C. elegans to stringently deplete embryos of Microprocessor activity. Using a combination of auxin-inducible degradation (AID) and RNA interference (RNAi), we achieved Drosha and Pasha/DGCR8 depletion starting in the maternal germline, resulting in Microprocessor and miRNA-depleted embryos, which fail to undergo morphogenesis or form organs. Using a Microprocessor-bypass strategy, we show that this early embryonic arrest is rescued by the addition of just two miRNAs, one miR-35 and one miR-51 family member, resulting in morphologically normal larvae. Thus, just two out of ∼150 canonical miRNAs are sufficient for morphogenesis and organogenesis, and the processing of these miRNAs accounts for the essential requirement for Drosha and Pasha/DGCR8 during the early stages of C. elegans embryonic development.MicroRNAs (miRNAs) are a class of post-transcriptional repressors with diverse roles in animal development and physiology . The Mic  •Depletion of Drosha and Pasha results in embryos that fail to undergo morphogenesis•The mirtron pathway enables expression of miRNAs in the absence of Drosha and Pasha•Two miRNAs are sufficient to rescue embryogenesis in the absence of Drosha and Pasha•miR-35 and miR-51 play an unexplored, likely conserved role in animal development C. elegans embryos causes failure to undergo morphogenesis. This can be overcome by re-introducing two miRNAs, miR-35 and miR-51, resulting in morphologically normal larvae. These conserved miRNAs account for the essential embryonic function of Drosha and Pasha.Dexheimer et al. show that depletion of maternal and zygotic Drosha and Pasha in  MicroRNAs (miRNAs) are short non-coding RNAs that act with Argonaute proteins to elicit translational repression and decay of specific target mRNAs . miRNAs C. elegans, we developed a conditional approach for stringent removal of Drosha and Pasha/DGCR8 and thus miRNAs. Microprocessor depletion in embryos is generally challenging, given the maternal contribution of mRNA and protein. In C. elegans in particular, the function of the Microprocessor has not been studied in embryos because homozygous Drosha (drsh-1) or Pasha/DGCR8 (pash-1) mutants derived from heterozygous mothers carry enough maternal RNA and/or protein to develop into adults, albeit sterile . B. BC. eleormation , we thusormation , yieldinpash-1 and drsh-1, animals were subjected to Cas-9 mediated genome engineering via RNP-microinjection following the Co-CRISPR approach described by [S. aureus, purified in-house), 500ng/μl TracerRNA, 100ng/μl crRNA targeting the Co-CRISPR marker gene dpy-10, and 15ng/μl dpy-10- > rol-6 repair template oligo. Additionally, 100 ng/μl crRNA against the locus of interest as well as 100-200ng/μl dsDNA repair template encoding the desired modification with around 50bp flanking homology arms were added to the mix. Note that for efficient degradation of DRSH-1 we needed to insert AID degrons at the N- and C-termini simultaneously as either degron alone was insufficient for depletion. Guide RNAs used and sequences inserted (in bold) were as follows:drsh-1 – N-terminal AID-tag – guide sequence used = 5′- TTAGATTTTCATTTAGATGT-3′ – locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGGGAGGAAGCGGAGGAGGAAGCGGTGGAGGAAGCGGAGGAGGAAGCGGAATGGAACAGAAACTCATCTCTGAAGAGGATCTGATGTCGGACGAAAAGATTTCAATGACGCTTAACTTCCCGAAACACAAGCGTAGTTAACGTTTTCATCTGAAATTGTAGACAGATTTAGATTTTCATTTAGdrsh-1 – C-terminal AID-tag - guide sequence used = 5′-GATACCAGCGACTAATTACG-3′ – locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGAACAGAAACTCATCTCTGAAGAGGATCTGTAGTTACGGGGTTATAATTATACTATGTCTGTTTGAATGTGATTCGGTTCTCAAGTGGTTTCAGAACATGCGCCGTCGTCTTGAACAAGATACCAGCGACGGAGGCAGCGGTGGTGGAAGCGGCGGGGGAAGCGGCGGTGGAAGCGGTGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGpash-1 – C-terminal AID-tag – guide sequence used = 5′-AGGTGAATATACTATTTGTG-3′ – locus post-edit:ATGCCTAAAGATCCAGCCAAACCTCCGGCCAAGGCACAAGTTGTGGGATGGCCACCGGTGAGATCATACCGGAAGAACGTGATGGTTTCCTGCCAAAAATCAAGCGGTGGCCCGGAGGCGGCGGCGTTCGTGAAGGAACAGAAACTCATCTCTGAAGAGGATCTGTAGGGAGGAAGCGGAGGAGGAAGCGGAGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTATATTCACCTCATATGTTTGTTGTTTTGTTGGTAGTTTTAATTTTTGGGGAAGACATGACGATTCATCATCCCCATCACATCAGAAACCACACAAAInserted sequences contained the TIR-1 recognition peptide , a FLAG (GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG) and a MYC (GAACAGAAACTCATCTCTGAAGAGGATCTG) tag, as well as GGSG linkers in between.To generate AID-tagged copies of ribed by , with moribed by . Brieflypash-1(ts) allele mj100 were kept constantly at the permissive temperature of 16°C. pash-1(ts) animals expressing the mirtron-51 transgene were selected every few generations for high levels of the co-expression marker elt-2p::dsRed, as this transgene was prone to undergo silencing. For experiments, L4 stage larvae were transferred to a fresh plate and shifted to 25°C approximately 16h later. In order to achieve maximal depletion of maternal miRNAs, mothers were kept at the restrictive temperature an additional 7h before harvesting early embryos (1-4 cell stage). Extending the time at the restrictive temperature was prohibited by the rapidly deteriorating health of gravid pash-1(ts) adults expressing mirtrons at 25°C. For backshift-experiments with mirtron-rescued pash-1(ts) animals, hatched L1s that developed under restrictive conditions were transferred to a fresh plate within 1h after hatching, allowed to develop at 16°C, and scored for their ability to reach adulthood within 7 days.Animals bearing the E. coli OP50 at 20°C. For combined AID and RNAi treatment, NGM plates were supplemented with 1mM Carbenicillin, 1mM IPTG and 4mM Auxin . RNAiD plates were seeded with E. coli HT115 expressing dsRNA which elicits an RNAi response against pash-1 mRNA  library, ORF-ID T22A3.5). To ensure efficient mRNA knockdown, RNAi bacteria were grown freshly for every experiment in liquid LB culture, the medium was brought to 1mM IPTG around 1h before pelleting bacteria, and plates were seeded with a 20X concentrate . For experiments, L4s were transferred to RNAiD plates, embryos were harvested 24h later, and assayed as described below. While the AID-system rapidly depletes proteins within minutes to hours [pash-1(ts) animals, eventually owed to the temperature difference and/or deleterious side-effects of PASH-1(TS) at 25°C. Efficiency of RNAi-mediated pash-1 mRNA knockdown was assessed via RT-qPCR. Briefly, 5-10 embryos in the 2-cell stage were transferred to about 1 μl of Lysis Buffer  using a thin glass needle. These samples were subjected to 10 min digestion at 65°C before heat inactivation of proteinase K for 1 min at 85°C. Next, crude lysates were reverse transcribed before performing qPCR using the GoTAQ qPCR Mastermix (Promega) according to the manufacturer’s instructions. Relative expression was calculated according to the ΔΔCq-method using cdc-42 as a reference gene, the following primer sequences were employed:pash-1-F: 5′-GCCTTCGAGAAAACGGGGAA-3′; pash-1-R: 5′-TGGCTCCCATTTCGGAGATT-3′;drsh-1-F: 5′-TGAGCTGGCTTTGGCTAATCT-3′; drsh-1-R: 5′-ACCCCGTAATTAGTCGCTGG-3′cdc-42-F: 5′-TGGGTGCCTGAAATTTCGC-3′; cdc-42-R: 5′-CTTCTCCTGTTGTGGTGGG-3′Animals were kept constantly on standard NGM plates seeded with To assess the extent of protein degradation of PASH-1:AID:MYC and DRSH-1:AID:MYC upon Auxin treatment, L4s were transferred to Auxin plates prepared as described above. 24h later, gravid adults were collected in Lämmli-buffer containing 2.5% β-Mercaptoethanol (v/v), and subjected to multiple cycles of snap-freezing followed by boiling until embryos were disrupted. Proteins were separated via SDS-PAGE and transferred onto a Nitrocellulose membrane. As primary antibodies either monoclonal mouse anti-myc  or a polyclonal rabbit anti-gamma tubulin  were used. As secondary antibody, anti-mouse IgG HRP-linked Antibody  or anti-rabbit IgG HRP-linked Antibody  was applied followed by visualization using ECL reagent (Thermo Scientific).Embryos were obtained from day 1 gravid adults reared as described above, by slicing mothers in a drop of M9 buffer on a microscopic slide. 2-cell embryos of normal size were transferred to standard NGM plates or RNAiD plates and allowed to develop at the respective temperatures. Hatched animals were collected for subsequent analysis  from plates within 1h after hatching. The final hatching rate was assessed > 24h after embryo collection by scoring the fraction of animals that successfully escaped the eggshell. To obtain samples at specific developmental stages for small RNA sequencing, 2-cell embryos were harvested via slicing, collected or allowed to develop on NGM plates before being selected manually by stage at given time points .myo-2prom::mCherry. Mean and range are plotted for each genotype; unpaired t test was used for statistical comparison.For phenotypic analysis of embryonic arrest or hatched L1 larvae, animals were mounted on a thin agar-pad on a microscopic slide sealed with a coverslip. Images were recorded at 400x magnification using an AxioImager Z2 (Zeiss) equipped with DIC and fluorescence optics, and analyzed via ImageJ. Arrest phenotypes were scored as follows: cell mass = embryo fails to initiate bean stage; morphogenesis = embryo begins to elongate but fails to reach 2-fold stage; elongation = embryo completes 2-fold stage but arrests before hatching. Body measurements are based on DIC micrographs. Body length was measured as the length of a segmented path running through the mid body axis from head to tail. Body width was assessed as the arithmetic mean of 3 independent width measurements in the anterior, middle, and posterior part of each animal. Pharynx length was determined by the length of a segmented path running through the pharynx middle axis in animals expressing To profile miRNA levels, a modified version of the small RNA sequencing protocol described in  was perf18-30nt-NNNNNNXXXXXAGATCGGAAGAGCAC-ACGTCT/3ddC/-3′. After sufficient PCR-amplification as observed by SYBR green derived qPCR signal (requiring around 16-20 cycles), libraries were size-selected on an agarose gel to remove adaptor dimers and sequenced on a HiSeqV4 platform (Illumina). Small RNA reads were mapped and processed as in [https://github.com/lengfei5/smallRNA_nf/tree/master/dev_sRBC. To accurately quantify sRNAs, both mapped sequences and associated random nucleotides (functioning as UMIs) within the raw reads were quantified for miRNAs and piRNAs as well as spike-ins. To alleviate PCR amplification bias, UMI counts were used and normalized to spike-in as described in [https://github.com/lengfei5/smallRNA_analysis_philipp/tree/master/scripts. Every round of library preparation included one sample of a comparable amount of Arabidopsis thaliana total RNA to assess the extent of potential contamination. The average counts for reads mapping to C. elegans miRNAs in contamination control samples (∼1% of the wild-type miRNA content) across four independent library preparations were subtracted from all samples as a background correction. For background-corrected, spike-in normalized, miRNA counts in molecules per embryo, see (XXXXX = barcode): 5′-ACACUCUUUCCCUACACGACGCUCUUCCGAUCUNNNN-(sRNA)ed as in . Modificed as in . The modribed in , allowinPlots were created using Prism, statistical analysis was also performed in Prism as described in the figure legends. For sRNaseq data statistical analysis was performed using DESeq2 . Figures


Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies. Chronic hepatitis B virus (CHB) infection affects roughly 250 million people worldwide  and is aImmunotherapy had already emerged in the 1990s as a promising option to treat CHB. T cell responses are considered essential for viral clearance but are scarce or exhausted in CHB patients –7. Still–8–in vivo. Others have explored strategies to predict HLA-I epitopes from Pol but focused exclusively on a single HLA type or assessed only a limited number of HBV sequences  or core antigen (HBcAg). However, the proteins X (HBx) and polymerase (Pol) also pose interesting targets, as both are vital for viral persistence –27 and i–30–equences –40. TakeIn addition to a limited epitope repertoire, there is another hurdle in the development of HBV-directed immunotherapy. Established epitopes might not all be equally effective, as they can be subjected to different levels of viral mutagenesis and subsequent immune escape. Indeed, previous reports clearly demonstrate that HBV is subject to immune pressure and that mutation of epitope sequences leads to immune evasion –43 or ev–46–Here, we have taken an effort to tackle the above-mentioned issues by integrating viral indispensability, genomic variation, HLA binding, and immunogenicity to identify the best HBx- and Pol-derived T cell epitopes for immunotherapy across 6 of the most prevalent HLA supertypes within the HBV-infected population. The results of this study pave the way for the development of globally effective HBV antigen-specific immunotherapies.n = 14) and Pol (n = 50)  , left, w , left, (n = 50)  Fig. 1,,n = 14 a = 50) 4648, 52–69We argued that epitopes in which the least conserved amino acid was still present in 80% of all sequences tested would be targetable in the majority of the population. Combining this criterion with the preference for functional association, we found 7 HBx-derived and 26 Pol-derived sequences reported as epitopes across HLA types to be preferential targets for global immunotherapy . We first predicted binders spanning 8 to 14 amino acids for supertype-representative HLA types using the established in silico prediction tool NetMHCpan to make a frequency distribution of predicted binders (P = 0.57 by a Mann-Whitney test). Predicted binders spanning 9 to 11 amino acids were subsequently aligned to our maps outlining conservation and function .The prediction yielded totals of 251 potential novel HLA binders for HBx and 1,655 for Pol , right, ed assay . For praed assay . For HLAin vitro binding assay. Peptides were classified as HLA binders when their binding capacity was higher than 25% of that of a known high-affinity peptide (Table S4). HLA-A*11:01 and HLA-A*03:01 were both tested as members of the HLA-A*03 supertype since many HBV-infected patients are Asian and HLA-A*11:01 is more prevalent in this population than the supertype representative HLA-A*03:01, which is more prevalent in Caucasians  from blood donors who had previously resolved an HBV infection were expanded in the presence of peptide pools, followed by single-peptide restimulation and an interferon gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA). As expected, IFN-γ production was detected in response to the well-established epitopes c18-27 and p549-557 . FurtherThe aim of this study was to rationally address two major hurdles in developing a generic antigen-based immunotherapy for HBV: (i) the lack of prioritization of epitopes in further studies toward clinical implementation and (ii) the shortage of non-HLA*02-restricted epitopes.To address the first issue, we ranked all currently described HBx- and Pol-derived epitopes according to conservation and association with viral indispensability. Conservation patterns were similar to those previously reported, with the most conspicuous observation being that the spacer domain of Pol is extremely variable , 42, 73.Four Pol-derived epitopes were more frequently described in acute or resolved infection than in chronic infection. Of these, p756-764 and p573-581 scored the highest in conservation. p756-764 contains at least 1 amino acid important for viral persistence, but no functional relevance has been described for p573-581. Since the DNA sequence coding for p573-581 completely overlaps that of HBsAg, we interrogated the literature for the functional relevance of amino acids in this overlapping part of HBsAg. However, no functional association was reported 7375. Altho76–Although we rationalized to prioritize conserved peptide sequences, we reckon that regions containing prevalent sequence variation could still be interesting if immunogenicity is preserved. Novel T cell responses may arise due to cross-reaction between the variant and the original sequence , 81. Thi+ epitopes, while HBx seemed more subject to immune-pressure-induced deletion of epitopes  vaccines. SLPs can be designed to harbor both HLA-I and HLA-II epitopes and are processed more efficiently by dendritic cells than whole proteins types covering the vast majority of the HBV-infected population that target the virus where it is most vulnerable. Collectively, the results of this study provide a valuable resource to guide future development of HBV-specific immunotherapies.n = 8,127) and Pol . Positions where a gap (indicated by “−”) was most frequent were deleted, after which the dominating amino acid at each position was determined. Percentages of sequences containing the dominant amino acid were calculated as the conservation score. Combining all dominant amino acids for Pol led to the consensus sequence MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPDIHLQEDIINRCQQFVGPLTVNEKRRLKLIMPARFYPNVTKYLPLDKGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGRLVFQTSKRHGDESFCSQSSGILSRSPVGPCIQSQLKQSRLGLQPQQGSLARRQQGRSGSIRARVHPTTRRSFGVEPSGSGHIDNSASSSSSCLHQSAVRKAAYSHLSTSKRQSSSGHAVELHNIPPSSARSQSEGPVFSCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCTEHGEHHIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARLSSNSRIINNQHGTMQNLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLYTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAELLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPDRVHFASPLHVAWRPP.A frequency table was downloaded from HBVdb V42.0 , 51 baseThe resulting consensus sequence for HBx was determined to be MAARLCCQLDPARDVLCLRPVGAESRGRPLSGPLGTLPSPSPSAVPADHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETTVNAHQVLPKVLHKRTLGLSAMSTTDLEAYFKDCVFKDWEELGEEIRLKVFVLGGCRHKLVCSPAPCNFFTSA.in vitro binding assay as described below.These sequences were loaded into NetMHCpan3.0  to prediin vitro binding assay as described previously (Synthetic peptides (Peptide 2.0 Inc.) of selected potential HLA binders were used in an eviously . In briein vitro HLA binding assay were assessed for immunogenicity. Briefly, PBMCs were isolated by Ficoll  density centrifugation from buffy coats of 9 donors who had previously resolved HBV infection. Buffy coats were provided by the local blood bank with corresponding 2-digit HLA types. Four-digit HLA typing was performed for 7 out of 9 donors using the global screening array (GSA)  (Peptides with >25% binding in the tterdam) . All donP value was <0.05. For the few epitopes with a response in at least 25% of HCC patients, patient groups could not be compared due to the lack of data for CHB and acute hepatitis patients.The Hepitopes initiative previously performed an extensive literature search of the Medline and Embase databases to collect all HBV-derived HLA-I epitopes from 112 papers , 49. All55–


Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake  During synaptic transmission, the synaptic vesicle fuses to the presynaptic membrane and its content is released into the synaptic cleft. During this event, vesicular pH rises and falls again after endocytosis1, which is consistent with proton release into the synaptic cleft. Taking into account the very small volume of a synaptic vesicle (2 × 10 −20 L) and the estimated pH before exocytosis (around 5.5), it has been calculated that there is less than one free proton per vesicle3. However, upon vesicle opening a process which includes deprotonation of proteins and neurotransmitters takes place and over 50 protons are released per vesicle3. Therefore, the synaptic cleft, which is a space 50 nM wide, is exposed to changes in pH due to the release of vesicle content and also to transmembrane fluxes owing to Ca2+/H+ exchange by the plasma membrane Ca2+ ATPase4. A transient drop in extracellular pH has been reported during synaptic activity in hippocampal sections5. It has been proposed that in photoreceptor terminals the liberation of vesicular protons results in an acidification of the synaptic cleft from 7.5 to 6.9, depending on the feedback inhibition of Ca2+ channels6. This study also showed that pH oscillations depend on the concentration of protons in the extracellular solution and that acidification is insignificant in acutely dissociated terminals, demonstrating that an intact synaptic cleft is essential.The content of synaptic vesicles has been shown to be acid1, a pH-sensitive green fluorescent protein, was fused to the extracellular domain of a postsynaptic membrane protein and used in lateral amygdala pyramidal neurons to report changes in extracellular pH at dendrite synapses7. After stimulation, the transfected pyramidal neurons transiently acidified neighboring dendrites and spines, a phenomenon which was followed by a slower increase in pH. The extent of pH fluctuations depended on stimulus frequency although quantification of the changes in proton concentration was not reported. In contrast to the established dogma, Stawarski et al. recently used genetically encoded fluorescent pH indicators to demonstrate that glutamatergic synaptic clefts alkalinize rather than acidify during neurotransmission at both the mouse calyx of Held and Drosophila neuromuscular junction (NMJ)8. Therefore, the exact changes in pH during synaptic transmission are not fully resolved.Recently, Super Ecliptic pHluorin (SEP)Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to SEP1 and efficiently expressed this fusion protein in Pichia pastoris. Using this tool, we were able to detect a drop in pH in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation.Changes in pH at the synaptic cleft at the mammalian NMJ have not been documented. With this aim, we developed a probe by fusing α bungarotoxin (BTX), a neurotoxin derived from the snake P. pastoris and purified, resulting in an active protein of the expected size for de fusion ) was fitted to the data, where y0 represents the offset, ymax the dynamic range, and pK the pH where fluorescence is half maximal, a value that corresponds to minus the logarithm of the equilibrium constant for protonation9. The pK from the fit was 7.36.The sensitivity to pH of the SEP-BTX fusion protein probe was investigated by measuring the fluorescence intensity of the NMJ incubated at different pHs in a high capacity buffer (HEPES buffer 10 mM). A fluorescence vs pH curve was constructed taken the value at pH 9 as the maximal fluorescent output Fig. A,B and t+ channel blocker diaminopiridine , known to increase the amplitude and prolong the time course of transmitter release10. As a result of DAP addition, fluorescence changes were significatively stronger  on neuromuscular transmission17. Activation of ASICs requires changes in pH of 0.5 units or more, a value not reached in our measurements. One possibility is that the sensor also binds to other acetylcholine receptors located outside the synaptic cleft. Since these regions would not be exposed to the release of synaptic vesicle content, this event could mask the changes in pH reported by our SEP-BTX sensor in the synaptic cleft. However, it has been established that extra synaptic acetylcholine receptors are not expressed in normal adult mouse muscle both, by iontophoretic application of acetylcholine19 and by using labelled bungarotoxin20. Moreover, we focused the detection of fluorescent changes at the synaptic spot where only synaptic acetylcholine receptors are located. Secondly, it is worth mentioning that our measurements are highly dependent on the distance of the probe in relation with the source of the protons, which in our case is the synaptic cleft width. The high mobility of protons21 and the presence of buffers in the media could result in a very fast transient non accumulative increase in proton concentration not resolved with our detection system. Furthermore, the rapid dissipating proton concentration during the intervals between evoked transmitter release (10 ms at 100 Hz and 3.3 ms at 300 Hz) will strongly down average the fluorescent signal.Our experimental results using SEP-BTX have shown that acidification takes place at the NMJ synaptic cleft, confirming previous reports in other synapses23, we were able to show for the first time an acidification process due to transmitter release at the mouse NMJ. Synaptic cleft acidification and alkalization are probably not mutually exclusive processes. While they could operate in different types of synapses, it is also feasible that they occur at the same synapsis but with different kinetics. For example, following a rapid drop in pH, an over compensating mechanism driven by the Ca2+/H+ antiporter activity of plasma membrane Ca2+ ATPase4 and aimed at maintaining proton homeostasis could result in transient alkalinization8. Further experimental development of sensors located at the presynaptic membrane close to the vesicle release site and a fast image recording system are needed to obtain more realistic measurements of changes in the pH at the synaptic cleft.In summary, using a novel approach, in contrast to other reports showing an alkalization at CNS synapses9 in frame with BTX and followed by a TEV protease site and a 6XHis tag was subjected to codon optimization for expression in P. pastoris and synthetized by Integrated DNA Technologies (IDT). The sequence of the cloned DNA fragment, including EcoRI sites, is as follows:The DNA fragment containing SEPEcoRI site of pPic9 (Invitrogen Life Technologies Inc.) in frame with Saccharomyces cerevisiae α-factor signal sequence.5′GAATTCTCTAAGGGTGAAGAGCTTTTCACCGGCGTGGTTCCTATTCTTGTGGAATTGGATGGTGACGTCAATGGTCACAAATTTTCAGTGTCAGGTGAAGGGGAGGGTGACGCCACTTACGGTAAATTGACTTTGAAGTTTATATGCACTACCGGCAAATTGCCAGTTCCATGGCCTACCTTGGTTACTACCTTAACTTACGGTGTTCAATGCTTTTCTAGATATCCTGACCATATGAAGAGACATGATTTTTTCAAGTCAGCCATGCCAGAGGGATATGTACAAGAACGTACAATTTTTTTTAAGGATGACGGTAATTACAAGACTCGTGCAGAAGTTAAGTTCGAAGGCGACACGCTGGTCAATCGTATTGAACTGAAGGGTATTGACTTCAAGGAGGATGGGAACATACTAGGACACAAATTAGAATATAATTATAACGATCACCAGGTTTACATTATGGCTGACAAACAAAAGAACGGCATTAAAGCTAACTTTAAAATTCGTCATAATATTGAGGATGGGGGAGTCCAATTAGCAGATCATTACCAACAGAATACACCAATAGGAGATGGTCCAGTGCTGTTGCCTGATAATCACTATCTTTTCACTACTTCTACTTTATCTAAAGACCCAAACGAGAAGAGGGACCATATGGTTTTGCTGGAATTTGTGACTGCAGCCGGGATTACTCACGGTATGGACGAGCTTTATAAAGGGCACGTCGGGATAGTTTGCCATACGACAGCCACCTCCCCCATTTCTGCTGTTACCTGTCCTCCAGGCGAAAACCTGTGCTACAGAAAAATGTGGTGTGATGCCTTCTGTTCTTCACGTGGTAAAGTTGTCGAGCTGGGATGCGCTGCCACCTGTCCTAGTAAGAAACCTTATGAAGAAGTAACATGTTGCTCTACAGATAAATGTAATCCACATCCTAAACAACGTCCTGGTTCGCGAGAGAACCTGTACTTTCAAGGACCCGGGCATCATCATCACCATCATTAAATTTAAATGAATTC3′. This fragment was cloned into the P. pastoris was previously described24. Basically, pPic9-SEP-BTX-TEV-6XHis vector was linearized with DraI and used for transformation of P. pastoris strain GS115 (Invitrogen Life Technologies) by electroporation, and recombinant clones reverting histidine auxotrophy were selected on minimal MD plates 2SO4, 2% dextrose and 2% agar). Selection of clones expressing and secreting SEP-BTX was performed by transferring colonies to a nitrocellulose membrane placed on plates of MM minimal medium 2SO4, and 2% agar) with methanol as sole carbon source, for induction of the AOX1 promoter . After 4 days of growth at 30 °C, the nitrocellulose membranes were washed with distilled water and the secreted protein was revealed by western blot using a polyclonal rabbit anti-HIS antibody . For SEP-BTX production, recombinant clones were grown in 50 mL of BMGY medium 2SO4, 400 mg/L biotin, 1% glycerol, 100 mM potassium phosphate buffer, pH 6.0) for 48 h at 30 °C and 220 rpm. Cells were harvested by centrifugation for 5 min at 1500 g and resuspended in BMMY medium 2SO4, 400 mg/L biotin, 200 µM CuSO4 and 3% sorbitol) to a final OD600 nm = 10 and cultivated in 1000 mL shake flasks at 28 °C and 220 rpm. Sterile methanol  was added every 24 h to maintain induction conditions.The protocol for protein expression in P. pastoris was previously described24. Briefly, P. pastoris cultures were harvested after 4 days of induction in BMMY and centrifuged at 1500 g for 10 min. The extracellular supernatant was concentrated by ultrafiltration  and buffer exchanged to 300 mM NaCl, 50 mM sodium phosphate buffer, pH 8, containing 1 mM PMSF. SEP-BTX was purified by gravity flow Ni–NTA affinity chromatography using a “His select” nickel affinity gel , eluted with 300 mM NaCl, 50 mM sodium phosphate buffer, pH 6.5, 250 mM imidazole, 1 mM PMSF. Fractions containing SEP-BTX were pooled and buffer exchanged to 1× PBS by ultrafiltration . Recombinant SEP-BTX was revealed by western blot. Culture supernatants and purification fractions were separated by reducing 12% SDS-PAGE and transferred to 0.45 μm nitrocellulose membranes . Western blot was performed by probing the membranes with 0.1 μg/mL of polyclonal rabbit anti-HIS antibody  or a 1:1000 dilution of mouse anti-GFP antibody (Abcam) followed by 1:15,000 dilution of alkaline phosphatase-linked goat anti-rabbit antibody or goat anti-mouse antibody . Phosphatase activity was revealed by a chromogenic reaction using 5 bromo-4 chloro-3 indolyl phosphate and nitroblue tetrazolium as substrates .The protocol for protein purification from Levator auris longus (LAL) muscle of male C57BL/6 J mice (https://www.jax.org/strain/000664) as previously described25. Briefly, animals were supplied by the animal house of the FCEN-UBA. Animals were cared for in accordance with national guidelines for the human treatment of laboratory animals, similar to those of the US National Institutes of Health. All experimental protocols were approved by the Institutional Commission for the Care and Use of Laboratory Animals (CICUAL), FCEN-UBA. Animals were anesthetized with an overdose of 2% tribromoethanol (0.15 mL/10 g body weight) injected in the peritoneal cavity and exsanguinated immediately.Experiments were carried out on the left 2, 1 MgSO4, 12 NaHCO3, 1 Na2HPO4 and 11 glucose; continuously bubbled with 95% O2/5% CO2; pH 7.3 as previously described25.The muscle with its nerve supply was excised and dissected on a Sylgard-coated Petri dish containing physiological saline solution of the following composition (in mM): 137 NaCl, 5 KCl, 2 CaClLAL muscles were incubated in Ringer´s saline solution with the SEP-BTX sensor for 2 h and washed for 30 min. For each batch of SEP-BTX produced, containing 30–50 ng/µL of purified recombinant protein, preliminary experiments were done to identify the dilution (1/5 to 1/20) necessary to obtain a low background strong labelled NMJ. The preparation was then transferred to a 1.5 mL recording chamber. Experiments were performed at room temperature (20–23ºC). NMJs were visualized with Nomarsky interference optics and illuminated with a 488 nM light source .–6 g/mL  was used during the experiment.The nerve was stimulated via two platinum electrodes isolated with vacuum grease  coupled to a pulse generator . Supra threshold high frequency  nerve stimulation was applied during 250 ms. To avoid using high concentrations of the reporter and to prevent any remaining muscle contraction during stimulation, curare up to 5 × 10For fluorescent image acquisition, we used a BX51WI upright microscope with a 60X objective lens (Olympus) and an electron multiplying CCD camera , together with cell^M System Coordinator/cell^R real time controller software.The only exception is the image shown in Fig. t-test.Data analysis was done using Clampfit 10.6 , Sigma Plot 10.0, SigmaStat 3.5 and Excel 2007 (Microsoft). Average data are expressed and plotted as mean ± sem. Statistical significance was determined using paired or unpaired Student’s


FOXC2 are predominately associated with lymphedema. Herein, we demonstrate a key role for related factor FOXC1, in addition to FOXC2, in regulating cytoskeletal activity in lymphatic valves. FOXC1 is induced by laminar, but not oscillatory, shear and inducible, endothelial-specific deletion impaired postnatal lymphatic valve maturation in mice. However, deletion of Foxc2 induced valve degeneration, which is exacerbated in Foxc1; Foxc2 mutants. FOXC1 knockdown (KD) in human lymphatic endothelial cells increased focal adhesions and actin stress fibers whereas FOXC2-KD increased focal adherens and disrupted cell junctions, mediated by increased ROCK activation. ROCK inhibition rescued cytoskeletal or junctional integrity changes induced by inactivation of FOXC1 and FOXC2 invitro and vivo respectively, but only ameliorated valve degeneration in Foxc2 mutants. These results identify both FOXC1 and FOXC2 as mediators of mechanotransduction in the postnatal lymphatic vasculature and posit cytoskeletal signaling as a therapeutic target in lymphatic pathologies.Mutations in the transcription factor  The lymphatic vasculature has a critical role in maintaining tissue homeostasis by returning interstitial fluid to the venous circulation, absorbing lipids from the digestive tract, and providing a network for immune surveillance and response . MutatioFOXC1 have primarily been dominantly associated with eye anterior segment defects, cerebellar malformation, and cerebral small vessel disease. In contrast, mutations in FOXC2 have been dominantly associated with lymphedema-distichiasis syndrome characterized by failure of lymph drainage in limbs, venous valve failure, and the growth of an extra set of eyelashes (FOXC2 mutations (FOXC1 and FOXC2 are closely related members of the forkhead box (FOX) transcription factor family with nearly identical DNA binding domains, similar expression patterns in mesenchymal tissues during development, and essential roles in cardiovascular developmental processes . Mutatioyelashes . Work fryelashes . Additioyelashes . Furtherutations . HoweverFoxc1 postnatally impairs valve maturation, while Foxc2 deletion impairs maturation and induces valve degeneration, as previously described  in vitro, which also improves LEC barrier integrity in vivo, while valve degeneration is partially rescued in only Foxc2 mutants. Finally, via generation of transgenic mice that express Foxc2 within the Foxc1 locus, we show Foxc2 is capable of functionally substituting for Foxc1 in lymphatic development and maturation. Together, our data show a complementary role for FOXC1 in addition to FOXC2 as key mediators of mechanotransduction in the postnatal lymphatic valves and implicate new mechanistic targets for therapeutics in the treatment of lymphatic-associated diseases.Here, we report an essential role for FOXC1 during lymphatic valve maturation and maintenance. Detailed comparison of FOXC1 and FOXC2 expression and roles in lymphatic valves suggests some overlap with a broader importance for FOXC2, but more subtle, key contribution for FOXC1. In mice, endothelial cell (EC)-specific deletion of escribed . HoweverOur group previously reported that FOXC1 and FOXC2 expression co-localizes with PROX1 in lymphatic valve-forming cells at E17 and later at P3 . HoweverFoxc1 during murine embryonic development impairs lymphatic valve maturation (flFoxc1 mice (ERT2Chd5-Cre mice (Foxc1 mutant (EC-Foxc1-KO) mice. Tamoxifen was administered from P1-P5 to induce Cre-mediated recombination and we confirmed deletion of Foxc1 via qPCR analysis of isolated CD31-positive cells from hearts of P6 individuals and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  protein ligand fibronectin-EIIIA to regulate the formation of the ECM core of valve leaflets  mice and administered tamoxifen postnatally. PROX1 immunostaining showed no significant differences in total valve number of LEC-Foxc1-KO mice  mice and administered tamoxifen from P1-P5 with inactivation confirmed by qPCR in ECs isolated from P6 hearts and by immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  mice and administered tamoxifen from P1 to P5 with inactivation in compound mutant individuals verified by qPCR and immunostaining of the mesenteric lymphatic vasculature with antibodies against FOXC1, FOXC2, and VEGFR-3  developed severe chylous ascites  compared to controls   , indicatFOXC1 knockdown in LECs impairs cell adhesion, we slightly modified our protocol: LECs were first seeded on fibronectin-coated surfaces, then transfected with scramble control, FOXC1, FOXC2, or combined FOXC1/FOXC2 (50%:50%) siRNAs and kept under static conditions for 2 days. Given that FOXC2 inactivation and exposure to OSS in LECs results in increased actomyosin contractility  suite of tools (https://ecrbrowser.dcode.org) -phosphorylated myosin light chain (pMLC) signaling pathway has been well established as a regulator of cytoskeletal contractility mechanisms . FurtherDKO mice . Recent DKO mice . Our RNADKO mice  revealed embryos . Further embryos  were notpression . Becausepression  and our of tools  was usedof tools  in activof tools . Then, wode.org)  tool to PRICKLE1 , ARHGAP2ARHGAP23  loci. ToPRICKLE1 , ARHGAP2P23 loci . We founin ECR-1 . Additio ECR-1–2 . Finally23 locus .ICAM1 that was not predicted to bind FOX transcription factors by in silico analysis  allele, in which the Foxc1 coding region has been replaced with the cDNA coding (from the start codon to the stop codon) for Foxc2    and FoxCrostomes . Althougnditions . TherefoPecam1 or Sdc4 and LEC-specific deletion of Cdh5 in vivo resulted in impaired valve morphogenesis phenotypes and poor orientation and elongation of LECs with the direction of flow in mesentery collecting vessels  for Foxc2 by using restriction enzyme digestion and PCR cloning. To facilitate the elimination of the selectable marker gene rNeo in mice, we introduced the ACN cassette flanked by two flox sites  mice , c2/+Foxc1 , and c2/c2Foxc1  individuals.SalI-linearized targeting vector (100 μg) was then electroporated into TL1 ES cells (129S6) as described . The cel/+) mice  were theGenotyping of mice for use in analysis was performed by Transnetyx Inc  using real-time PCR.Dissection, immunostaining, and imaging of the lymphatic vasculature in mesentery tissue from 4 week old C57Bl6 mice for whole-mounts was performed as previously described . BrieflyThe lymphatic collecting vessel vasculature of neonates was analyzed by whole mount immunostaining of mesentery tissue harvested from pups at the indicated time. Briefly, mesentery tissue was dissected from the intestinal tract, laid out in plastic dish and left until it was firmly attached. Following fixation with 2% PFA in PBS, tissues were washed with PBS, then permeabilized and blocked in PBS solution containing 0.5% BSA, 5% serum, 0.3% Triton X-100, and 0.1% Sodium Azide. Tissues permeabilized/incubated with blocking buffer were then incubated with primary antibodies listed in Imaging was performed using a Zeiss AxioVision fluorescence microscope and Zeiss Axiovision software, Zeiss LSM-510 Meta, LSM-800 and LSM 880 confocal microscopes and Zeiss Zen Blue acquisition software, or using a Nikon A1 Confocal Laser Microscope System NIS-Elements software. Images were processed with Imaris and Adobe Photoshop software. Imaris colocalization function was used to produce pictures showing p-MLC2 , vinculiPpia) or 18S was used as an internal standard for mRNA expression. Primer sequences are provided in Hearts from neonatal mice were digested in collagenase Type I solution (2 mg/mL) for 40 min at 37°C with gentle agitation. Cells were then filtered through a 70 μm cell strainer and the pellet was resuspended in Buffer 1 . The cell suspension was then incubated with magnetic Dynabeads (Invitrogen) pre-coated with CD31 antibody to isolate the endothelial cell population. After several washes with Buffer 1, RNA was extracted from endothelial cells using RNA STAT solution (Tel-Test) followed by phenol-chloroform treatment. Extracted RNA was subjected to DNAse I treatment and concentration was determined using a NanoDrop machine (Thermo Scientific). cDNA was synthesized using an iScript reverse transcriptase kit (Bio-Rad) according to the manufacturer’s instructions. Triplicates of cDNA samples for qPCR analysis were performed using a Fast qPCR machine (Applied Biosystems), Fast SYBR reaction mix (Applied Biosystems), and gene specific primer sets. Peptidylprolyl isomerase A  coated with 40 µg/ml human fibronectin, cultured for 24 hr and then subjected to LSS (4 dyn/cm2), OSS (4 dyn/cm2 and flow direction change every 4 s) using Ibidi Pump system, or kept in static conditions for an additional 24 hr prior to fixation, immunostaining, and mounting using Ibidi Mounting Medium (ibidi GmbH).Flow experiments with cultured LECs were performed as described previously . Brieflyhttps://genome.ucsc.edu/ENCODE/)(2012). Putative sites in the human genome were then searched against the mm10 mouse genome using the ECR Browser (https://ecrbrowser.dcode.org) (Putative FOX-binding sites were determined first by using the Hypergeometric Optimization of Motif EnRichment (HOMER)  suite ofode.org)  and rVishttps://www.promocell.com/product/human-dermal-lymphatic-endothelial-cells-hdlec/) were cultured and used according to the manufacturer's protocol. The cells were cross-linked with 1% formaldehyde, followed by sonication. The sheared chromatin was immunoprecipitated with dynabeads  conjugated with anti-FOXC2 antibody , anti-FOXC1 antibodies , or control IgG . DNA extraction and PCR were performed as previously described  from juvenile foreskin  (escribed  with priPRICKLE1 ECR-1.>hg19 chr12:42876176–42876523TGTTGGCTCCTGAACAACACTCCTCTTTGTGAAACTTACAGCACCCATTTGAAACAAGTTTCCAGAGAAAACACTTCAAGAAAGTGTTTGAGAAGTCACAAGACTCGAGTTGTAAAAACAAATTCCACACATAGCCTGGCTTATAAGGACACAGACTAACACCACACAAGGCGGTGCTCTAATGAGCCCATTATTTTCCATAATGGGGGATGCAGATATTTTCTCAAAATCGTGTTCTCCTCAGTCTTCTATTGATTTTTTGGATTTCTATTTTCAACAGTGGCCCGAGGAAACGGCAGCCAGACTTGACTCCAATGTACACACAGACTCAGGTTTCGCCCCGTCACCPRICKLE1 ECR-2.>hg19 chr12:42878066–42878454GAGCACCTACCGCCGCCCGCCCGCTCCATTCTCCCGAGCCCAGTGAGTGAAGCCGCTAAGATGCAAATACCCTAGGACGCTTATGTAAACTTCCCCCTCCCGCAGGTGCACGCGCGGGCCACGAAACGCTGGGAGAATATGAAAGGCCACCTCTTAAAGAAATCATCTCCACTCTGCCCATAACAATGATGTCAGCAAATGGCACATTTAAGCAAGTTCTCACTTAGAAGGGCTCATTAGCATATGAATTCTCTTAGGACTTTCCCTGCATTTCGGAGTGATTCCTACTGCTTAGCGCAGGAGATTTATTTTTATCAGTAAATAACAGCAAAGAAAAGGAGCCAGGTCACGCGATGTACTAACTCAAGTACACTACTGAGAGTTTTTACPRICKLE1 ECR-3.>hg19 chr12:42879624–42879943AGTTACTTGTAAATACGTTTTGTTATATTTTCAACAGGTACTGTCATGGTTTATTACCCATTGTAAGCGTTTTTAGTGATGAAGCAGTGTGGAGACAGTTGCAAGCTTCCTACAAAGGTTCAATCTCAATGAAACAGACTTTAGTCTGGTCTGAAAGGGGTTGTTATTGTCAAGGGTGAACTTATGCAGAATGGAGAGCAAGGCCCCCAACCAGCATCCTTTTGTTTCAGCCAGGTGGAATATTCATGCTTGCAGATCACACTTTAGGGGCCAGTTGAGAAAGGAAGCCCAAAAATTCACAGGGCTTGGTTCCCTCGCTCPRICKLE1 ECR-4 and ECR-5.>hg19 chr12:42981201–42982292TACATTAGCCATGACTTATTAAACTTGGAGATTTTCAAGTTCATCAGCAAGTGTGCAAACCCTTAACTGTGGGTAACATCCATTTATTTGTAGCACCTTTCGGTTTTAATATGTAGAGCACATGCATTGTTAACCTCTAAATCCTTTGT(ATAAACA)TTTCTGGAAGAGCTGGTAAAATATCTCCTTCTGTGTTTCCTGACTCGCCAGTTGATGGCATTTAGAAACCCTCTGGTACCAGCAGGTGCTGTATTTTGCTTTCTTCAGGCTCCAGCTGGGCTACAATGACAGATTCCTGTCCCAGGCCAAGCCTAGCCACCAAGGCTAGGACCACATTGGAGGCAAACTGAACCAGGCTCCACCAGACAGCAAGATGAATGGCTGCTGTTTAAGTTTAAAATCCCCTGGTGGGA(GTAAATATTGTTCCAGAGAAAAGCCTTGACAAATA)CTGCGTCATCCTTACAGAACTGTCTTGATTAAAGCAGAATCTTTGGATTAAGTTGATGCTCAATTCAAAATGTATCTATCTTGCTGTCATGGGATTTTTTTTTGTTTTTTTTTTCCTTTCTAGAGTCTGAAAACAGACATAATGTTGGGGACGGTCAAACAAGGCTGCCGGCTCCCAAGGGGCTAGAGTCCACTCCTGATAATAGAAGGCGGCTGAACACTGACACTTCACTGAGGATAATGGAGACAGCAAAGGCTTAGTGGGAAAGGGCCAGTTGTCACCTAAGTGACAGGCAACAGCTGAGCTCACACATCTGGAGCCGGACTACGGCAAACATTAGCAACCCTCACCAGTCTACACCTTGGGCCTGTCTGAAAAGACAGATGGAAGTTCCCTCTACTCCTAAAGTACATTAAAAAATGTCTGATGGTGAACCACATCAATTATATAACATCAACTGCAGGCACAGCCTTCCAAAGTACTGATTAAGACGAGGCAGTAGACAACACTGTATGCATGAACAGATACAAGATACCATTTCAGTGATTTGTCATTCATAAAACTTATCCTAAAAGACACATATACATGCATCCATTTGATAGCACAAATGCATGTTAACTCTGCAGGAGAGGCAGATTTTTACATGTPRICKLE1 ECR-6.>hg19 chr12:42982328–42982743CCTTTAGGCTGGTTCCCGTGGTGTGTTTGCCTTGTTCATGGTCTCAGTTCTGCCGCTGATACCCTTTTAAAAATCAGCAACCAAACGCGTTCGGCTTGTGATCCTGAACCCCCTTAGGCAAGCTGGAACTAAGCGTGATGCAGCCGTCCTCCCTCTCTCCCAACCCCCAACCTCGTTCTTCAGCCTCCTGAAGACAATCTGTGAACAATTTTCCCAAAGTCCCAAGAATAACACAGCACTGCCAATAGTCACTGGCGATGCCGTTTGTTTTTCTTAGAGGGTAATGAAAATTTAACAGCTTTCTGCTGCATCCTGAGTCCCGCTCCTAATAACTATTAACATGCCTAGTTTCTTCAACTTTTTCTACCTCAAGAGAGGAAGACGCTCCCATTTTTTCCCTATATCTGTGCTACATARHGAP21 ECR-1 and ECR-2.>hg19 chr10:25009076–25009514CTGTCATTGTTAATAAAAGCCAAGTGTGCAACAAACTGGAAATACTGCTTGCTAGCAAGGACAAGATGTGTCTAAATTCTTGGTTTCAGGACATCTCTTAAATGACCAAAAAAAAAAAAAAAAATCAAATAACATTAGTTCTT(GCAAACAGAATGCAAATACAATGCTAATTAAAGTATTCACAAGACAATGACAAATA)AGGCTTCAGGACCACAATACATATTATTATGTAACTGCAATACACATTAAGCAATCACCAGGTTGCAGGTAGGCCTTCCAAAAGGAGTTATTATGGTTTACCGTGATCAGAGGATTGTGGTGTTCCACTTAATCATGCTTTTGCCTGCAAGCAGGTGTTTACAGATGTCAAAAAGTAAAACACTGATTCTAAATGTAAACCTACAGTTCTGCCTAATAAATTGTACAGTAATAGCACACARHGAP21 ECR-3.>hg19 chr10:25017295–25017667CTATATTAATAAAAACTACAAGAAAGCTTTATACACTAAATCTAGGCAAGACATTTATGAAGATGAGAACTGTATCCTTAAAAGGTAAGTGTTGGCTTTGCTAATGACATAATATTGTTTTGGTGAACCAATATCAAGGGAAAAAATGTCAAAGCCAAAAATAGAGGCAAAGTATCCCAGCCCCTGGTGTGGAAGGCCATTCTATGATAATCTATGAATGATTTCTACTCTGAATATGTTAACAGAAGCTGGCACATCTGAGAAGCACAAGTGTTTGCTAGTGAATCCACAAATGAAATTTGCAATTTGGGTTGAAGCCTTTGCAAAACTACGTTAAGACCAATAGCCCTCAGAAGAGTAAGGGGTTTTGTTTARHGAP23 ECR-1.>hg19 chr17:36585363–36585767TTCTAGACACAGGCCCAGGACCCCGGGCTCTGCCGGCGAGGCTGCCCTCCCCTCTGCCCTCTCCGACCGGCTGTGGGTGGGTCAGAGCGCGGGGTGCCAGGGGCATTACTCAGCGCTGGGCTGCTCTGCCTGGGTTCTTTCATCTGCCAGCTGCTGAGGCTGGGGAGGGGCCAGCAGGGGCCTCCCAGCCCCATCCCCCCATCAGGGCCATTCCCTTACCTCTGAGCCTGGCTGCCCGCCCTGCAGGAGCCCCCCAGCAGGCCTCCCTGCTCCTAAGTTGAAGGGTTGAACACTGTCAGGCCAACAGTTTCCCTGAGCTCGGAAAAGAAATTCCCCGGGGTCCAGGTTGAGGTCAAGGCCAGGGCTGAGGCCTGTTCCTCTTTAGACAGGGCTGAAAGACTTGGGTo identify lymphatic valves, mesentery tissue was stained with PROX1 antibody and areas of high expression were quantified as mature (visible leaflets) or immature (no visible leaflets) on four lymphatic collecting vessels per individual. Total number of valves were determined and percent of mature valves was normalized to the total counted per individual. For assessment of apoptosis in lymphatic collecting vessels, mesentery tissue was immunostained with PROX1 and active caspase-3 antibody and the percentage of PROX1/caspase 3-positive LECs was quantified from 20X high-power fields generated from confocal z-stacks using Fiji software to determine the total number of LECs per field using thresholded PROX1 immunostaining. Quantification was completed from three biological replicates.in vitro quantifications, relative nuclear intensity levels were measured using Fiji software. Nuclei were considered as regions of interest from thresholded Hoechst staining pictures. FOXC1 or FOXC2 intensity was then measured in each nuclei using RawIntDen function. For quantification of F-actin and p-MLC2 area per cell, LECs were fist manually segmented and defined as individual regions of interest, then F-actin (or p-MLC2) staining was thresholded using similar parameters for all pictures and F-actin+ (or p-MLC2+) area was measured per each region of interest. Quantification was completed from three independent experiments.For in vivo experiments was performed using GraphPad Prism v5 or v8. P values were obtained by performing a 2-tailed Student’s t test or one-way ANOVA and Tukey’s test. Data are presented as mean ± standard deviation of representative experiments from at least three biological replicates. For quantification of apoptosis in LECs of compound Foxc1; Foxc2 mutant mice, the ROUT method with Q set to 1% was used to identify outliers in the data set that were then excluded from statistical analysis using student’s t-test. P values less than 0.05 were considered statistically significant. Statistical analysis for in vitro experiments was performed using GraphPad Prism v8 to perform mixed-effects analysis and calculation of correlation coefficients. P values less than 0.05 were considered statistically significant for mixed-effects analysis.Statistical analysis for All procedures and animal studies were approved by Northwestern University’s IACUC or by the Animal Ethics Committee of Vaud, Switzerland. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study provides new insight into the mechanically regulated activation of, and distinct roles for, FOXC1 and FOXC2 in lymphatic vessel valve development. The identification of unique roles for these closely related transcription factors is intriguing and will pave the way to a deeper understanding of the transcriptional mechanisms regulating their distinct activities. The study also implicates FOXC1 as a gene that may potentially underlie primary lymphoedema.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Natasha L Harvey as the Reviewing Editor and Reviewer #2, 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: Mark Kahn (Reviewer #3).Thank you for submitting your article "Shear stimulation of FOXC1 and FOXC2 differentially regulates cytoskeletal activity during lymphatic valve maturation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Here, Norden, Sabine and colleagues dissect the roles of transcription factors FOXC1 and FOXC2 in lymphatic vessel valve development, revealing that FOXC1 primarily controls focal adhesions in lymphatic endothelial cells, while FOXC2 primarily controls adherens junction integrity. The work builds on previous studies by the authors that have demonstrated (1) the importance of FOXC2 and shear stress for lymphatic vessel valve development and , (2) important roles for both FOXC1 and FOXC2 in embryonic lymphangiogenesis via regulation of RAS/ERK signalling . Aspects of this current study overlap with some of the authors' prior work and this should be clearly articulated in the manuscript where relevant. Distinctions and extensions of the work should also be clearly articulated. There are key novel and interesting findings described in this manuscript that address important questions regarding the distinct mechanisms by which FOXC1 and FOXC2 activity are regulated, together with the mechanisms via which these transcription factors regulate cytoskeletal architecture.Essential revisions:Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90°) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as α5-laminin and/or α9-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3—figure supplement 1?1) The phenotype caused by early postnatal deletion of Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image  aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: "Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration." The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1;Foxc2 double mutants. Figure 3—figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3—figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3—figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example – Figure 3—figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3—figure supplement 3H show low PROX1 levels overall in Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If 5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?6) Figure 1—figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.7) Figure 3—figure supplement 2: A lower power image depicting the broader profile of α9-integrin expression throughout multiple valves would enable the reader to determine how α9-integrin deposition is affected more globally in EC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.Foxc1c2/c2 mice. What is the remaining signal in FOXC1 stained c2/c2Foxc1 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in  Essential revisions:1) The phenotype caused by early postnatal deletion of Foxc1 seems modest, and is characterized by a larger proportion of immature valves in mesenteric lymphatic vessels. It thus becomes important to know how the authors define immature valves. It is not obvious why those shown in Figure 2E, F are defined as immature, and not representing valves that are imaged from a different (90 degrees) angle compared to valves that show the typical v-shape. Staining of the valve leaflets would demonstrate this more clearly. The inclusion of staining for markers of valve maturation such as α5-laminin and/or α9-integrin would help to demonstrate this point. To more convincingly demonstrate that Foxc1 modulates the Foxc2 KO phenotype, side-by-side comparison of Foxc2 single and Foxc1;Foxc2 double mutants should be shown. Of note, deletion of Foxc2 seems to be incomplete . Considering that the authors show that Foxc2 can compensate for Foxc1 function, Foxc1 deletion in combination with the incomplete Foxc2 deletion may just decrease total Foxc signaling sufficiently but not necessarily indicate distinct functions of the two genes as proposed. The question is whether Foxc1 deletion would affect the phenotype of Foxc2 mutants if Foxc2 is efficiently deleted. Why is Foxc1 staining apparent on the cell membrane and present in the Foxc1 mutant in Figure 3—figure supplement 1?Foxc1, Foxc2, and compound Foxc1; Foxc2 mutant mice. To address the first part of this comment, we have included new representative images of α9-integrin/VE-Cadherin immunostaining of lymphatic valve regions side-by-side with PROX1/CD31 immunostained lymphatic valves in Figure 2. Inset images of only the α9-integrin channel more clearly show the presence of mature, intraluminal bi-leaflet valve structures in Control and EC-Foxc1-KO mice as well as valve regions that are considered immature that are not characterized by a distinct intraluminal leaflet structure by α9-integrin immunostaining. In EC-Foxc2-KO mice, we have observed that overall, α9-integrin expression is reduced compared to littermate control mice. However, there are regions characterized by the presence of intraluminal leaflets, as depicted by PROX1, CD31, and VE-Cadherin immunostaining, that are shortened in length. We describe these observations in the subsection “FOXC1 and FOXC2 are required for postnatal lymphatic valve maturation and maintenance”.The authors thank the reviewers for this comment and careful assessment of our described phenotypes in our Foxc2-KO mice and EC-Foxc1; Foxc2-DKO mice. Figure 2H and N depict representative images of a mesentery collecting vessel immunostained with PROX1/CD31 and Figure 2R and T depict 10X power images of collecting vessels immunostained with PROX1 to show the difference in phenotypes more clearly.To address the second part of this comment, we have also updated Figure 2 to include representative images showing side-by-side comparison of EC-Foxc1-KO, EC-Foxc2-KO, EC-Foxc1; Foxc2-DKO mice and respective littermate controls and included representative images in Figure 2—figure supplement 1. In Figure 2—figure supplement 1G, we show that FOXC2 expression is markedly reduced in EC-Foxc2-KO mice and FOXC1 expression is reduced compared to littermate controls, likely as a result of perturbed flow contributing to the reduced induction of FOXC1 expression. In contrast, Figure 2—figure supplement 1E shows strong reduction of FOXC1 expression but no discernible difference in FOXC2 expression. Thus, the maintenance of FOXC2 expression in EC-Foxc1-KO mice may in part explain the differences in phenotype severity observed in Foxc2 mutant mice compared to Foxc1 mutant mice. We have revised our Discussion section (second paragraph) to note this important difference. The difference between our qPCR data, depicted in Figure 2—figure supplement 1B, and our immunostaining evidence is likely attributable to the fact that CD31-positive cells were isolated from cardiac tissue to use for qPCR analysis. It is likely that FOXC2 expression levels are different between lymphatic endothelium in mesentery collecting vessels and the cardiac vasculature. Thus, this may explain the differences in deletion efficiency. The repeated immunostaining of collecting vessels with FOXC1, FOXC2, and VEGFR-3 antibodies also now shows clear expression of FOXC1 in the nucleus of both lymphatic endothelial cells and smooth muscle cells of arterioles. As shown in Figure 2—figure supplement 1E, G, and I, there is no membrane signal detected. We suspect that there may be some cross-reactivity with our secondary antibodies used for the immunostaining combination of FOXC1 and VE-Cadherin, thus warranting our use of VEGFR-3 antibody to address this issue and verify the reduction of FOXC1 expression in the endothelium of collecting vessels.To address the third part of this comment, we performed immunostaining of FOXC1, FOXC2, and VEGFR-3 in mesentery collecting vessels of EC-2) The conclusion 'Cell elongation and junctional integrity is markedly impaired in compound EC- specific Foxc1; Foxc2 mutants resulting in increased apoptosis in lymphatic collecting vessels' requires more supporting evidence. We see one image  aiming to support the claim that cell elongation and junctional integrity is impaired but it is not clear how representative this image is. It is surprising that such a high number of apoptotic cells is detected in control vessels, drawing into question the specificity of the caspase-3 staining, and whether the signal is indeed detected in the LECs. No evidence is provided for cell elongation defects leading to increased apoptosis. This statement should also be demonstrated: "Of note, apoptotic bodies were more frequently observed at branched areas of the lymphatic collecting vessels potentially indicating areas of valve degeneration." The authors should expand on the cellular mechanisms underlying the presumed rupture of lymphatic vessels and accumulation of chylous ascites in EC-Foxc1;Foxc2-DKO pups. Ultrastructural analyses could be informative here.Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels’ as the previous statement was not accurate. Our group previously reported that valves of LEC-Foxc2-KO mutant mice were characterized by increased apoptosis and apoptotic cells were often arranged in doublets with symmetrically organized PROX1-high apoptotic bodies, suggesting that apoptosis may occur in dividing cells. Additional evidence identified that half of the dying cells in LEC-Foxc2-KO mice were PROX1/Ki67-positive, demonstrating that valves of LEC-Foxc2-KO mice were characterized by abnormal activation of cell proliferation and increased cell death. This phenotype was associated with improper activation of TAZ signaling in the absence of FOXC2 . We have included a summary of this finding in the subsection “Cell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutants and is accompanied by increased apoptosis throughout lymphatic collecting vessels”. We then sought to characterize junctional integrity and apoptosis in compound Foxc1; Foxc2 mutant mice to assess if there was a synergistic effect of inactivation of both Foxc1 and Foxc2 in the lymphatic endothelium.We thank the reviewers for this helpful comment and careful critique of our data. We have revised our conclusion to more accurately state, ‘Cell elongation and junctional integrity is markedly impaired in compound EC-specific Foxc1; Foxc2 mutant littermates that show apoptotic bodies are in closer proximity to branched regions.To further address this reviewer comment, we carefully re-evaluated our caspase-3 immunostaining and repeated analysis to characterize the percent of PROX1/cleaved caspase-3 positive lymphatic endothelial cells in 20X high-power field images. Figure 2—figure supplement 4I-K has been updated to include new representative images and quantitative analysis. In addition to representative images of maximum intensity projections of PROX1/cleaved caspase-3 immunostained collecting vessels, we also show panels of z-sections from selected regions in higher magnification where PROX1/cleaved caspase-3 positive cells were identified. We also now show images from two compound 3) Inconsistent quality of staining between different experiments and even panels in the same figure (as if staining and imaging is done at different time points and not using littermate mice) make assessment of the phenotypes difficult, especially when a single high magnification image is provided as a supporting evidence. For example – Figure 3—figure supplement 2 VE-cadherin staining, Figure 2E-F CD31 staining. Figure 3 and Figure 3—figure supplement 3H show low PROX1 levels overall in Foxc1;Foxc2 double mutants. Figure 3—figure supplement 3D shows instead extremely high PROX1 levels, along with 'discontinuous junctions', making it difficult to judge if this is a representative image. The junctional phenotype presented in Figure 3—figure supplement 3D is indeed dramatic , but the phenotype is seemingly different in Figure 3—figure supplement 2D where the phenotype is not obvious at all. 'Discontinuous junctions' in the Foxc1;Foxc2 double mutants should be demonstrated more convincingly.Foxc1; Foxc2-DKO mice more clearly depict that collecting vessels in these mice are characterized not only by discontinuous junctions, but the increased presence of overlapping junctions as well. We have also generated videos of 3D reconstructions of the collecting vessels depicted in Figure 2—figure supplement 4A-H (Videos 1 – 4), characterizing this observation.We thank the reviewers for this helpful comment and careful critique of our representative images. To address the first part of the reviewer comment, we have repeated immunostaining of tissues and re-evaluated the post-processing of our representative images for several figures to address issues with inconsistencies. This includes new images for PROX1/CD31 immunostaining in Figure 2C, E, I, and K and new images for PROX1/VE-Cadherin immunostaining in Figure 2—figure supplement 4A-H that now show consistent PROX1 expression levels with representative images in Figure 2. Additionally, our new representative images for Figure 2—figure supplement 3E-H and Figure 2—figure supplement 4A-H show consistent VE-Cadherin expression levels among different tissue samples. Furthermore, our new images of VE-Cadherin immunostaining in EC-4) The authors should provide further evidence to demonstrate how relevant the described in vivo phenotypes are to those described in vitro. If Foxc1 and Foxc2 have distinct functions in regulating the actin cytoskeleton, it is not clear why Foxc2 is capable of functionally substituting for Foxc1 in vivo.Foxc2-KO and EC-Foxc1; Foxc2-DKO mutants in Figure 2—figure supplement 4A-H that shows differences in the presence of discontinuous junctions in collecting vessels of EC-Foxc2-KO mice, but both discontinuous and overlapping junctions present in collecting vessels of EC-Foxc1; Foxc2-DKO mice. This new data recapitulates our in vitro evidence depicting the same observations in FOXC2-KD and FOXC1; FOXC2-KD cultured lymphatic endothelial cells as represented in Figure 4—figure supplement 1, thus providing additional support for the relevancy of our in vivo and in vitro observations. In regard to the second part of the reviewer comment, although FOXC1 and FOXC2 are functionally similar, as characterized by their similar DNA-binding capacity, their transcriptional targets are likely to be different under oscillatory or laminar shear stress. For example, our group previously reported that loss of FOXC2 leads to distinct phenotypes in lymphatic endothelial cells under oscillatory shear stress and laminar shear stress . Thus, as FOXC1 is induced only by laminar shear, but FOXC2 is induced by both laminar and oscillatory shear, their downstream targets are likely different within the collecting vessels of lymphatic endothelial cells. We have also updated the fourth paragraph of the Discussion section to elaborate on these differences.We have now included new representative images of VE-Cadherin immunostaining in EC-5) Previous studies have demonstrated that FOXC2 is regulated by oscillatory shear forces in LECs, and that such forces likely play a key role in valve development. In Figure 4 the authors use in vitro flow studies to conclude that FOXC1 expression is regulated primarily by laminar shear while FOXC2 primarily by oscillatory shear. However, the endothelial cells shown in Figure 4A do not appear aligned with flow as would be expected for laminar shear and the authors fail to identify a clear mechanism that would account for such a distinct regulation. This theory appears to rely primarily on antibody detection of nuclear FOXC1 which is not as robustly detected as FOXC2. In vivo studies, especially Figure 10, suggest that these transcription factors are functionally redundant at the protein level as replacement of FOXC1 with FOXC2 does not impair valve maturation. Much of the data shown are carefully analyzed, but these findings and their connection to where FOXC1 is best detected in vivo in the valve tip seems like an over-reach. More specific mechanistic data identifying how such remarkable distinctions in shear regulation are achieved should be provided.As indicated in the Materials and methods section, the flow experiment presented in Figure 3  was run for only 24 hours (subsection “Cell transfection and immunostaining”). This shorter time – 24-hour flow instead of 48-hour flow presented in our previous publications  – is not sufficient to drive an important cell elongation in the direction of flow. In In addition, we also provide the reviewer with the quantification of length:width ratio for static, LSS and OSS conditions presented in Figure 3, which shows that there is a significant elongation of cells under LSS in average . To clarThe observation that FOXC1 and FOXC2 expression are already different in LECs after only 24 hours under laminar shear stress is interesting as it suggests that FOXC1 upregulation by laminar flow is an early response of LECs to directional flow. How expression levels of FOXC1 and FOXC2 are distinctly regulated by the directionality of flow is a very interesting question that addresses the more general one of how LECs sense flow direction and are able to respond differently to LSS and OSS. Answering such a question would require further investigation and, in our opinion, is beyond the scope of this paper in which the central message is that each factor is differentially regulated by flow and plays slightly distinct but complementary roles in regulating the cell cytoskeleton in response to shear stress.6) Figure 1—figure supplement 1: What is the level of FOXC2 protein present in LEC of EC-Foxc2-KO compared to mRNA? Foxc2 mRNA appears to be reduced by approximately 50% compared to control EC Foxc2 levels. Does this level of reduction in EC-Foxc2-KO animals explain the partial penetrance of the phenotype of chylous ascites?Foxc2-KO mice compared to littermate controls. We also suggested that the differences observed in reduction of mRNA levels compared to what we observe in regard to protein expression may be attributable to the different vascular beds analyzed. The difference in the penetrance of the phenotype of chylous ascites in the current study may be attributable to both the timeline of experimental analysis as well as the differences in Cre-drivers used. In our group’s previous study, tamoxifen was administered to ERT2; Foxc2fl/flProx1-Cre mice beginning at P4 with approximately 50% of mutants developing chylous ascites 4 days after tamoxifen administration and nearly all mutants presenting chylous ascites 6-8 days after tamoxifen administration . In the current study, tamoxifen administration started at P1 and we primarily investigated tissues at P6. We believe it is likely that our ERT2; Foxc2fl/flCdh5-Cre mice would present chylous ascites at later time points.As previously described in our response to reviewer comment #1, Figure 2—figure supplement 1F and G show a marked reduction of FOXC2 protein within collecting vessels of EC-7) Figure 3—figure supplement 2: A lower power image depicting the broader profile of α9-integrin expression throughout multiple valves would enable the reader to determine how α9-integrin deposition is affected more globally in EC-Foxc2-KO compared to EC-Foxc1;Foxc2-DKO mice.Foxc2-KO mice at P6, but the collecting vessels of EC-Foxc1; Foxc2-DKO mice are absent of α9-integrin-positive valve regions.We thank the reviewers for this suggestion. Figure 2—figure supplement 3A-D now depicts 10X low power images of α9-integrin/VE-Cadherin immunostaining to show that a few, α9-integrin-positive degenerating valve regions are detected in collecting vessels of EC-8) Figure 6: How do the changes in Prickle1, Arhgap21 and Arhgap23 levels compare to other gene expression changes in LEC-Foxc1;Foxc2-DKO LEC? It would be informative to provide more detail as to why these three genes were selected for further analysis. Were they the only genes involved in RhoA/ROCK signalling that were changed in expression? It would also be informative to know whether RAS/ERK signalling is affected in mutant LEC at this stage of development, given the authors' previous work which demonstrated regulation of this key pathway by FOXC1 and FOXC2 in embryonic LEC.Foxc1; Foxc2-DKO mice and littermate controls in panel K. Figure 5—figure supplement 1K now shows the changes in expression for RhoA, Rock1, and Rock2, and several GTPase activating proteins associated with regulation of RhoA signaling and endothelial barrier function and lumen maintenance . Here, we see that there is a modest, yet significant increase in Arhgap18 expression and a significant reduction of Arghap20, although its expression was generally lower compared to other GAPs. Because of the previously reported evidence of the formation of a physical complex between Prickle1 and Arhgap21/23  and our observation that all three genes were significantly downregulated in our RNA-seq analysis, we focused our investigation on these putative downstream targets. In regard to changes in RAS/ERK signaling, we attempted to perform whole-mount immunostaining of pERK using the same antibody utilized in our group’s previous study  but we were not successful in identifying a positive signal in control or mutant lymphatic valves at P6, testing the antibody at various concentrations. While we are interested in characterizing whether RAS-ERK signaling may be affected in our mutants during postnatal development in other lymphatic vascular beds, the authors believe that this is not of focus for the mechanism described in this study and is beyond its scope.We agree with the reviewers that providing additional details regarding the choice to focus on Prickle1, Arhgap21, and Arhgap23 would be helpful. We have now updated Figure 5—figure supplement 1 in the revised manuscript to include additional data from our RNA-seq analysis for LECs isolated from the dorsal skin of embryonic LEC-9) Figure 6D: Additional controls are required in which ChIP of both FOXC1 antibodies at a negative control region of the genome (e.g. an enhancer region demonstrated or predicted not to bind FOXC1) is assessed. This should demonstrate the selectivity of FOXC1 antibodies in terms of their specific versus non-specific/background binding to DNA and may help to explain the distinct results observed with each antibody in different regions of the genome.ICAM1 promoter that is not predicted to bind to FOX transcription factors. Panel J then shows data from three separate experiments showing no detection of band signals from ChIP with our FOXC1 or FOXC2 antibodies. Within the text of the manuscript, we have also included relevant literature that has identified variations in immunohistochemistry related work for antibodies recognizing the same immunogen peptide that are dependent on the source of the manufacturer. This information is included in the second paragraph of the subsection “FOXC1 and FOXC2 regulate LEC expression of negative RhoA signaling regulators PRICKLE1, ARHGAP21, and ARHGAP23”.We thank the reviewers for this important suggestion. We have now included Figure 5—figure supplement 1I and J to directly address this comment. Panel I depicts the location of primers used to amplify a transcriptionally active region of the c2/c2 mice. What is the remaining signal in FOXC1 stained Foxc1c2/c2 tissue?10) Figure 10D-F: It is not clear from the images presented that a call can be made regarding FOXC2 levels being modestly increased in Foxc1c2/c2Foxc1 mice, we have also included new representative images of FOXC1/VEGFR-3 immunostaining in Figure 9D and E. As previously stated in our reply to reviewer comment #1, we suspect that the remaining signal in the FOXC1 channel of Figure 9H is non-specific and may be related to a cross-reactivity issue of secondary antibodies. However, we believe that Figures 9F-H clearly depict a reduction in nuclear FOXC1 expression as expected.We agree with the reviewers that it is likely not accurate to describe that FOXC2 levels are modestly increased. Thus, this statement has now been removed from the revised manuscript. To verify that FOXC1 protein expression is absent in collecting vessels of


Serratia marcescens TKU011 with the highest yield of 4.62 mg/mL at the optimal conditions of liquid medium with initial pH of 5.65–6.15 containing 1% α-chitin, 0.6% casein, 0.05% K2HPO4, and 0.1% CaSO4. Fermentation was kept at 25 °C for 2 d. Notably, α-chitin was newly investigated as the major potential material for PG production via fermentation; the salt CaSO4 was also found to play the key role in the enhancement of PG yield of Serratia marcescens fermentation for the first time. PG was qualified and identified based on specific UV, MALDI-TOF MS analysis. In the biological activity tests, purified PG demonstrated potent anticancer activities against A549, Hep G2, MCF-7, and WiDr with the IC50 values of 0.06, 0.04, 0.04, and 0.2 µg/mL, respectively. Mytomycin C, a commercial anti-cancer compound was also tested for comparison purpose, showing weaker activity with the IC50 values of 0.11, 0.1, 0.14, and 0.15 µg/mL, respectively. As such, purified PG displayed higher 2.75-fold, 1.67-fold, and 3.25-fold efficacy than Mytomycin C against MCF-7, A549, and Hep G2, respectively. The results suggest that marine chitins are valuable sources for production of prodigiosin, a potential candidate for cancer drugs.Marine chitins (MC) have been utilized for the production of vast array of bioactive products, including chitooligomers, chitinase, chitosanase, antioxidants, anti-NO, and antidiabetic compounds. The aim of this study is the bioprocessing of MC into a potent anticancer compound, prodigiosin (PG), via microbial fermentation. This bioactive compound was produced by  Chitin, an abundant material, has been widely produced from fishery processing byproducts. Of the natural chitin-containing materials, shrimp shells, squid pens, and crab shells have the highest chitin content , and as Serratia marcescens and some other Gram-negative bacterial strains [Prodigiosin (PG), a red pigment is a typical alkaloid constituent produced by several bacterial genus,  strains . PGs hav strains ,20,21,22 strains  activiti strains . S. marcescens. However, numerous scientific parameters were not investigated in our previous studies, such as the kind of marine chitin (α or β), protein sources, chitin/protein ratio, and supplementary minerals for the best PG productivity production by S. marcescens. All those previously unknown items were newly investigated in this study, and the PG produce from the medium containing marine chitin was also evaluated for its effect on four cancerous cell lines—A549, Hep G2, MCF-7, and WIDR—in this report.Due to the wild range of unique applications of PG, the production studies on this bioactive compound have been received with great interest ,24, and Serratia marcescens TKU011, compared to other materials; SPP was reported to contain approximately 60% chitin and 40% protein [S. marcescens, the chitins obtained from SPP (β-chitin) and shrimp shells (α-chitin) by using the method reported by Wang et al., 2006 [w/w) and used as the sole carbon and nitrogen source for fermentation by S. marcescens TKU011; SPP was also used as the control for comparison purpose. The results in w/w) give higher PG yield production (2.73 mg/mL) than that of SPP (2.45 mg/mL) fermented by S. marcescens TKU011, while α-chitin mixed with free protein at the ratio of 5/3 (w/w) reach the greatest PG yield production of 3.23 mg/mL. In addition to the use of α-chitin providing higher PG yield, and α-chitin could be more abundantly obtained from vast resources  than β-chitin (mainly obtained from squid pens); thus, α-chitin was chosen for our further investigation. Based on the recent literature review, PG has been produced by S. marcescens with various types of carbon/nitrogen sources [Carbon source has been proven to play an important role in PG production via microbial fermentation . In prev protein . Thus, cl., 2006  were mix sources ,31,32,33 sources  as the cS. marcescens strains, a total four strains including S. marcescens TKU011, S. marcescens CC17, S. marcescens TNU01, and S. marcescens TNU02 were conducted for fermentation. As shown in the S. marcescens TKU01, S. marcescens TNU01, and S. marcescens TNU02 showed their same level in PG production with the PG yield of 325–335 mg/100mL, and 236–243 mg/100mL when the medium contained newly designed C/N source (0.94% α-chitin and 0.56% Casein), and 1.5% squid pens, respectively. S. marcescens CC17 demonstrated the lowest production of PG yield.For the comparison of the PG producing by different S. marcescens TKU011 strain reached 335 (mg/100mL). However, two previous studies reported that with 2.0% sesame seed [S. marcescens TKU011 in this study. In these above cited reports [To date, PG has been produced from many carbon sources with multiple designed media ,31,32,33ame seed  and the  reports ,33, the S. marcescens TKU011, some chitinous materials, including chitosan (a derivative of chitin), N-acetyl-glucosamine (monomer of chitin), glucosamine (mono of chitosan), and some other carbon sources, such as cellulose and starch, were used for fermentation. As shown in S. marcescens with the greatest yield of 3.21 mg/mL, followed by its monomer N-acetyl-glucosamine with the PG yield production of 1.81 mg/mL, and all other tested carbon source give low yield PG production (≤ 0.96 mg/mL). Thus, α-chitin was chosen as an excellent substrate for further investigation. To further investigate the effect of the combination of α-chitin and free protein source, a total of five protein sources—beef extract, casein, nutrient broth, yeast extract, and peptone—were combined with α-chitin used as sole C/N source for fermentation by Serratia marcescens to produce PG. The experimental results in w/w). This designed medium also reached the high PGs yield of 3.21 mg/mL, and as such used for next investigation. To investigate the effect of C/N sources on PG production by 2HPO4 was found to be the most suitable phosphate salt for PG biosynthesis by S. marcescens. Further experiments investigated the optimal added K2HPO4 was 0.05% 2SO4, MgSO4, CaSO4, CuSO4, (NH4)2SO4 as the basal salt solution. CaSO4 demonstrated good effect on PG production with the highest yield of 4.32 mg/mL  in liquid medium with initial pH of 5.65–6.15 containing 1% α-chitin, 0.6% casein, 0.05% K2HPO4, and 0.1% CaSO4, fermentation was kept at 25 °C for 2 d. To achieve maximum production of prodigiosin, some parameters, including cultivation temperature e, initiaw/w) and used as the sole C/N source at the concentration of 1.5% (w/v) for fermentation by S. marcescens TKU011. PG was primary extracted from the cultured broth by ethyl acetate. The PG from the cell pellet extracted with acetone was mixed with the ethyl acetate layer. After evaporation to dry crude PG, this compound was further purified via silica gel column, and then finally isolated by thin layer chromatography. The procedure is summarized in α-chitin was mixed with casein with the ratio of 5/3 , A549 (Human lung carcinoma), Hep G2 (Human hepatocellular carcinoma), and WiDr (Human colon adenocarcinoma). The means of inhibition (%) with the same letter are not significantly different based on Duncan’s multiple range test . CV (%) = 1.979533.50 value. IC50 value is a concentration of sample that may reduce 50% of cancerous cells; therefore, the smallest this value of the sample, the strongest anticancer activity it displayed. As shown in 50 values of 0.04, 0.06, 0.04, and 0.20 against A549, Hep G2, MCF-7, and WiDr, respectively . Four cancerous cell lines—MCF-7 (Human breast adenocarcinoma), A549 (Human lung carcinoma), Hep G2 (Human hepatocellular carcinoma), and WiDr (Human colon adenocarcinoma)—were purchased from the Bioresources Collection and Research Centre . Mitomycin C and Silicagel  were obtained from Sigma Chemical Co.  and Mitsubishi Chemical Co. , respectively. Reagents, solvents and common chemicals were used at the highest grade available.us study . S. marcus study , S. marcSerratia marcescens TNU01: 16S gene sequence of TGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGC.Serratia marcescens TNU02: 16S gene sequence of CTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTG C.w/w) and used as the sole carbon and nitrogen source with the concentration of 1.5% (w/v) for fermentation. The medium containing 1.5% carbon and nitrogen source, 0.1% K2HPO4 and 0.1% FeSO4(NH4)2SO4 was fermented by S. marcescens TKU011 at 30 °C in 1 d, and then 25 °C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). α-chitin mixed with casein at the ratio of 5/3 (w/w) reached the greatest PG yield production; as such, α-chitin/casein was used for comparison in the following experiments evaluating other carbohydrate sources  and proteinous sources. α-chitin/casein at the ratio of 5/3 (w/w) finally proved best and was used for fermentation in the subsequent investigation, including the effect of added salts and some parameters.α-chitin and β-chitin obtained from shrimp shells and squid pens were mixed with casein with 6 ratios of 1/7, 2/6, 4/4, 5/3, 6/2, and 7/1 2SO4, 0.1% phosphate salt, and 1.5% C/N source were fermented at 30 °C in 1 d, and then at 25 °C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). KH2PO4 was found to be the most suitable phosphate salt; as such, it was used to investigate its optimal concentration added to the medium. 0.025, 0.05, 0.1, 0.125, 0.15, 0.175, and 0.2% K2HPO4 and combined with 0.1% FeSO4(NH4)2SO4; the fermentation procedure was conducted at 30 °C in 1 d, and then 25 °C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v).The effect of phosphate salts and its optimal concentration added to culture medium on PG production: four kinds of phosphate salts—KH4(NH4)2SO4, MgSO4, CaSO4, CuSO4, and (NH4)2SO4 were used as sulfate salts. The medium containing 0.05% K2HPO4 and 0.1% sulfate salt, 1.5% C/N source were fermented at 30 °C in 1 d, and then 25 °C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v). CaSO4 was found to be the most suitable sulfate salt; as such, it was used to investigate its optimal concentration added to the medium. 0.025, 0.05, 0.1, 0.125, 0.15, 0.175, and 0.2% CaSO4 and combined with 0.1% K2HPO4; the fermentation was conducted at 30 °C in 1 d, and then 25 °C over the next 2 d, shaking speed of 150 rpm, and a ratio volume of medium:flask of 1:2.5 (v/v).The effect of sulfate salts and its optimal concentration added to culture medium on PG production: FeSOThe effect of some parameters on PG production: some parameters including temperature programs , initial pH  and period of cultivation time . w/v) hydrated potassium aluminum sulfate was added into this mixture, mixed, and then centrifuged at 1400 g for 5 min. The harvested supernatant was then mixed with a solution of methanol/0.5 N HCl at the ratio of 1/9, v/v. The final solution optical density was measured at 535 nm. PG purified from the culture broth was used as the standard to convert OD535 nm measurement to mass concentration via an appropriate calibration. PG was purified by the method previously described [g for 15 min. The supernatant was collected and mixed with ethyl acetate with the ratio 1/1. The mixture was kept in a funnel for 3 h and immediately shaken every 30 min. The PG dissolved in ethyl acetate layer was collected. The PG from the cell pellet was extracted with acetone, and centrifugated at 10000× g for 15 min. The ethyl acetate layer and acetone containing PG were mixed, concentrated by evaporation of the solvent and then dissolved in ethyl acetate for further air oven drying at 55 °C to get dry crude PG powder. The crude PG was further purified by loading onto a silica open column  for column chromatography, size: 0.040–0.063 mm) and eluted with methanol in chloroform with a ratio of 0/10–2/8 (v/v). The PG was finally isolated by thin layer chromatography (TLC) with the mobile phase system using methanol in chloroform with a ratio of 2/8 (v/v). After TLC separation, the lane contained PG was cut into small pieces, and methanol was used to dissolve PG. Then PG was concentrated in a rotary evaporator  at 60 °C under vacuum. Finally, all the residue solvent was removed by keeping the sample in the oil pump in 12 h at 60 °C. The isolated PG was used to detect UV, MALDI-TOF MS and biological activities. PG concentration was determined according to the method previously described by Wang et al., 2012 . A mixtuescribed  with mod50 values, were analyzed with the use of Statistical Analysis Software (SAS-9.4) provided by the SAS Institute Taiwan Ltd. .Four cancerous cell lines: A549, Hep G2, MCF-7, and WIDR were conducted to evaluate the anticancer activities. The bioassay was done according to the methods described in detail in our previous report . The sig2HPO4, and 0.1% CaSO4 for efficient biosynthesis of bioactive prodigiosin. The fermentation was maintained at 25 °C for 2 d. The prodigiosin was purified, qualified via UV and Mass. The purified prodigiosin was also evaluated for its anticancer properties. Notably, the purified PG displayed high inhibition on four cancerous cell lines. The results in this study suggest that the purified prodigiosin newly biosynthesized may be a potential candidate for cancer drugs.The current study established the novel designed medium containing 1% α-chitin, 0.6% casein, 0.05% K