phenotypes resulting from specifically disrupting the long isoforms, and by the lack of rescue of null alleles by UAS-Fhos-PA, when driven by armadillo-GAL4. We now show this result in figure panels 3D-D’, as was our original intention. We fully agree with the reviewer that over-expression of the short form results in partial rescue- as we show (Figure 3—figure supplement 1B-B”), such rescue is likely the result of “ectopic” localization of the short form to the Z-disc, and, in fact, corresponds to our view that Z-disc localization is critical for Fhos function.

2) The experiment in Figure 4 shows that the Fhos-i knockdown (which reduces long Fhos isoforms specifically) reduces GFP-actin incorporation at Z lines and at the myofibril periphery, but does not affect the GFP-actin incorporation at the M line. This is a very nice experiment. However, since the short Fhos-PA localizes to the M lines (Figure 3 H-H'), and the Fhos-PA does rescue the myofibril disruption phenotype to some extent (Figure 3D), this could mean that the short Fhos-PA functions at M lines to promote GFP-actin incorporation. However, this was not tested. Depending on the results, the model in Figure 7 may need to be modified.

The Fhos-i knockdown in this figure was based on an siRNA construct targeting ALL isoforms. In any case, we now find (upon specific elimination of the short forms) that the fully functional (endogenous) long isoforms localize to both the Z-disc and M-line regions, while Fhos-PH, an isoform that localizes strictly to the Z-disc and contains only part of the large N-terminal region, provides only partial rescue of null alleles. All this leads us to conclude that Fhos may well function at sites other than the Z-disc (where it is essential), including the M-line. While the nature of these non-Z disc roles is currently unknown, we have modified the text to reflect this notion.

3) The GFP-actin (act88F) incorporation in the sals RNAi experiment (Figure 5E-E') is not convincingly qualitatively different from the control in Figure 5D-D'. I can see both M and Z line incorporation, just that some M lines appear fainter than others, and overall the image appears to have reduced/fuzzier GFP-actin incorporation. (Note that the shorter sarcomeres due to reduction of sals are convincing (Figure 2—figure supplement 2) and repeat the previous study from Bai et al.) However, I wonder whether the sals is actually affecting pointed end actin elongation directly. Do the authors have stronger data for the GFP-actin incorporation locations? Can the relative M and Z line incorporation be quantified by line scans to compare the WT and the knockdown?

We now use fluorescence intensity measurements to provide quantification of both the sarcomere shortening (Figure 5F) and reduced M/Z incorporation ratio (Figure 5G) phenotypes associated with sals knockdown.

4) Along these same lines, the study of Mardahl-Dumesnil and Fowler implied that Tmod reduces actin pointed end incorporation and thus prevents elongation. However, these authors never tested a Tmod knockdown or deletion, or actin incorporation. Can the authors perform a knockdown of fly Tmod in the IFM to determine whether this affects GFP-actin incorporation at the pointed end? This would fill in the picture, and strengthen the unique role(s) of the Fhos isoforms.

Tmod is indeed a logical candidate for an element involved in pointed end elongation. As recommended by the reviewer, we assessed and analyzed the effects of knockdown in similar fashion to the study of sals. In general the tmod knockdown phenotypes are quite mild, and possibly indicate an early role for Tmod in thin-filament array assembly. The relevant observations are shown in Figure S5.

5) In Figure 6, the overall thin/thick filament organization appears normal by TEM of cross sections in the small myofibrils that form in the Fhos I966A point mutant (Figure 6C). However, I noticed a few areas of discontinuity in the lattice (upper left of myofibril) in which there are missing or extra thin filament surrounding the thick filament. In Figure 6B, there is an impression that the M line is not perfectly straight and that H zone is not perfectly even, so that some thin filaments may be shorter and some longer. I wonder whether there may be some subtle thin filament length or organization defects. Can the authors comment on this?

We thank the reviewer for pointing out these imperfections in the Fhos I966A mutant arrays. However, given that these were obtained from 1 day old adult flies, following several days of IFM growth arrest, they may well be the result of secondary effects. We therefore prefer not to speculate about their significance, and to limit our interpretation to the general resemblance they bear to intermediate stage IFMs.

6) In Figure 1, can higher magnification images of the GFP-actin88F for the 45 h incorporation be shown to help identify the location of the actin "patches"? It appears that they do not colocalize with the Z line marker, Zasp. Do they colocalize with the pointed end marker, Tmod? Similarly, in the FhosMI-GFP experiment in Figure 3 F-F', where are the FhosMI-GFP patches? The colocalization with α-actinin is not shown at high magnification. (The regular sarcomeres at later times make it easier to figure out what is going on).

Following these comments, we have extended our analysis of the actin “patches”, by adding magnified panels and fluorescence intensity profiles for 45 hr IFMs stained for GFP-actin, phalloidin and the Z-disc and M-like markers Zasp and Obscurin (Figure 1—figure supplement 1B-E). Further quantification clearly demonstrates that the patches tend to localize to the ends of the arrays (Figure 1—figure supplement 1F).

Reviewer #3:

The study describes 3 stages of actin monomer incorporation during Drosophila indirect flight muscle differentiation. Initial bulk polymerisation is followed by ordering into units, which are then elongated and widened to match the uniform sarcomere size. The formin FHOD is implicated in organising and thickening the sarcomere, which requires its long isoforms with N-terminal Z-disk localisation domain. Sals is required for pointed end elongation (as had been shown previously). In both these areas actin88F is incorporated, while at the barbed end, a different actin isoform, actin5c is being built in in a FHOD independent manner.

FHOD seems not to require its barbed-end association (and any associated nucleation, elongation and capping function) until about 60h APF as a conserved mutation I966A can still form organised sarcomeres. Thus the authors argue that FHOD might use its bundling activity during the early stages. However, such a bundling activity is neither shown for FHOD in the present study, nor in the two cited papers.

We feel that our study covers considerable ground in breaking down the process of thin-filament array assembly into a discrete series of continuous events, some of which overlap temporally, and in assigning functional roles to several key elements that mediate these processes. Admittedly, we do not provide biochemical data that tests the actin-associated activities of Fhos (not for lack of trying- Fhos expression at useful levels has proven to be very difficult). However, we feel compelled to use the ample descriptive and genetic data provided to speculate on molecular mechanisms, bearing in mind established molecular capacities of the proteins involved. That said, we respect the reviewer’s expert assessment that some of the statements come across as over-interpretation of the data, and have tried to reorganize the text and “tone down” the language (in the main text and the relevant mention in the abstract) accordingly.

With regard to FHOD formins acting as microfilament bundling proteins- here we feel fully justified in building upon this notion, which was clearly demonstrated by Schonichen et al. (JCS 2013) using a number of assays, and is treated as an established capacity of FHOD proteins in various papers including the study from Kutscheidt et al. (NCB 2014) which we cite. However, we agree that initial mention of this specific activity was granted excessive prominence, and has now been moved from the Results section to the Discussion. Further mention of a bundling capacity and its significance are now phrased in a less dogmatic fashion.

It is slightly puzzling why FHOD needs to localise to the barbed end, but doesn't need to actually bind to the barbed end. Any mechanistic explanations provided are speculative as no data as to the activity of the mutant or mechanistic action of FHOD or Sals itself are contained in the paper. Thus interpretations rest on the assumption that these would function as other formins. Interestingly, FHOD3, the closest mammalian homologue of FHOD inhibits barbed end elongation, rather than accelerating it (Taniguchi 2009) – a fact that seems not to be discussed in this study.

We fully agree that the critical nature of Fhos localization to the Z-disc region does not readily correspond with its presumed molecular capacities. As brought up in the Discussion, Z-disc region localization appears to be a general feature of FHOD proteins in striated muscle, and resolving this matter is a priority for future studies on sarcomeric FHOD/Fhos function. Clearly, determination of the microfilament-regulating properties of Fhos should be a central aspect of such investigations. Our data only allows us to speculate regarding possible mechanisms, but we feel compelled to do so. Again, we have revised the tone of our discussion to properly reflect its speculative nature. The Taniguchi et al. study is now cited as a reference for the “poor nucleation” capacity of FHOD-family proteins.

While the N-terminal extension of the longer FHOD isoforms seem to be of functional importance to organise sarcomeres and the longer isoforms localise specifically to the Z-disk, a causal relationship cannot be drawn, i.e. it is not clear that the localisation itself is required or that the localisation is sufficient to enable the canonical elements of FHOD to fully function. This would need to be tested with more subtle mutations that affect localisation (rather than the removal of 120kD!) and/or using means to bypass the requirement for the N-terminal domain by a more direct recruitment of the shorter FHOD isoforms to Z-disks. In the absence of such mechanistic experiments, the observations are difficult to interpret and some of the statements would need to be altered or stated as being purely speculative.

We think that we have provided a considerable body of data supporting the notion that Z-disk localization is critical nature:

a) The active long isoforms- which are absolutely necessary for sarcomeric Fhos fuction- localize to this site;

b) The short forms do not localize there under physiological conditions, and completely fail to provide function (although they are fully sufficient for Fhos fuction in all other tissues);

c) Partial rescue by the short form is provided by its ectopic localization to the Z-disc region, under circumstances of over-expression.

We believe that this set of results justifies our conclusions regarding the significance of Z-disc localization. We agree that identification and manipulation of a minimal localization sequence within the long isoform is warranted, and would serve as an important step in refining and understanding the nature of Fhos localization within the sarcomere, but this must be left to future studies.

To my understanding, no data show that radial size increase occurs through recruitment of peripheral filaments – instead incorporation of actin monomers suggest their new synthesis. Also, the barbed end-binding activity of FHOD seems to be required for this step, whether nucleating or inhibitory.

We are in agreement with the reviewer on this point- namely, that newly-synthesized microfilaments constitute a major portion (all?) of the filaments added to the periphery of the sarcomere “core”, and stated as such in the Discussion text. We agree and similarly state that barbed-end binding by Fhos is required for its involvement in radial growth of the array. The question we try to grapple with is why a FHOD-type formin, unlikely to be responsible for synthesis, is still essential for radial growth. Bundling is suggested as a possible mechanism.

Overall a beautifully illustrated descriptive story identifying temporal and spatial patterns of actin incorporation and formin activity. However, the authors have over-interpreted their findings and make mechanistic claims that are not substantiated by data in the study (or elsewhere).

We thank the reviewer both for the positive assessment of our experimental work and for pointing out the shortcomings of our interpretation, which we have tried to revise accordingly.

[Editors’ note: the author responses to the re-review follow.]

The additional referee has kindly provided a detailed critique within a few days. While this referee sees the value of your description of actin polymerization and thin filament assembly in Drosophila IFM, the referee has raised a number of points, all of which seem very reasonable and geared towards tightening controls and improving imaging quality.

Reviewer #4:

Major points:

1) The data presented regarding the unsuccessful rescue of the Fhos deletion phenotype by FHOS-PA is not entirely convincing. While there is an apparent lack of rescue in Figure 3 supplement 1A,A', the stronger overexpression of Fhos-PA results in clearly thicker and more organized sarcomeres in Figure 3D, suggesting that the short isoform can, at least at high expression levels take over the function and even localize to the Z-disc (Figure 3—figure supplement 1C,C'). The data from targeting the 5'coding exon provides further evidence for the involvement of the large isoforms (Figure 3 E,E'), however, the authors should perform rescue experiments with the PH isform as well, to show that it can fully restore the myofibril integrity.

These are all good points, some of which were raised by Reviewer #2 as well (please also see our answers there). With regard to the function of the short isoform- we agree, and now better present the data, which we believe to show that rescue by the short forms is possible, but requires localization to the Z-disc. As for the rescuing capacity of the long forms- the CRISPR-based mutation we have now generated, specifically disrupting the short forms, demonstrates that the long forms are both necessary (as shown before) and sufficient. Rescue by the PH form (Figure 3—figure supplement 1C-C’’’) is partial, but while this construct localizes strictly to the Z-disc region, rescue by the endogenous long isoforms is associated with localization to both the Z-disc and M-line regions (Figure 3—figure supplement 1D). We conclude and now state that while Z-disc localization is critical, M-line localization may be of functional importance as well.

2) Further, regarding the isoforms and the conclusions that the authors draw from their data:

a) The authors suggest that PA and PH are the main isoforms, but no data (qPCR or similar) is presented, or studies cited, to this regards.

The use of the term “main” is in error- it has been changed to “representative”, and we thank the reviewer for pointing it out. Form PA is identical in coding sequence to most other short isoforms, and was used by Bogdan and colleagues in their study of Fhos function, to which we refer throughout. PH was used as a proxy for the long forms, since a corresponding cDNA was available to generate reagents.

b) Disregarding the expression levels, a rescue with the π isoform and localization of the π isoform should be tested as well to exclude major involvement of this isoform.

Our new results now suggest, in fact, that the longer forms (PI and PJ) are the ones to provide full functionality, but unfortunately, we were not successful in attempts to generate matching constructs.

3) The authors state that the short isoform is inactive, due to its localization to the vicinity of the M-band (3H,H'). This is however not supported by the presented data since the authors find 1) addition of actin to M-Bands, and thickening of myofibrils at late stages of development (Figure 1E); 2) the authors argue, referencing the data from Schoenichen et al. for FHOD1, that Fhos has an important actin bundling activity, which requires filament side binding, however not necessary barbed end binding. 3) stage specific localization changes have been reported for FHOD3 as well, with early localization to the vicinity of the M-Band (Iskratsch et al., 2013). 4) the data from Figure 4D-G demonstrates a patchy incorporation of actin at 60hr, not only at the Z-disc, which is lost after knockdown of Fhos. This demonstrates that localization to the Z-disc is not necessarily the single determinant of actin assembly activity actin filament turnover could be contributing to differing localization, especially since it was shown in living zebrafish that YFP-actin is incorporated all along the I-Band(Sanger et al., 2009). The authors could test this by performing FRAP experiments to investigate Actin turnover at the Z-disc, around the M-Band and the periphery of the sarcomeres in presence or absence of the various isoforms.

As noted above, our genetic data now supports the notion that Z-disc/barbed-end associated function is not the entire story. While we do not have solid ideas regarding M-line function of Fhos (pointed-end elongation appears to be Fhos-independent), we stress this additional layer of functional complexity in the text.

4) Several of the images comparing the control and Fhos deletion/knock downs are difficult to compare due to varying quality, with fuzzier staining also present in the other channels (especially F-actin and zasp in Figure 4 H vs I and J vs K, and F-actin vs Obscurin Figure 5 D vs E). The authors should provide better comparable images (or if not possible, discuss the reason for this difference). Also the authors should provide quantifications of the intensities (e.g. of the M and Z-band intensities normalized to the background).

We have now improved data presentation according to the reviewer’s comments:

a) In Figure 4H’, I’ and J we quantified the distribution of actin intensity across the arrays, demonstrating enhanced wildtype incorporation at the periphery, which is gone following Fhos knockdown.

b) In Figure 4M we quantified the width of the incorporation band at the Z-disc, an analysis that demonstrates that this turnover is Fhos independent

In this context we wish to note that- as the reviewer observed- the GFP incorporation signal is generally “fuzzier” reviewer upon fhos knockdown, possibly due to accumulation of excess free GFP-tagged actin monomers in the array vicinity.

5) The GFP-act88F localization around the M-bands is narrower in the sals-i, but still present. The authors should provide quantifications for the width (i.e. does this account for the difference in thin filament length) and test or at least discuss what other proteins could be responsible for the ongoing pointed end assembly in absence of sals.

As requested, we have added quantifications in Figures 5 and Figure 5—figure supplement 1, and have examined the possible contribution of Tropomodulin to pointed end elongation (Figure 5—figure supplement 1). Please also see our answers to Reviewer #2 (points 3 and 4).

Author details

1. Arkadi Shwartz

   Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   AS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Nagaraju Dhanyasi

   Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   ND, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Eyal D Schejter

   Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   EDS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   eyal.schejter@weizmann.ac.il

   Competing interests

   The authors declare that no competing interests exist.

4. Ben-Zion Shilo

   Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

   Contribution

   B-ZS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   benny.shilo@weizmann.ac.il

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-4903-8889

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