HGF and Met mutants do not lead to hernias. While these regions are likely more susceptible to CDH, muscle loss is not sufficient to cause CDH.The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, congenital diaphragmatic hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors and effects on phrenic nerve have not been explicitly tested. Using conditional mutagenesis in mice and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue in the diaphragm, are the source of HGF critical for diaphragm muscularization. PPF-derived HGF is directly required for recruitment of MET+ muscle progenitors to the diaphragm and indirectly (via its effect on muscle development) required for phrenic nerve primary branching. In addition, HGF is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Localization of HGF at the diaphragm’s leading edges directs dorsal and ventral expansion of muscle and regulates its overall size and shape. Surprisingly, large muscleless regions in  The diaphragm is an essential skeletal muscle and a defining feature of mammals . ContracDefects in the development of the diaphragm cause congenital diaphragmatic hernias (CDH), a common (1 in 3000 births) and costly ($250 million per year in the US) birth defect . CDH comRecruitment of muscle progenitors and targeting of phrenic nerve axons to the nascent diaphragm are essential first steps for correct diaphragm development. The receptor tyrosine kinase signaling cascade initiated by the binding of the ligand hepatocyte growth factor (HGF) to its receptor MET is a promising candidate pathway for regulating these steps of diaphragm development as HGF/MET signaling has been implicated in multiple aspects of muscle and motor neuron development  10.5 when muscle progenitors are migrating from cervical somites to the nascent diaphragm and progenitors have already populated the forelimb  and in the limb bud mesoderm  of HGF/MET signaling in diaphragm development, we investigated the expression of forelimb . At thismesoderm , while Mlimb bud . At E11.limb bud . During dal PPFs , arrows,and Pax7 . These eMet  mice were stained for neurofilament  or partially muscularized (n = 7/12). While the phrenic nerves reach the muscleless diaphragm, they lack primary and secondary branches , tumor protein Trp53 (which encodes p53), and autophagy marker Map1l3ca in BMS777607 and vehicle-treated PPFs. Both Fas and Map1l3ca are significantly upregulated following treatment with BMS777607, while Trp53 is unaffected . Cells were significantly more circular with BMS777607 treatment, which is consistent with compromised survival and motility  and results in a loss of the dorsal-most and ventral-most diaphragm muscle.Our in vivo studies show that HGF/MET signaling is required for the development of the dorsal and ventral-most regions of the diaphragm muscle, and our in vitro studies find that MET is required for muscle progenitor survival and motility. Based on these data, we hypothesized that in vivo loss of dorsal and ventral muscle regions is due to fewer muscle progenitors and/or myoblasts at the dorsal and ventral leading edges of the diaphragm when it is expanding. To test this, wild-type embryos were treated with BMS777607 daily E7.5–E11.5, harvested at E12.5, and stained for myogenic cells with a cocktail of PAX7, MyoD, and Myosin antibodies as well as for EdU, cleaved Caspase-3, and neurofilament. We found that the PPFs  were more variable in size, but not significantly decreased in size from control diaphragms. We also found, consistent with our analysis of nhibitor ; Video 1nhibitor  and an inhibitor . To exclreatment . Thus, tThe diaphragm is an essential mammalian skeletal muscle, playing a critical role in respiration and serving as a barrier that separates the thorax from the abdomen. Not only is the diaphragm a functionally important muscle, but it serves as an excellent system to study muscle patterning and morphogenesis, since it is a flat muscle that largely develops in two dimensions. Development of the diaphragm, like other skeletal muscles, requires the integration of muscle, connective tissue, and nerve that arise from different embryonic sources. Our study establishes that the connective tissue fibroblasts are the source of a molecular signal, HGF, that directly controls the recruitment, survival, and expansion of MET+ muscle progenitors and indirectly, via muscle, regulates phrenic nerve branching .Met is required for diaphragm development  muscle progenitors is Hgf-dependent and occurs contemporaneously. Recruitment of shoulder muscle progenitors to the nascent diaphragm has been proposed as important for the evolutionary origin of the mammalian diaphragm  must be involved in the evolutionary recruitment of the phrenic nerves. Also, still unknown are the developmental and evolutionary mechanisms driving formation and morphogenetic expansion of the PPFs.Our study also provides insights into how the diaphragm might have evolved. The diaphragm muscle is unique to mammals and how it evolved in mammals is a major unanswered question . Evolutiiaphragm . Thus, oHgf in the PPFs or pharmacological inhibition of MET signaling do not herniate. We previously conducted a detailed analysis of mice in which the transcription factor Gata4 was deleted in the PPFs  and the ventral-most diaphragm  . Our anathe PPFs . In thes hernias  develop TgPrrx1Cre -embedded tissues were sectioned to 10 μm thickness and fixed for 5 min in 4% paraformaldehyde (PFA). Tissue sections were blocked for 60 min in 5% goat serum in phosphate-buffered saline (PBS), incubated overnight at 4°C in primary antibodies. Sections were washed in PBS, incubated with secondary fluorescent antibodies  for 2 hr at room temperature (RT), washed with PBS, stained for 5 min with Hoechst to label nuclei, post-fixed in 4% PFA, rinsed in water and mounted with Fluoromount-G (Southern Biotech). Primary antibodies are listed in Key resources table. Sections were imaged on an Olympus BX63.For immunofluorescence on PPF cell cultures, cells were fixed in 4% PFA for 20 min at RT, washed in PBS, blocked for 60 min in 5% goat serum with 0.1% Triton X-100 in PBS, and stained overnight for primary antibodies (see Key resources table). Cells were then washed in PBS, incubated for 2 hr in secondary antibodies, washed in PBS, incubated in Hoechst to label nuclei, washed in PBS, and rinsed in water and mounted in Fluoromount. EdU (Life Technologies) was applied to cells 1 hr prior to fixation and detected after secondary labeling based on the manufacturer’s instructions with Alexa647 picolyl azide. Stained cells were imaged with ImageXPress Pico automated cell imager (Molecular Devices).2O2:Dent’s fix) and stored in Dent’s fix (1:4 DMSO:methanol) for at least 5 days at 4°C. Embryos were washed in PBS, blocked for 1 hr in 5% goat serum and 20% DMSO, incubated in primary antibodies (see Key resources table) for 48 hr, washed in PBS, incubated in secondaries for 24–48 hr, washed in PBS, and cleared BA:BB  at RT. Embryos labeled with AP-conjugated anti-Myosin heavy chain were heat-inactivated at 65°C for 1 hr, incubated in primary antibody for 48 hr, and detected with 250 μg/ml NBT and 125 μg/ml BCIP (Sigma) in alkaline phosphatase buffer. For detection of HRP-conjugated secondary antibodies, embryos were incubated in 10 mg diaminobenzidine tetrahydrochloride in 50 ml PBS with 7 μl hydrogen peroxide for approximately 20 min.Whole-mount embryos were fixed for 24 hr in 4% PFA at 4°C, dissected, incubated for either 2 hr at RT or overnight at 4°C in Dent’s bleach , and stained for 16 hr at 37°C in X-gal staining solution .Whole-mount in situ hybridization was performed as previously described . For whoWhole-mount fluorescent images were taken on a Leica SP8 confocal microscope. Optical stacks of whole-mount images were rendered and structures highlighted using FluoRender . To highmTmG/mTmGRosa26 or nTnG/nTnGRosa26 females mated with Cre/+Pax3 males. Embryos and PPFs were dissected as previously described  in 70% PEG-300 in PBS was administered via oral gavage. Vehicle alone (1% DMSO in 70% PEG300 in PBS) was administered to control pregnant dams. For cell culture experiments, BMS777607 was used at 10 μM concentration in 0.001% DMSO, and vehicle controls were treated with 0.001% DMSO.Cre/+; Rosa26nTnG/+Pax3 nuclei, GFP and phase images were taken every 8 min for 14 hr. Tracking, cell velocity, and displacement of peripheral cells  were determined using TrackMate  software was then used to count GFP+ cells per time point to calculate growth of myogenic cells over time. To control for differences in initial number of myogenic cells per well, fold changes of cell growth were calculated by dividing each treatment by the initial cell number at time 0. For cell motility analysis of rackMate . For celCreERt2/+Rosa26Pham/+Pax7 mice   primary antibody 1:50 dilution was added to the samples for 20 min at RT. Samples were washed with PBS, centrifuged at 800 × g for 5 min at 4°C, aspirated supernatant, and resuspended in PBS for analysis on BD FACSCanto II HTS (BD Biosciences). Median PE frequency from FloJo was used for statistical analysis.Sorted GFP+ cells were washed with Ham’s F12 media, centrifuged at 800 × Quick-RNA Microprep Kit  was used to extract total RNA according to the manufacturer’s protocol. Applied Biosystems High-Capacity RNA-to-cDNA kit (Thermo Fisher) was used to synthesize cDNA from purified RNA according to the manufacturer’s protocol. qRT-PCR was used to analyze expression of Met, Pax7, Hgf, MyoD1, and Gata4 using pre-validated primer sets . 10 μl reaction volumes were prepared using TaqMan Fast Advanced Master Mix (Thermo Fisher). The following conditions were used for amplification: 20 s at 95°C followed by 40 cycles at 95°C for 1 s, and 60°C for 20 s. Gene expression levels were normalized against 18S ribosomal RNA for each sample and fold changes calculated using 2-Δ/ΔCt method  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Carmen Birchmeier-Kohler as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Thomas Braun (Reviewer #2).Thank you for submitting your article "Fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.SummarySefton et al., analyze 'Fibroblast-derived HGF integrates muscle and nerve development during morphogenesis of the mammalian diaphragm'. The role of Met in the development of the muscle has been previously investigated by many papers, and roles in delamination of progenitor cells form the somites, in migration, survival, proliferation and differentiation have been described. The novel finding of the paper is a in depth analysis of the development of the diaphragm muscle, and the role of Met and HGF in the process. The authors provide evidence for the expression of HGF in pleuroperitoneal folds and for its requirement for muscle progenitor expansion and maintenance during diaphragm muscle formation. The reviewers found the manuscript clearly written and the experimental approach overall of high standard. However, there were several deficiencies noted that need to be addressed. The data supporting that HGF/Met function during development of the phrenic nerve was found to be less well supported and requiring additional experimental data to be included in the manuscript.Essential revision1) A major concern is the limited data on the role of Met in the development of the phrenic nerve. While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons has been highly debated. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development. Despite the weakness of the data on the role of Met in phrenic development, the role of HGF/Met in is strongly emphasized in abstract /intro/discussion. Data relying on motor neurons specific ablation of met need to be included in order to strengthen this point. Additional suggestions how to improve the analysis of phrenic development in Met-/+ animals are provided in the individual reviews appended below.2) The authors should exclude a connective tissue phenotype in conditional HGF/Met mutants. Why is Prx1Cre used instead of PDGFRaCre to trace PPFs? In addition, the reason for the differences in phenotypes regarding dorsal/ventral diaphragm muscles after either HGF inactivation or Met-inhibitor treatment should be explained, or at least discussed. Is this due to timing of the mutation/inhibition, or to efficacy of ablation/inhibition?3) The authors propose a role of Met in myogenic commitment, based on the co-culture experiments. Myogenic commitment implies a major role in myogenic lineage progression or differentiation. The evidence for that claim is weak and the reduced differentiation might be the consequence of increased cell death, reduced proliferation or other causes that change myoblast density. It is mandatory to address this issue appropriately, by analyzing both, fibroblast and myoblast cell numbers, proliferation, apoptosis, myogenic differentiation (i.e. ratio of MyoD/MyoG-positive cells).4) A potentially interesting conclusion form the manuscript is hat hernia does only develop when both, connective tissue and muscle of the diaphragm are affected. Although this explanation is intriguing, the authors have to make sure that body wall muscles are fully developed, to exclude that the abdominal pressure is not reduced in mice conditional Met/HGF mutations. The images in Figures 1 and 2 seem to indicate normal development of body wall muscles but a dedicated statement in this respect would be helpful.5) Treatment of WT embryos from E7.5 will impact on myogenic progenitor delamination, and this is also expected for Pax3CreKIMetnull/fl mutation. The authors have to dissociate the Met-related somite delamination, a migration of the progenitors to the diaphragm and the potential additional role of Met signaling in the colonization and expansion of myogenic progenitor cells in the anlage of the diaphragm.6) Validation for the drug BMS777607 effectiveness in inhibiting MET downstream pathway should be performed and shown.7) Comments referring to missing controls, scale bars, statistics need to be addressed.Additional points: The authors should address wherever possible the other comments raised by the three reviewers (see below).Reviewer #1 (Recommendations for the authors):It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.The data show that fibroblasts provide HGF signals received by Met in muscle progenitor cells that is essential for diaphragm development. The PdgfraCreERT2 line was used to demonstrate that HGF produced by fibroblasts but not by muscle progenitors is essential for diaphragm development. Moreover, development of dorsal and ventral regions of diaphragm muscle requires continuous MET signaling. Thus, HGF is not only required for the delamination of progenitors, but also for proliferation and survival of those muscle progenitors that reached the anlage of the diaphragm.My major concern is the limited data on the HGF-dependent development of the phrenic nerve (defasciculation). While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons was highly debated due to the fact that some changes observed in Met or HGF mutant mice in vivo are also present in other mutants that lack the muscle groups derived from migrating muscle progenitors. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development, i.e. a non-cell-autonomous function of Met during the recruitment of motor neurons to PEA3-positive motor pools .Sefton et al., provide an analysis of a single time point, one histological picture  that indicate that in Met+/- animals defasciculation of the phrenic nerve does not occur correctly. This is accompanied by a quantification that barely reaches significance . Data shown in Figure 7 using Met inhibitors show a major change in phrenic nerve branching, which is presumably due to the major change in diaphragm development, as conceded by the authors.Despite this weakness on the experimental side, the role of HGF/Met in phrenic nerve development is strongly emphasized in abstract /intro/discussion . The data need to be strengthened in order to conclude that HGF coordinates both, diaphragm muscle and phrenic development. I expect that the defasciculation of the phrenic nerve is highly dependent on the developmental stage. The authors should provide data that show different stages of phrenic nerve development, i.e. the time course of the of defasciculation in wildtype animals, explain how the staging was done, and compare different stages in the Met mutants and analyze whether the defasciculation is resolved at later stages. Met mutations also affect placental development, resulting in developmental delays that in turn might lead to an apparent small change in the time course of defasciculation. The authors should exclude that indirect effects cause the small change in phrenic nerve morphology, for instance by examining conditional Met mutations that are restricted to motor neurons. Good Cre lines that target motor neurons are available.Reviewer #2 (Recommendations for the authors):Since the authors observed a correlation between loss of should muscle and loss of diaphragm muscularization, which is related to the timing of migration of shoulder muscle and diaphragm muscle progenitors, they claim a "closer relationship" between should muscles and diaphragm, which was further extended in the discussion. I was not convinced by this conclusion. Is there a "closer relationship" between the muscles just because the progenitor cells migrate roughly at the same time? They authors may modify or delete this statement, although I agree that a broader expression of HGF may facilitate enhanced recruitment of muscle progenitor cells, required for formation of the diaphragm.Based on co-cultures of PPFs and myoblasts the authors describe a function of MET in myogenic commitment. In my opinion the evidence for such a function is weak. The authors observed no change in proliferation but reduced motility and a higher rate of apoptosis after pharmacological inhibition of MET. Increased apoptosis and reduced aggregation of myoblasts at distinct locations may easily interfere with myogenic differentiation. A general function of MET in myogenic commitment does not seem very likely, since myoblasts that do not undergo prior long-range migration differentiate normally in the absence of Met.Pharmacological inhibition of MET increases the rate of apoptosis in numerous cell types, which has been studied extensively in cancer. It seems appropriate to explore the mechanism of increased apoptosis in myoblasts following MET inhibition more closely. Previous reports suggest increased expression of p53, increased sensitivity to Fas-mediated apoptosis or increased autophagy, among others, as potential causes for increased apoptosis after MET inhibition. Which pathway is relevant in myoblasts?Obviously, it would be great to learn more about the mechanisms that control Hgf expression in fibroblasts within and derived from PPTs. Such knowledge may also help to better understand the reasons leading to Congenital Diaphragmatic Hernias (CDH). Unfortunately, the authors did not to go any further in this direction.The authors observed that branching of the phrenic nerve was reduced in heterozygous Met mutants with normal diaphragm musculature, suggesting a direct role of Met in phrenic nerve branching. Timed inactivation of Met specifically in motoneurons would greatly increase the impact of this finding and allow a more specific analysis of the role of MET in phrenic nerve development and branching.Surprisingly, the authors did not observe CDH in mutant mice with muscle-less diaphragms, from which they conclude that additional defects in the connective tissue are necessary to allow hernia formation. Although this explanation is intriguing, the authors have to make sure that abdominal pressure is not reduced in mice without muscularized diaphragms, e.g. demonstrate that body wall muscles are fully functional. The images in Figures 1 and 2 seem to indicate normal development of body wall muscles but a dedicated statement in this respect would be helpful.Scale bars are missing in some panels.According to the methods part, Student's t-test was used for the statistical analysis shown in Figure 3. A pairwise comparison of WT, heterozygous and homozygous Met mutants is not appropriate when all three genotypes are compared with each other. An ANOVA test should be used as in Figure 5F.Reviewer #3 (Recommendations for the authors):The present manuscript addresses questions on the role of HGF/MET signaling in diaphragm formation once myogenic progenitors have already migrated to the PPFs. In addition, it identifies the PPFs as the source of HGF. The study is interesting and developed with rigor. However, the role of HGF is not clearly dissociated from the presence/absence of the fibroblasts/connective tissue itself. Also, the authors do not link in the results, for example, how the muscleless diaphragms and HGF itself relate to the hernia phenotype mentioned in the abstract.Figure 11C) Co-staining for Hgf (PPFs) and Met (migrating progenitors) should be provided for a clear visualization of ligand-expressing cells and receptor-expressing cells.1F) Co-staining for Met and migrating progenitors (PAX3 or LBX1 for example) should be provided for a clear visualization of migrating progenitors versus general Met expression in other cell types.1M) The claim that fibroblast-derived HGF is required for diaphragm muscle development cannot be addressed with only this experimental analysis. What is the connective tissue phenotype in PDGFaCreER;HGFnull/fl embryos? Lack of connective tissue could affect muscle migration and development rather than HGF expression on its own. Are fibroblasts still present?1G, I, K, M) Control genotypes should be analyzed and added in the Figure or as Supplementary Data.1I) Pax3CreKIMetnull/fl originates a muscleless diaphragm but this could be associated with lack of delamination and migration from the somites rather than a specific MET requirement for diaphragm muscle formation once progenitors have colonized this area as suggested by the authors.Figure 3It is not clear whether HGF controls phrenic nerve formation independently of muscle. In the end of this section the authors mention that Met heterozygous embryos have normal muscles referring to Figure 1K (which is not a picture referring to a Met het embryo). The authors should confirm if in Met null embryos there is a direct effect on the nerve bifurcation and brunching or if the lack of muscle is leading to this observation. Is the phenotype dose dependent for muscle formation? This is not properly shown by the authors. Could the authors perform a conditional KO of Met in the nerves?This concern is further supported by the Figure 4 data where phrenic nerve only extends to regions with muscle.Figure 4The role of HGF versus the presence of fibroblasts/connective tissue remains vague. Is the lack of ventral muscles associated with lack of migration of progenitors within the forming diaphragm towards an HGF source? Or due to the lack of connective tissue scaffold itself? Why is Prx1Cre used instead of PDGFRaCre  to trace PPFs?4E) Please provide a control with a Cre allele to compare putative secondary effects for the presence of the Cre in the cKO embryos.Figure 5Validation for the drug BMS777607 effectiveness in inhibiting MET downstream pathway should be performed and shown.The phenotype with the inhibition of Met signaling  is distinct to the one observed in Figure 4 . How do the authors explain the dorsal phenotype when inhibiting Met (since migration should have not been affected at this time-point) and Hgf inhibition in Figure 4 is not leading to this phenotype?Figure 6What is the time point after treatment in B and C? What is the total nuclei number in the cultures (since these are co-cultures)?The authors should provide pictures together with nuclear staining for a global view of the cell density in the cultures. Also T0 time point pictures should be added for comparison, in particular in C.In these experiments the authors performed co-culture (fibroblasts + myogenic progenitors) derived from PPfs. However, fibroblast phenotype is not addressed. If there is less GFP+ cells is there more fibroblasts in the culture? Could this impact on the phenotype observed and linked by the authors to the Met inhibitor treatment? The only piece of evidence is Gata4 qPCR which is not sufficient to address fibroblast phenotype in the culture.Figure 7Treatment of WT embryos from E7.5 will impact on myogenic progenitor migration. The authors have to dissociate the Met-related somite migration phenotype and the potential additional role of Met signaling in the diaphragm muscle formation.HGF controls recruitment and expansion of muscle during morphogenesis of the mammalian diaphragm" for further consideration by eLife. Your revised article has been evaluated by Didier Stainier (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Fibroblast-derived The manuscript has been much improved but there are a few remaining issues that need to be addressed, as outlined below:Reviewer #1 (Recommendations for the authors):The majority of my concerns were appropriately addressed by the authors. There are a few additional points that need to be addressed, and most of these concern the text/wording.Unfortunately, the changes in the text have not been highlighted in red in the uploaded version of the manuscript, as it was mentioned in the rebuttal letter.1. While the quality of the figures is overall good, many of the panels in Figure 2 are very dark. It should be easy to modify this.2. While the authors show very convincingly that the effects on the phrenic nerve observed are caused indirectly by the loss of muscle, and not directly by the loss of HGF/Met signaling, this is not always made clear in the text. Furthermore, the mechanism that causes branching deficits should be clearly stated in the Abstract.Line 41-42...and indirectly required for phrenic nerve primary branching.Please mention the specific indirect mechanisms, i.e. via the effect on muscle development.Line 81…HGF is also critical for innervation.The reports cited do not distinguish between the effect of the nerve/muscle or report in vitro experiments. The text should take this into account.238: Thus, PPF-derived HGF is necessary for phrenic nerve defasciculation.Additional experiments shown in the next paragraph indicate that it is the absence of the muscle that causes defasciculation. Please rephrase.3) Met inhibition using BMS777607BMS777607 acts as an AXL, RON and Met tyrosine kinase inhibitor. This should be mentioned. Are effects of RON/AXL on skeletal muscle development described?4) Antibody specificity: Antibody specificity should be tested by analysis of the phrenic nerve on Met mutants.Reviewer #2 (Recommendations for the authors):Sefton et al., have submitted a revised version of a study, in which the role of fibroblast-derived HGF for recruitment and expansion of muscle during morphogenesis of the mammalian diaphragm was investigated. The authors have changed the title to cope with new findings, indicating that reduced primary branching of the phrenic nerve in Met-mutants is not due direct effects of HGF on the nerve but most likely caused by indirect effects resulting from reduced muscle formation in Met-mutants.The authors did an excellent job to deal with the reviewers' criticisms. In particular, by specifically deleting Met in motoneurons and by analysis of splotch mutants they demonstrate that Met does not intrinsically regulate phrenic nerve branching. Analysis of splotch mice, which show normal expression of HGF PPFs but display muscle-less diaphragms, revealed reduced branching of the phrenic nerve, similar to Met-mutants, clearly suggesting a critical role of muscle fibers in the diaphragm for phrenic nerve branching. Furthermore, Sefton et al., now demonstrate that fibroblasts expand and populate the diaphragm in the absence of muscle, which essentially excluded a connective tissue phenotype in conditional Hgf/Met mutants. They also clarified the statement about what I understood was meant to claim a role of Hgf/Met in myogenic commitment. Additional controls  were done as requested, providing additional interesting insights into a role of Met for ventral expansion of the diaphragm at relatively late developmental timepoints.The authors argue that "PPF-derived HGF, via muscle, controls phrenic nerve defasciculation". Well, this is formally correct but exaggerates the role PPF-derived for phrenic nerve defasciculation in my view. Probably, deletion of any gene that prevents diaphragm muscle formation will have similar effects on phrenic nerve defasciculation. I would prefer a more neutral statement: "Muscle formation, requiring PPF-derived HGF, controls phrenic nerve defasciculation", or something similar. The authors may consider modifying the statement.I do not have any further objections. Essential Revision1) A major concern is the limited data on the role of Met in the development of the phrenic nerve. While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons has been highly debated. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development. Despite the weakness of the data on the role of Met in phrenic development, the role of HGF/Met in is strongly emphasized in abstract /intro/discussion. Data relying on motor neurons specific ablation of met need to be included in order to strengthen this point. Additional suggestions how to improve the analysis of phrenic development in Met-/+ animals are provided in the individual reviews appended below.Δ/ ΔMet  and Δ/+Met  compared with +/+Met embryos. In the Δ/ ΔMet  embryos, no muscle is present in the diaphragm. Therefore, the greatly reduced branching in these embryos is likely a secondary effect of the requirement of Met in muscle progenitors for diaphragm muscularization. Of particular interest is the reduced branching in the Δ/+Met  embryos. Because the diaphragm is muscularized in these embryos, this suggested that Met may be required intrinsically in the phrenic nerve. One reviewer suggested that the reduced branching in the Δ/+Met  embryos could be due to a developmental delay in the whole embryo. However, we found that Δ/ ΔMet  and Δ/+Met  embryos are not overall delayed relative to +/+Met embryos . Also, to increase the robustness of these data, we added additional embryos to the analysis. We then extended our analysis of Δ/ ΔMet , Δ/+Met  and +/+Met embryos to E12.5  to see whether the branching phenotype persisted; we found that while the of Δ/ ΔMet  embryos continue to have very few branches, the number of branches in Δ/+Met  embryos recovers and matches that of +/+Met embryos.In response to comments from the reviewers, we have more thoroughly investigated the role of Met in the development of the phrenic nerve and include two new sets of genetic experiments. In our first submission, we found a decreased number of phrenic nerve branches at E11.5 in Cre/+Olig2to conditionally delete Met. This line was chosen for its early expression in motor neurons . We examined Cre/+;MetOlig2/floxΔ embryos compared to Cre/+; Metflox/+Olig2 embryos. We chose to include CreOlig2 in our controls because the CreOlig2 is a knock-in/knock-out and Olig2 has important roles in nerve development. However, deletion of Met did not affect the number of branches at E11.5  or E12.5 (data not shown). These data suggest that Met does not intrinsically regulate phrenic nerve branching. This suggests that PPF-derived HGF regulates phrenic nerve branching indirectly via muscle. To test if HGF is sufficient to promote early stages of nerve branching in the absence of muscle, we turned to SpD/SpDPax3 mutants in which a point mutation in Pax3 prevents migration of muscle progenitors into the diaphragm . In these embryos, the diaphragm is muscleless, but the PPFs still express HGF. In these diaphragms the number of branches at E11.5 is severely reduced. These data demonstrate that in the absence of muscle the presence of HGF in the PPF fibroblasts is not sufficient to support diaphragm branching.To explicitly test whether Met is required within the phrenic nerve, we used Δ/+Met  data demonstrate that Met controls phrenic nerve branching at E11.5 in a dose-dependent manner, but this effect is lost by E12.5. Although we see no obvious defects in muscle of Δ/+Met  diaphragms at later stages, the most parsimonious explanation of the reduced phrenic nerve branching at E11.5 is that this is due to fewer muscle progenitors at this time point.Altogether our data demonstrate that PPF-derived HGF, via its regulation of muscle, controls the primary branching of phrenic nerve. The We thank the reviewers for prompting us to look at the role of HGF/Met in the phrenic nerve more closely. Our revised conclusions are presented in the Results and Discussion. We show that PPF-derived HGF is critical for integrating both muscle and phrenic nerve development, but now demonstrate that HGF’s regulation of phrenic nerve branching is via muscle, which is well-known to express multiple trophic factors required by motor neurons.2) The authors should exclude a connective tissue phenotype in conditional HGF/Met mutants. Why is Prx1Cre used instead of PDGFRaCre to trace PPFs? In addition, the reason for the differences in phenotypes regarding dorsal/ventral diaphragm muscles after either HGF inactivation or Met-inhibitor treatment should be explained, or at least discussed. Is this due to timing of the mutation/inhibition, or to efficacy of ablation/inhibition?Prx1Cre and CreERPDGFRa both label connective tissue fibroblasts in the developing diaphragm. We have included additional panels to Figure 4—figure supplement 1 of CreER/+; HGFdelta/flox; RosamTmG/+PDGFRaembryos demonstrating the presence of GFP+ fibroblasts in the muscleless regions. Thus, fibroblasts do expand and appropriately populate the diaphragm even in the absence of muscle.HGF via Prx1Cre or CreERPDGFRa and after MET-inhibitor treatment is quite similar . Genetic HGF deletion or MET inhibition early leads to ventral muscleless regions and two bilateral dorsal muscleless regions . Genetic HGF deletion or MET inhibition later consistently leads to ventral muscleless regions and less commonly to dorsal muscleless regions . We have provided additional clarification in the text and in model in Figure 8.The phenotype after genetic deletion of 3) The authors propose a role of Met in myogenic commitment, based on the co-culture experiments. Myogenic commitment implies a major role in myogenic lineage progression or differentiation. The evidence for that claim is weak and the reduced differentiation might be the consequence of increased cell death, reduced proliferation or other causes that change myoblast density. It is mandatory to address this issue appropriately, by analyzing both, fibroblast and myoblast cell numbers, proliferation, apoptosis, myogenic differentiation (i.e. ratio of MyoD/MyoG-positive cells).We apologize for the misunderstanding here and have altered the text to indicate that we do not propose a role for Met in myogenic commitment, but rather that Met regulates the number of MyoD+ cells by promoting their survival. Using our co-culture methods, unfortunately we are unable to quantify fibroblast cell numbers, as they are too dense to distinguish using either our software or manual counting.4) A potentially interesting conclusion form the manuscript is hat hernia does only develop when both, connective tissue and muscle of the diaphragm are affected. Although this explanation is intriguing, the authors have to make sure that body wall muscles are fully developed, to exclude that the abdominal pressure is not reduced in mice conditional Met/HGF mutations. The images in Figures 1 and 2 seem to indicate normal development of body wall muscles but a dedicated statement in this respect would be helpful.We have added a statement in the Results section indicating the body wall muscles are normal.5) Treatment of WT embryos from E7.5 will impact on myogenic progenitor delamination, and this is also expected for Pax3CreKIMetnull/fl mutation. The authors have to dissociate the Met-related somite delamination, a migration of the progenitors to the diaphragm and the potential additional role of Met signaling in the colonization and expansion of myogenic progenitor cells in the anlage of the diaphragm.In Figure 5, we have delivered the Met inhibitor at various stages to determine when Met is required. Delivery at E7.5-E12.5 gives the most pronounced phenotype; muscleless regions in the dorsal and ventral regions of the diaphragm. To exclude that inhibition of Met is affecting the initial delamination of progenitors from the somites and migration to the PPFs, we also delivered the inhibitor at E11.5-E12.5 . Previously we showed that migration to the PPFs is completed by E11.5 . Thus the appearance of dorsal and ventral muscleless regions in these diaphragms demonstrates that Met has an additional later role.Our original analysis of the mechanistic effects of Met inhibition in vivo  were based on experiments administering BMS777607 E7.5-E12.5 because this dosage gave the most highly penetrant and consistent phenotype. To exclude possible effects due to early delamination and migration defects, we have repeated these experiments by administering inhibitor at E11.5-E12.5 and analyzing the ventral-most expansion of diaphragm muscle at E15.5 . We find that Pax7/MyoD/Myosin+ cells are substantially reduced in the ventral leading edge of the diaphragm following BMS777607 treatment. This demonstrates that Met is required at later timepoints for the ventral expansion of the diaphragm.6) Validation for the drug BMS777607 effectiveness in inhibiting MET downstream pathway should be performed and shown.Due to limited number of cells in our PPF cultures, quantitative validation of pathways is difficult in the embryonic diaphragm. As such, we have validated the effectiveness of BMS777607 in inhibiting phosphorylation of MET, which is essential for downstream pathway signaling, using adult skeletal muscle stem cells, where there are sufficient cells to perform phospho-specific flow cytometry . Moreover, the drug BMS777607 has been previously validated in downregulating pAkt, pERK, and pPLC by Western blot . BMS777607 from Selleck Chem has been cited by 47 publications.7) Comments referring to missing controls, scale bars, statistics need to be addressed.We have addressed comments about controls, scale bars, and statistics below and thank reviewers for their careful and thoughtful reviews.Reviewer #1 (Recommendations for the authors):It was previously shown that HGF and Met controls development of the diaphragm muscle. In particular, the signal induces delamination and migration of muscle progenitor cells that colonize the diaphragm. The present manuscript by Sefton and coworkers confirms and extends these observations using (i) conditional mouse lines in which the HGF gene was targeted by Cre/loxP recombination in the pleuroperitoneal folds (Prx1-cre) and at other sites PdgfraCreERT2, and of (ii) Met inhibitors. Overall, the technical quality of the data on diaphragm muscle development is excellent; the conceptual advance over previous work is not exceptional; the evidence for Met/HGF-dependent development of the phrenic nerve is marginal and needs to be strengthened.The data show that fibroblasts provide HGF signals received by Met in muscle progenitor cells that is essential for diaphragm development. The PdgfraCreERT2 line was used to demonstrate that HGF produced by fibroblasts but not by muscle progenitors is essential for diaphragm development. Moreover, development of dorsal and ventral regions of diaphragm muscle requires continuous MET signaling. Thus, HGF is not only required for the delamination of progenitors, but also for proliferation and survival of those muscle progenitors that reached the anlage of the diaphragm.My major concern is the limited data on the HGF-dependent development of the phrenic nerve (defasciculation). While it is well documented that HGF acts as a trophic factor for motor neurons in culture, its role in development of motor neurons was highly debated due to the fact that some changes observed in Met or HGF mutant mice in vivo are also present in other mutants that lack the muscle groups derived from migrating muscle progenitors. Moreover, careful genetic analyses previously demonstrated indirect mechanisms of Met during motor neuron development, i.e. a non-cell-autonomous function of Met during the recruitment of motor neurons to PEA3-positive motor pools .Sefton et al., provide an analysis of a single time point, one histological picture  that indicate that in Met+/- animals defasciculation of the phrenic nerve does not occur correctly. This is accompanied by a quantification that barely reaches significance . Data shown in Figure 7 using Met inhibitors show a major change in phrenic nerve branching, which is presumably due to the major change in diaphragm development, as conceded by the authors.Despite this weakness on the experimental side, the role of HGF/Met in phrenic nerve development is strongly emphasized in abstract /intro/discussion . The data need to be strengthened in order to conclude that HGF coordinates both, diaphragm muscle and phrenic development. I expect that the defasciculation of the phrenic nerve is highly dependent on the developmental stage. The authors should provide data that show different stages of phrenic nerve development, i.e. the time course of the of defasciculation in wildtype animals, explain how the staging was done, and compare different stages in the Met mutants and analyze whether the defasciculation is resolved at later stages. Met mutations also affect placental development, resulting in developmental delays that in turn might lead to an apparent small change in the time course of defasciculation. The authors should exclude that indirect effects cause the small change in phrenic nerve morphology, for instance by examining conditional Met mutations that are restricted to motor neurons. Good Cre lines that target motor neurons are available.Δ/ ΔMet  and Δ/+Met  compared with +/+Met embryos. In the Δ/ ΔMet  embryos, no muscle is present in the diaphragm. Therefore, the greatly reduced branching in these embryos is likely a secondary effect of the requirement of Met in muscle progenitors for diaphragm muscularization. Of particular interest is the reduced branching in the Δ/+Met  embryos. Because the diaphragm is muscularized in these embryos, this suggested that Met may be required intrinsically in the phrenic nerve. One reviewer suggested that the reduced branching in the Δ/+Met  embryos could be due to a developmental delay in the whole embryo. However, we found that Δ/ ΔMet  and Δ/+Met  embryos are not overall delayed relative to +/+Met embryos . Also, to increase the robustness of these data, we added additional embryos to the analysis. We then extended our analysis of Δ/ ΔMet , Δ/+Met  and +/+Met embryos to E12.5  to see whether the branching phenotype persisted; surprisingly we found that while the of Δ/ ΔMet  embryos continue to have very few branches, the number of branches in Δ/+Met  embryos recovers and matches that of +/+Met embryos.In response to comments from the reviewers, we have more thoroughly investigated the role of Met in the development of the phrenic nerve and include two new sets of genetic experiments. In our first submission, we found a decreased number of phrenic nerve branches at E11.5 in Cre/+Olig2to conditionally delete Met. This line was chosen for its early expression in motor neurons . We examined Cre/+;MetOlig2/floxΔ embryos compared to Cre/+; Metflox/+Olig2 embryos. We chose to include CreOlig2 in our controls because the CreOlig2 is a knock-in/knock-out and Olig2 has important roles in nerve development. However, deletion of Met did not affect the number of branches at E11.5  or E12.5 (data not shown). These data suggest that Met does not intrinsically regulate phrenic nerve branching. This suggests that PPF-derived HGF regulates phrenic nerve branching indirectly via muscle. To test if HGF is sufficient to promote early stages of nerve branching in the absence of muscle, we turned to SpD/SpDPax3 mutants in which a point mutation in Pax3 prevents migration of muscle progenitors into the diaphragm . In these embryos, the diaphragm is muscleless, but the PPFs still express HGF. In these diaphragms the number of branches at E11.5 is severely reduced. These data demonstrate that in the absence of muscle the presence of HGF in the PPF fibroblasts is not sufficient to support diaphragm branching.To explicitly test whether Met is required within the phrenic nerve, we used Δ/+Met  data demonstrate that Met controls phrenic nerve branching at E11.5 in a dose-dependent manner, but this effect is lost by E12.5. Although we see no obvious defects in muscle of Δ/+Met  diaphragms at later stages, the most parsimonious explanation of the reduced phrenic nerve branching at E11.5 is that this is due to fewer muscle progenitors at this time point.Altogether our data demonstrate that PPF-derived HGF, via its regulation of muscle, controls the primary branching of phrenic nerve. The We thank the reviewers for prompting us to look at the role of HGF/Met in the phrenic nerve more closely. Our revised conclusions are presented in the Results and Discussion. We show that PPF-derived HGF is critical for integrating both muscle and phrenic nerve development, but now demonstrate that HGF’s regulation of phrenic nerve branching is via muscle, which is well-known to express multiple trophic factors required by motor neurons.Reviewer #2 (Recommendations for the authors):Since the authors observed a correlation between loss of should muscle and loss of diaphragm muscularization, which is related to the timing of migration of shoulder muscle and diaphragm muscle progenitors, they claim a “closer relationship” between should muscles and diaphragm, which was further extended in the discussion. I was not convinced by this conclusion. Is there a “closer relationship” between the muscles just because the progenitor cells migrate roughly at the same time? They authors may modify or delete this statement, although I agree that a broader expression of HGF may facilitate enhanced recruitment of muscle progenitor cells, required for formation of the diaphragm.This statement has been modified (lines 206-211).HGF at this later developmental time point may facilitate recruitment of muscle cells necessary for development of diaphragm and shoulder muscles.”“However, based on their similar temporal sensitivity to HGF/MET signaling, the shoulder acromiodeltoid and spinodeltoid progenitors migrate at a similar time as the diaphragm progenitors. Thus continued expression of Based on co-cultures of PPFs and myoblasts the authors describe a function of MET in myogenic commitment. In my opinion the evidence for such a function is weak. The authors observed no change in proliferation but reduced motility and a higher rate of apoptosis after pharmacological inhibition of MET. Increased apoptosis and reduced aggregation of myoblasts at distinct locations may easily interfere with myogenic differentiation. A general function of MET in myogenic commitment does not seem very likely, since myoblasts that do not undergo prior long-range migration differentiate normally in the absence of Met.We apologize for the misunderstanding here and have altered the text to indicate that we do not propose a role for Met in myogenic commitment, but rather that Met regulates the number of MyoD+ cells by promoting their survival.Pharmacological inhibition of MET increases the rate of apoptosis in numerous cell types, which has been studied extensively in cancer. It seems appropriate to explore the mechanism of increased apoptosis in myoblasts following MET inhibition more closely. Previous reports suggest increased expression of p53, increased sensitivity to Fas-mediated apoptosis or increased autophagy, among others, as potential causes for increased apoptosis after MET inhibition. Which pathway is relevant in myoblasts?Fas, Trp53, and autophagy marker Map1l3ca. We found increased expression of Fas and Map1l3ca in PPFs following treatment with the Met inhibitor, while Trp53 did not change .We have performed qPCR on vehicle and BMS777607-treated PPFs for Obviously, it would be great to learn more about the mechanisms that control Hgf expression in fibroblasts within and derived from PPTs. Such knowledge may also help to better understand the reasons leading to Congenital Diaphragmatic Hernias (CDH). Unfortunately, the authors did not to go any further in this direction.Understanding the mechanisms regulating HGF expression in the PPFs is critical for understanding why in development and in evolution muscle is recruited to the nascent diaphragm. This will be explored in future studies.The authors observed that branching of the phrenic nerve was reduced in heterozygous Met mutants with normal diaphragm musculature, suggesting a direct role of Met in phrenic nerve branching. Timed inactivation of Met specifically in motoneurons would greatly increase the impact of this finding and allow a more specific analysis of the role of MET in phrenic nerve development and branching.SpD/SpDPax3 mice.Please see response to Essential Revision Point 1. We have now included a more detailed analysis of the role of Met in phrenic nerve branching, including conditional deletion in motor neurons and analysis of Surprisingly, the authors did not observe CDH in mutant mice with muscle-less diaphragms, from which they conclude that additional defects in the connective tissue are necessary to allow hernia formation. Although this explanation is intriguing, the authors have to make sure that abdominal pressure is not reduced in mice without muscularized diaphragms, e.g. demonstrate that body wall muscles are fully functional. The images in Figures 1 and 2 seem to indicate normal development of body wall muscles but a dedicated statement in this respect would be helpful.We have added a dedicated statement to the manuscript indicating that no defects in abdominal wall muscle development were observed.Scale bars are missing in some panels.Thank you for this observation and we have checked for scale bars in figure panels. Please note that some figures contain one scale bar for multiple panels.According to the methods part, Student’s t-test was used for the statistical analysis shown in Figure 3. A pairwise comparison of WT, heterozygous and homozygous Met mutants is not appropriate when all three genotypes are compared with each other. An ANOVA test should be used as in Figure 5F.We had used a one-way ANOVA for the statistical analysis used in Figure 3, but did not specifically state this in the methods. This has now been added to the methods and Figure legend.Reviewer #3 (Recommendations for the authors):The present manuscript addresses questions on the role of HGF/MET signaling in diaphragm formation once myogenic progenitors have already migrated to the PPFs. In addition, it identifies the PPFs as the source of HGF. The study is interesting and developed with rigor. However, the role of HGF is not clearly dissociated from the presence/absence of the fibroblasts/connective tissue itself.We have added to a supplemental figure demonstrating that fibroblasts are indeed present in muscleless regions following loss of HGF .Also, the authors do not link in the results, for example, how the muscleless diaphragms and HGF itself relate to the hernia phenotype mentioned in the abstract.We now discuss how the partially muscleless diaphragms relate to herniation in the abstract. While loss of muscle is necessary for development of hernias, it is not sufficient; additional defects in the connective tissue are necessary to weaken the diaphragm and lead to CDH.Figure 11C) Co-staining for Hgf (PPFs) and Met (migrating progenitors) should be provided for a clear visualization of ligand-expressing cells and receptor-expressing cells.HGF and Met in E11.5 PPF.We have included an additional panel in Figure 1—figure supplement 1 showing coexpression of 1F) Co-staining for Met and migrating progenitors (PAX3 or LBX1 for example) should be provided for a clear visualization of migrating progenitors versus general Met expression in other cell types.CreKI/+; RosamTmG/+Pax3 embryos in cross section .We have previously shown this in a supplemental figure analyzing the migration pathway of diaphragm muscle progenitors, co-staining MET and GFP abelling in 1M) The claim that fibroblast-derived HGF is required for diaphragm muscle development cannot be addressed with only this experimental analysis. What is the connective tissue phenotype in PDGFaCreER;HGFnull/fl embryos? Lack of connective tissue could affect muscle migration and development rather than HGF expression on its own. Are fibroblasts still present?We have demonstrated that fibroblasts are still present in muscleless regions .1G, I, K, M) Control genotypes should be analyzed and added in the Figure or as Supplementary Data.We have added a supplemental figure with control embryos stained for myosin .1I) Pax3CreKIMetnull/fl originates a muscleless diaphragm but this could be associated with lack of delamination and migration from the somites rather than a specific MET requirement for diaphragm muscle formation once progenitors have colonized this area as suggested by the authors.Yes, we agree. It is the primary reason we chose to use the pharmacological inhibitor BMS777607 to control the timing of MET signaling inhibitions.Figure 3It is not clear whether HGF controls phrenic nerve formation independently of muscle. In the end of this section the authors mention that Met heterozygous embryos have normal muscles referring to Figure 1K (which is not a picture referring to a Met het embryo). The authors should confirm if in Met null embryos there is a direct effect on the nerve bifurcation and brunching or if the lack of muscle is leading to this observation. Is the phenotype dose dependent for muscle formation? This is not properly shown by the authors. Could the authors perform a conditional KO of Met in the nerves?This concern is further supported by the Figure 4 data where phrenic nerve only extends to regions with muscle.SpD/SpDPax3 mice.Please see response to Essential Revision Point 1. We have now included a more detailed analysis of the role of Met in phrenic nerve branching, including conditional deletion in motor neurons and analysis of Figure 4The role of HGF versus the presence of fibroblasts/connective tissue remains vague. Is the lack of ventral muscles associated with lack of migration of progenitors within the forming diaphragm towards an HGF source? Or due to the lack of connective tissue scaffold itself? Why is Prx1Cre used instead of PDGFRaCre  to trace PPFs?PDGFRa-derived fibroblasts are present in the muscleless regions. The Prx1Cre lineage is also used in supplemental experiments for HGF deletion and in previous work from the lab. We believe it is useful to demonstrate the finding that fibroblasts are present with multiple lines of evidence and have left this in the supplemental figure .We have lineage data demonstrating that 4E) Please provide a control with a Cre allele to compare putative secondary effects for the presence of the Cre in the cKO embryos.The panel in 4E has been replaced with a control that has the Cre allele.Figure 5Validation for the drug BMS777607 effectiveness in inhibiting MET downstream pathway should be performed and shown.Due to limited cell number, this is difficult to directly measure in embryonic diaphragms either via Western or phospho-flow cytometry. As such, we took a strategy to measure phospho-Met levels in adult skeletal muscle satellite cells. We have demonstrated that phospho-Met levels are downregulated in skeletal muscle satellite cells following treatment with BMS777607 .The phenotype with the inhibition of Met signaling  is distinct to the one observed in Figure 4 . How do the authors explain the dorsal phenotype when inhibiting Met (since migration should have not been affected at this time-point) and Hgf inhibition in Figure 4 is not leading to this phenotype?CreER/+; HGFnull/flPDGFRa embryos and we do indeed often find dorsal muscleless regions in CreER/+; HGFnull/flPDGFRa embryos given tamoxifen at E9.5 . We suspect that slight differences in the timing of Cre-mediated deletion of HGF deletion likely have a substantial impact on the extent of muscle loss and result in the variable muscle phenotypes. We have also clarified the summary of the phenotype of CreER/+; HGFnull/flPDGFRa diaphragms in the model in Figure 8.There is quite a bit of variability in the Figure 6What is the time point after treatment in B and C? What is the total nuclei number in the cultures (since these are co-cultures)?The timepoint after treatment is 72 hr. This has been added to the figure legend. With our co-culture method, we have not been able to maintain myogenic progenitors after passaging. As such, these experiments are done on freshly explanted PPFs that are in a pyramid shape on the plate. Because the fibroblasts are tightly packed together in 3-dimensions prior to passaging, we cannot count total nuclei accurately with the automated software or evenly manually. The myogenic progenitors are less dense and can be accurately counted. As such, unfortunately we do not have total numbers of nuclei.The authors should provide pictures together with nuclear staining for a global view of the cell density in the cultures. Also T0 time point pictures should be added for comparison, in particular in C.We have added photos of nuclear staining of a 72 hr culture to a new supplemental figure . While we cannot get accurate quantitative data from this, it does give a qualitative picture of overall nuclear density. T0 pictures, using GFP fluorescence of live muscle progenitors have also been added to this supplemental figure.In these experiments the authors performed co-culture (fibroblasts + myogenic progenitors) derived from PPfs. However, fibroblast phenotype is not addressed. If there is less GFP+ cells is there more fibroblasts in the culture? Could this impact on the phenotype observed and linked by the authors to the Met inhibitor treatment? The only piece of evidence is Gata4 qPCR which is not sufficient to address fibroblast phenotype in the culture.Although it is possible that fibroblasts are affected by Met inhibition, our in vivo deletion of Met in fibroblasts  does not suggest that Met inhibition would negatively impact PPF fibroblasts in culture. Because of technical limitations (see above), it is difficult to explicitly quantify the number of fibroblasts unless we were to passage the fibroblasts.Figure 7Treatment of WT embryos from E7.5 will impact on myogenic progenitor migration. The authors have to dissociate the Met-related somite migration phenotype and the potential additional role of Met signaling in the diaphragm muscle formation.Our original analysis of the mechanistic effects of Met inhibition in vivo  were based on experiments administering BMS777607 E7.5-E12.5 because this dosage gave the most highly penetrant and consistent phenotype. To exclude possible effects due to early delamination and migration defects, we have repeated these experiments by administering inhibitor at E11.5-E12.5 and analyzing the ventral-most expansion of diaphragm muscle at E15.5 . We find that Pax7/MyoD/Myosin+ cells are substantially reduced in the ventral leading edge of the diaphragm following BMS777607 treatment. This demonstrates that Met is required at later timepoints for the ventral expansion of the diaphragm.Reviewer #1 (Recommendations for the authors):The majority of my concerns were appropriately addressed by the authors. There are a few additional points that need to be addressed, and most of these concern the text/wording.Unfortunately, the changes in the text have not been highlighted in red in the uploaded version of the manuscript, as it was mentioned in the rebuttal letter.1. While the quality of the figures is overall good, many of the panels in Figure 2 are very dark. It should be easy to modify this.Thank you for this observation. We have brightened many of the panels in Figure 2.2. While the authors show very convincingly that the effects on the phrenic nerve observed are caused indirectly by the loss of muscle, and not directly by the loss of HGF/Met signaling, this is not always made clear in the text. Furthermore, the mechanism that causes branching deficits should be clearly stated in the Abstract.Line 41-42...and indirectly required for phrenic nerve primary branching.Please mention the specific indirect mechanisms, i.e. via the effect on muscle development.We have explicitly noted the indirect effect is mediated by muscle in the abstract.Line 81…HGF is also critical for innervation.The reports cited do not distinguish between the effect of the nerve/muscle or report in vitro experiments. The text should take this into account.A sentence has been added indicating the reports do not distinguish between effect on muscle and nerve.238: Thus, PPF-derived HGF is necessary for phrenic nerve defasciculation.Additional experiments shown in the next paragraph indicate that it is the absence of the muscle that causes defasciculation. Please rephrase.This has been rephrased as “Thus, loss of PPF-derived HGF leads to both muscle defects and phrenic nerve defasciculation defects.”3) Met inhibition using BMS777607BMS777607 acts as an AXL, RON and Met tyrosine kinase inhibitor. This should be mentioned. Are effects of RON/AXL on skeletal muscle development described?We have added a note that BMS777607 also inhibits AXL and RON in addition to MET. There is limited information about the role of RON/AXL on skeletal muscle development. A recent study found that Axl is a survival and growth receptor in mouse myoblasts during skeletal muscle regeneration . A published meeting abstract states that double knock out Gas6-Axl mice have reduced hindlimb skeletal muscle mass (doi: https://doi.org/10.1096/fasebj.2020.34.s1.09757).4) Antibody specificity: Antibody specificity should be tested by analysis of the phrenic nerve on Met mutants.D/DMet mutants using the R&D antibody, we still see labeling  which was generated against a recombinant protein that encompasses Glu25-Asn929. This region includes the extracellular semaphorin domain, furin cleavage site, plexin semaphorin domain, and the four IPT domains. The mutant Met allele we are using is from Huh et al.,  and deletes exon 16 which contains a critical ATP-binding site in the intracellular tyrosine kinase domain. Western analysis from Webster et al.,  and the Huh et al., 2004 paper  show that the MET pTyr 1234/1235 domain and downstream pAKT are inactivated with this mutant allele, respectively. However, the pre-processed MET protein is still present in these mutants . The R&D antibody that we and others use recognizes the extracellular domain that is present in the preprocessed protein . Thus when we examine ling see . TherefoReviewer #2 (Recommendations for the authors):Sefton et al., have submitted a revised version of a study, in which the role of fibroblast-derived HGF for recruitment and expansion of muscle during morphogenesis of the mammalian diaphragm was investigated. The authors have changed the title to cope with new findings, indicating that reduced primary branching of the phrenic nerve in Met-mutants is not due direct effects of HGF on the nerve but most likely caused by indirect effects resulting from reduced muscle formation in Met-mutants.The authors did an excellent job to deal with the reviewers' criticisms. In particular, by specifically deleting Met in motoneurons and by analysis of splotch mutants they demonstrate that Met does not intrinsically regulate phrenic nerve branching. Analysis of splotch mice, which show normal expression of HGF PPFs but display muscle-less diaphragms, revealed reduced branching of the phrenic nerve, similar to Met-mutants, clearly suggesting a critical role of muscle fibers in the diaphragm for phrenic nerve branching. Furthermore, Sefton et al., now demonstrate that fibroblasts expand and populate the diaphragm in the absence of muscle, which essentially excluded a connective tissue phenotype in conditional Hgf/Met mutants. They also clarified the statement about what I understood was meant to claim a role of Hgf/Met in myogenic commitment. Additional controls  were done as requested, providing additional interesting insights into a role of Met for ventral expansion of the diaphragm at relatively late developmental timepoints.The authors argue that "PPF-derived HGF, via muscle, controls phrenic nerve defasciculation". Well, this is formally correct but exaggerates the role PPF-derived for phrenic nerve defasciculation in my view. Probably, deletion of any gene that prevents diaphragm muscle formation will have similar effects on phrenic nerve defasciculation. I would prefer a more neutral statement: "Muscle formation, requiring PPF-derived HGF, controls phrenic nerve defasciculation", or something similar. The authors may consider modifying the statement.I do not have any further objections.We appreciate the positive response to our revisions. We have altered the sentence as suggested.


The aim of this work is to propose two different and integrated sensors for the structural health monitoring of concrete beams. In particular, a diffused sensing element and a split ring resonator network are presented. The first sensor is able to detect the variations in the dielectric properties of the concrete along the whole beam length, for a diffuse monitoring both during the important concrete curing phase and also for the entire life cycle of the concrete beams. The resonators instead work punctually, in their surroundings, allowing an accurate evaluation of the permittivity both during the drying phase and after. This allows the continuous monitoring of any presence of water both inside the concrete beam and at points that can be critical, in the case of beams in dams, bridges or in any case subject to a strong presence of water which could lead to deterioration, or worse, cause serious accidents. Moreover, the punctual sensors are able to detect the presence of cracks in the structure and to localize them. Civil infrastructure systems such as bridges, buildings, dams, and pipelines play a central role in the economic and industrial wealth of society . MonitorThe most widely used construction material in the world is the concrete, thanks to its strength, durability, versatility, and cost effectiveness. Concrete is a mixture of Portland cement, water, aggregates, and in some cases, admixtures; its properties depend on the relative amounts and characteristics of the individual components. Even a perfect mix can result in poor quality concrete if the curing phase is not properly monitored . The conIndeed, a premature removal of formworks may lead to inadequate structure strength and to the presence of cracks, produced by internal possible strain, that can cause the collapse of the structure at a later time. Moreover, the ex-post monitoring also has great importance, since it allows an immediate response in case of anomalies, decay phenomena, and also in the case of structures in contact with water such as dams, riverbanks, etc.Recently, the monitoring of moisture content in concrete structures and their consequent deterioration has triggered the interest in electromagnetic sensors ,8,9,10.In particular, in one study , the desOn the bases of the results presented in , the numThe present work is organized as follows. An SRR is made up of metal tracks on a dielectric substrate; these tracks consist of circular crowns with cuts along a diameter, hence the name “Split Ring” resonator. There are various types of SRR which differ in geometry . Startinr = 2.55; loss tangent = 0.0013 @ 10 GHz; t = 0.035 mm, where t is the thickness of the copper cladding [In particular, the substrate thickness affects both the losses and the practical needs for measurement. For the latter, it is obvious that the greater the thickness, the greater the strength of the resonator leading to a good repeatability of the measurements and, especially, to a high accuracy. To determine the suitable substrate and to optimize the geometric parameters of the SRR with reference to the desired resonance frequency and optimal quality factor, parametric simulations were performed with EM CAD CST Microwave Studio , using vcladding .Four similar SRRs have been designed and optimized, using the same material and varying the geometric characteristics  for the four sensors operating in air.rf and the permittivity ε for the SRR1, while Subsequently, the SRRs were simulated with the numerical CAD, embedded in a medium with a permittivity ε that has been varied from 1 to 8 in step 1. To feed the four SRRs, the design of a power divider has been conducted with Microwave Office (MWO) by AWR . In part21 of the entire sensor network.21 of the entire sensor network  to material permittivity (ε) can be obtained. As reported in the literature [In this way, calibration curves relating resonance frequency , the corresponding permittivity of the material surrounding the sensor can be obtained.The diffused sensing element consists of two cylindrical conductors, parallel to each other and mutually isolated by a plastic covering. This type of sensor, embedded in the system under test (SUT) has one end accessible to perform measurements, allowing a diffused monitoring of SUT and providing information on the entire profile of the structure.In particular, the microwave reflectometry electromagnetic (EM) measurement technique is used, employed in many monitoring and diagnostic applications ,23. An Eapp ; it is given by:rv (t) is the amplitude of the reflected signal and incv (t) is the amplitude of the incident signal.The EM signal, usually a step-like signal, propagates along the sensing element and whenever it encounters an impedance variation, linked to the changes in the dielectric characteristics of the SUT, a partial reflection of the signal is generated. The TDR instrument acquires the reflection coefficient ρ, that is displayed as a function of time or the traveled apparent distance drL) the material effective dielectric constant can be achieved estimating the apparent length (aL) from the reflectogram:app, and reflects the interaction between the EM signal and the SUT.From the sensing element physical length , namely the nanoVNA  is employed for frequency domain measurements. The nanoVNA is a compact miniaturized VNA, with dimensions 15 cm × 10 cm × 6 cm, it is low-cost and it has 50 kHz–3 GHz frequency range. It was connected to the four split ring resonators through the power divider and combiner allowing the network SFor the TDR measurements a Campbell-Scientific TDR200  is used, a low-cost, portable TDR measuring instrument, with dimensions 22 cm × 5 cm × 11 cm. The TDR200 generates a step-like voltage signal, with a 200 ps rise time, which corresponds to a frequency bandwidth of approximately 1.7 GHz, that propagates through the sensing element embedded in the concrete beam ,26.In this way, measurements were performed over a 28-day period, every day, allowing an accurate monitoring of the concrete curing phase.First, the network transmission coefficient measurements were carried out though the nanoVNA in air, before embedding the network in the concrete beam. After inserting the four SRRs and the sensing element inside the beam during the concrete pouring, measurements on both types of sensors were repeated during the 28 days of hardening of the concrete.21 which looks like a large bell in which the peaks merged together due to the high losses dramatically decreasing the quality factor. On the second day, however, much of the water has evaporated and the four peaks begin to be clearly visible and shift to the right, as expected when permittivity begins to drop.As regards the SRRs, the first day, the presence of a large quantity of water produced a SOn the two-wire sensing element it is noted that as the days pass, the apparent length decreases see . In partTo extrapolate the permittivity of the concrete, the inversion of the measurements in the time domain was carried out using a Matlab algorithm, using a step as an incident pulse. By applying (3) the permittivity can be achieved. 21 has a bell-shaped trend and the four resonances of the SRR network are not clearly visible; this is the reason it was not possible to extrapolate permittivity data with the SRR network in It is worth noting here that, as already mentioned, the first day the SAnother important aspect is that the difference in concrete permittivity was deliberately made. In fact, in order to verify if it is possible to notice variations in the surroundings of the planar sensors and therefore to allow precise monitoring, a different mixture of cement was placed around the SRRs in the top layer of the beam structure, with a slightly lower cement-to-aggregate ratio, resulting in a lower permittivity.Even though the reported experimental results referred only to monitoring of the curing phase, a monitoring of this type will also allow controling the presence of water inside the beam ,30,31,32In this paper, two sensors for concrete monitoring were presented, a diffuse SE and a network of SRRs for punctual monitoring. The first sensor is able to monitor both the curing phase and possible anomalies arising during the lifespan  of the concrete structure, which might also be several hundreds of meters long. The second sensor typology, instead, is able to monitor both the presence of water inside the beam and any cracks around the sensor and determine its location thanks to the different working frequencies of resonators; however, the SRR network is only able to cover localized portions of the structure so as to achieve a detailed and sensitive monitoring of possible critical sections of the structure with a distributed localized approach.The integration of these two structures makes it possible both to monitor concrete during the curing phase, which is very important for the future structural strength of the beam, and to highlight the presence of water that could cause deterioration, especially in the cases of dams, bridges and structures that are constantly subjected to an important presence of water.For future developments it is planned to test the possibility of monitoring the presence of water near one of the SRRs and the localization of artificial cracks, specifically created with mechanical stress in some points of the beam.Although the present study was limited to a frequency range compatible with portable low-cost instruments, a very interesting future development could regard the adoption of miniaturized THz-band sensing structures, thus allowing for a finer distributed sensor network as well as a more efficient detection on micro-cracks occurring during the lifespan of the concrete structure. Such THz structures were recently proposed in the literature ,36,37,38


Upon excitation at 400 nm, transient absorption spectroscopy unveils photoinduced electron transfer from the host's chromophores to the guest, as indicated by characteristic spectral features assigned to the oxidized donor and reduced acceptor. The structure of the host–guest complex was characterized by NMR spectroscopy, mass spectrometry and single-crystal X-ray analysis. Spectroelectrochemical experiments and DFT calculations both agree with the proposed light-induced charge separation. A kinetic analysis of the involved charge transfer channels reveals, besides a guest-independent LMCT path, 44% efficiency for the host–guest charge transfer (HGCT).A [Pd Light–powered host–guest charge transfer (HGCT) is shown for a coordination cage based on electron-rich phenothiazines, containing an anthraquinone acceptor as guest. Transient absorption spectroscopy and spectroelectrochemistry data is presented. However, connecting multiple donors/acceptors via covalent bonds under precise control over their optimal distance and spatial orientation can be synthetically challenging and tedious. Recent progress in supramolecular chemistry provides alternative approaches to conventional multistep synthesis by connecting donor- and acceptor-functionalized building blocks through noncovalent or coordinative interactions, allowing the simple and high yielding assembly of D–A combinations in a modular fashion with determined stoichiometry and spatial arrangement.11–13 Such supramolecular D–A systems have been explored to act as artificial photosynthetic systems that harvest light energy, as electron-transfer systems, hydrogen evolution catalysts, and further photo-redox-functional devices.9,13–19In nature, photosynthesis is the fundamental reaction cascade that transforms light into chemical energy, involving light-induced charge separation within precisely arranged chromophore assemblies. Taking this as an inspiration, the quest to build efficient artificial molecular photosystems that harvest solar energy and transform it into an electrical potential difference or power the formation of high-energy fuel compounds is a key scientific challenge.20–24 featuring a confined yet accessible inner space, have been of interest as a nanoscopic container for guest encapsulation,25–27 as a molecular transport vehicle,28,29 as a stabilizer of reactive compounds30 and as a supramolecular catalytic center.31 Furthermore, the interaction between cages and guest molecules in terms of photophysical processes was also explored before.32–40 Self-assembled supramolecular systems represent an ideal platform for realizing photoinduced electron transfer (PET) processes due to the possibilities to arrange donor and acceptor functionalities in short through-space distance, achieving maximum local concentration, and defined orientation.18,35,36,38,41–44Self-assembled coordination cages,45 As a popular combination, PTZ and AQ derivatives are often implemented as D–A pairs owing to their suitable redox potentials and HOMO–LUMO energy gap characteristics.46,47The choice of the D–A pair and the spatial arrangement of the D and A moieties play crucial roles to effectively achieve a charge separated state with long lifetime, a prerequisite for application of such systems as a basis for active materials in photovoltaic devices. As donor components, electron-rich aromatics such as phenothiazines (PTZ), arylamines as well as thiophene and carbazole derivatives are frequently chosen. Anthraquinones (AQ), benzothiadiazoles, squaraines and various cyano- and keto-functionalized heterocycles have been employed as complementary partners to construct a D–A system.4L8] cage assemblies.48,49 We first demonstrated that the self-assembly of banana-shaped PTZ- or AQ-based bismonodentate pyridyl ligands  with Pd(ii) cations yielded homoleptic catenated double-cages [3BF4@Pd4L8]5+ (L = D or A).48,50 Further, we showed that the equimolar mixture of these ligands (D : A = 1 : 1) with Pd(ii) ions resulted in a statistical combination of mixed-ligand interpenetrated cages [Pd4DmAm8−]8+ (m = 8…0), containing all possible stoichiometries and stereoisomeric combinations. By far most of the components of the resulting mixture were mixed-ligand double-cages, containing at least one donor or one acceptor component, that contain densely packed PTZ and AQ functionalities in the immediate vicinity within the highly entangled supramolecular assemblies. Upon irradiation with light of 385 nm wavelength, electron transfer from the excited state phenothiazine donor onto the anthraquinone acceptor to obtain a charge-separated state could be unambiguously corroborated by a combination of transient absorption experiments in the UV-Vis and IR spectral regions. However, the fact that the examined samples in this study consisted of a statistical mixture of mixed-ligand cages hampered interpretation of the photophysical kinetics of the decisive species and restricts the understanding of structure–function relationships and the establishment of design principles for next-generation D–A systems.Previously, we introduced a self-assembly approach to amalgamate PTZ- and AQ-based building blocks in a D–A system based on interpenetrated (BF4)2 in 2 : 1 ratio resulted in quantitative formation of desired donor cage [Pd2L4](BF4)41 in DMSO solution  were downfield shifted compared to the free ligand, characteristic of the metal-mediated self-assembly. Signals corresponding to di-, tri-, and tetra-cationic complexes of the cage 1 with a differing number of BF4− counter anions were identified in the ESI mass spectrum , anthraquinone-2,7-disulfonate (G2), and anthraquinone-2-sulfonate (G3) were examined as suitable guest molecules for the donor-functionalized host 1 in DMSO. From 1H NMR studies it was observed that only G1 and G3 were encapsulated inside cage 1  are found in important compound classes such as natural pigments and redox carriers, anticancer drugs, and pesticides. They are also extensively studied in the context of redox flow batteries and photocatalysis, owing to their advantageous acceptor properties, reversible redox behaviour, and fast electrochemical kinetics. In this study, sodium salts of anthraquinone-2,6-disulfonate  of the host are most affected after the addition of the guest molecule G1. Proton Hi, which points inside the cavity, is downfield shifted by about 0.64 ppm, suggesting strong interactions with the guest's sulfonate groups. In contrast, phenothiazine backbone signals  along with signal Hd were found somewhat upfield shifted. In the case of [G3@1]3+, only proton Hi is downfield shifted by about 0.1 ppm and broadened compared to free cage 1. A competitive 1H NMR experiment was performed by combining G1, G2, and G3 in equimolar ratio (1 : 1 : 1) with cage 1 (1 equiv.) to check guest preference and selective uptake of G1 was observed  is superior to monoanionic G3  over a period of four weeks resulted in tiny needle-shaped single crystals that proofed suitable for synchrotron X-ray diffraction. The host–guest assembly crystallized in triclinic form, showing space group P1̄ (2). The asymmetric unit contains two host–guest assemblies [G1@1]2+ besides two unbound G1 dianions and fourteen DMSO molecules  centers, the LUMO for each host–guest assembly is spread over the acceptor guests G1 and G3, respectively. The calculated energy gaps between HOMO and LUMO are 1.95 eV for [G1@1]2+ and 1.97 eV for [G3@1 + BF4]2+, while it is 2.92 eV for [1 + 2BF4]2+ . The oxidation and reduction signals for the PTZ moieties in the host–guest assembly [G1@1]2+ are shifted to slightly lower positive potentials . Free guest G1 has its reduction and oxidation potentials at about −1.23 and −1.17 V, respectively. In the host–guest assembly [G1@1]2+, the reduction potential (about −1.14 V) for the acceptor moiety is shifted towards a more positive potential in comparison to free G1 (ΔEred = 0.09 V).The electrochemical properties of the D–A assemblies and their individual components were studied by cyclic voltammetry in DMSO solution at 298 K, with 0.1 M of TBAPFL, 1, G1, and [G1@1] were performed in a thin cell  containing a Pt gauze and a reference electrode (Ag/AgNO3) in combination with a diode array spectrophotometer. The resulting difference spectra are depicted in L(˙+) and [Pd2L4(˙+)] are similar, and a sharp absorption peak around 518 nm and a very broad peak around 691 nm is apparent in both cases. The reduced guest G1 revealed a broad signal around 574 nm. In the case of host–guest assembly [G1@1], the resulting spectra shows both signals for the oxidized donor and the reduced acceptor at respective voltages, similar to the free cage and guest at comparable potentials. Thus, donors and acceptor were found to maintain their characteristic electrochemical behaviours in the self-assembled host–guest complex and did not show any significant redox interplay in the ground state.Spectroelectrochemical measurements of L as well as the host cage [Pd2L4]4+, with and without the presence of equimolar amounts of guest molecules G1 or G3. All compounds were investigated in DMSO solution. They were excited at λexc = 400 nm (where G1 and G3 do not absorb) and probed in the UV-Vis at 390–730 nm using a white-light continuum.Femtosecond pump–probe spectroscopy was applied to get insight into the dynamics following photoexcitation of the bare donor ligand L  appears, however, its lifetime is only ∼0.7 ps. The minimum at 505 nm, indicating stimulated emission from LS1, is less pronounced and gets even weaker and more red-shifted with time compared to the free ligand transients , exhibiting absorption bands at 500 and 700 nm  to the bound Pd(ii), followed by immediate back electron transfer (BET).48,492L4]4+, for which photo-induced LMCT leads to the intermediate [PdIIPdIL3L(˙+)]4+. In L(˙+) characteristic absorption at 500 nm appears rather weak because (i) it is superimposed by the stimulated emission of the donor's S1 state and (ii) BET is so fast that the [PdIIL3PdIL(˙+)]4+ population is always low.In the [Pd Fig. S46. In addi2L4]4+ cage was found to be not affected by addition of the electron acceptor G3 to the cage at the same concentration used as for the host alone  being formed by electron transfer from one of the phenothiazine ligands in the coordination cage. The difference in peak position compared to the electrochemically generated spectrum of reduced G1  ions. Formation of G1(˙−) by host-to-guest charge transfer (HGCT) is also supported by a closer inspection of the decay kinetics of the 613 nm band. The grey data points in G1 the decay of this band is first faster and then turns over to a slower decay than without the guest (black points). Also for other probe wavelengths, time traces were found to be altered upon encapsulation of G1 ]4+-G1(˙−) couple is significantly slower than for the [PdIIL3PdIL(˙+)]4+ intermediate.The spectral evolution of the photo-excited  which is just the sum of the spectra of 1 and G1, indicating that there is no special interaction between host and guest that alters the electronic structure of the cage. In particular, there is no close π–π-interaction between host and guest aromatic parts. The main driving force for establishing decent host–guest affinity is the perfect arrangement of the guest's sulfonate groups close to both of the cage's inner Pd(pyridine)4 faces, thereby replacing loosely bound BF4− counter anions. Also, the crystal structure of the host–guest complex and DFT-based geometry-optimized models of the cage without guest are almost superimposable, i.e. the Pd–Pd distance is similar in both cases.Based on the reaction scheme depicted in Fig. S48a. Then, iFig. S48b. The relG1@1]2+ confirmed the presence of the guest in the central cavity of the cage. Theoretical studies of the host–guest assemblies suggested that HOMOs are spread over the donor cage components while the LUMOs are localized on the guest molecules with an energy gap of about 188 kJ mol−1. Spectroelectrochemistry results revealed the characteristics of the radical ions of the donor and acceptor components to be maintained in the host–guest assembly and the absence of electronic communication between donor and acceptor in the ground state. However, the femtosecond transient UV-Vis absorption spectroscopy unambiguously shows the formation of a cage-based PTZ radical cation and a guest (G1) radical anion upon excitation of the host–guest assembly. Thus, we have demonstrated that the donor cage built from electron-rich PTZ moieties is proficient in transferring electrons to the incarcerated acceptor G1 guest. The rational design of supramolecular donor–acceptor assemblies that promote photoinduced electron transfer will be of interest in the field of organic photovoltaics, given that modern self-assembly strategies allow to create complex and anisotropic molecular materials with potential to control the morphology of photo-active layers. Furthermore, photo-redox catalytic applications can benefit from host–guest chemistry via tuning substrate affinity and exchange kinetics with respect to the excited state life-time of a catalytic nanoconfinement.Herein, a self-assembled donor-functionalized cage based on electron-rich PTZ moieties is reported. The cage is capable of encapsulating electron-deficient acceptor molecules such as anthraquinone-2,6-disulfonate and anthraquinone-2-sulfonate. The crystal structure of one of the host–guest assemblies [S. Ganta and G. H. Clever conceived and designed the study. S. Ganta performed the synthesis and characterization of the materials. J. J. Holstein performed the X-ray structures analysis. C. Drechsler conducted the computational studies. J.-H. Borter and D. Schwarzer performed and analyzed femtosecond pump–probe spectroscopy experiments. S. Ganta wrote the initial draft and all authors reviewed and edited the paper.There are no conflicts to declare.QO-009-D2QO01339H-s001QO-009-D2QO01339H-s002