Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using  Proper integration of the cardiac and pulmonary systems begins during early embryogenesis and is essential for terrestrial life. A key feature of cardiopulmonary (CP) development is evolutionarily conserved bi-directional paracrine signaling between the foregut endoderm, which gives rise to pulmonary epithelium, and the cardiogenic mesoderm . The intFgf8, Fgf10, and Tbx1 and generates the right ventricle, portions of the outflow tract, and pharyngeal mesoderm  that give rise to lung mesenchyme, pulmonary vasculature, and myocardium of the inflow tract , termed the first and second heart fields, respectively  signaling is a strong candidate for the Tbx5-dependent signal that activates endodermal  foregut  and mani foregut . RA, a d foregut . Aldh1a2SHF fate . How theShh expression. We further define the molecular basis by which Tbx5 drives RA signaling and by which RA signaling drives Shh expression. Specifically, Tbx5 directly maintains expression of Aldh1a2 in pSHF via an evolutionarily conserved intronic enhancer, and Tbx5/Aldh1a2-dependent RA signaling directly activates Shh transcription in the foregut endoderm via an evolutionarily conserved MACS1 endoderm enhancer. We conclude that Tbx5 coordinates CP development by controlling expression of the RA-producing enzyme Aldh1a2, and that this RA signal initiates a mesenchyme-epithelial signaling cascade that controls both Hh/Wnt-dependent lung induction and SHF patterning. Hh/Gli and Tbx5 then cooperate to promote Wnt2/2b expression and lung induction. This work unifies previously unconnected observations to resolve the molecular basis of a mesoderm-endoderm-mesoderm signaling network that coordinates pulmonary induction and SHF cardiac patterning.In this study, we demonstrate that RA signaling is the link between mesodermal Tbx5 activity and endodermal −/Tbx5− mouse embryos at E9.5 (Tbx5 (≥1.5 fold change and 5% FDR) , Hh-targets (Hhip and Gli1), the lung-inducing Wnt2/2b ligands, and pulmonary progenitor marker Nkx2-1, indicated a loss of pulmonary fate in −/Tbx5− mutant CPP tissue    micro-dissected from wild-type (WT) and  at E9.5 . Differe 5% FDR) . ReducedP tissue . We examor cells . We founst, HGT) . On the 01, HGT) . The aSHnd Dusp6 . Gene seP tissue . Thus, T reports .−/Tbx5− CP tissue, including Aldh1a2, Crabp2, which promotes nuclear shuttling of RA, and Rbp1, a cytosolic chaperone of the RA precursor retinol  in Xenopus, either by CRISPR/CAS9-mediated mutation or morpholino (MO) knockdown, phenocopies the mouse −/Tbx5− phenotype with severe cardiac hypoplasia, a failure to induce Nkx2-1+ lung progenitors and the foregut tube fails to separate into distinct trachea and esophagus  (Tg(WntRE:dGFP)   . Quantifse E9.5) . AnalysiRE:dGFP) , confirmRE:dGFP) . Importaelopment . A time s (NF15) . These r−/Tbx5− CP tissue were also misregulated in Xenopus  into cardiac fate  inducible iac fate  was suff0 levels . Howeveraldh1a2 transcription in Xenopus. We injected RNA encoding a dexamethasone (DEX) inducible Glucocorticoid receptor (GR)-Tbx5 fusion protein  to block secondary protein synthesis  and 60% reduction (p=0.0031) of the mouse and frog reporter activity in the foregut respectively, and also significantly blunted their response to ectopic Tbx5 in the hindgut (Aldh1a2 expression via multiple T-box motifs found in an evolutionarily conserved first intron enhancer.We next tested Tbx5 regulation of the enh1 enhancer by combining reporter assays with LOF or gain-of-function (GOF) experiments. Tbx5-MO knockdown resulted in a dramatic reduction of the mouse and TBX5 RNA . Moreoveous tbx5 . Mutatio hindgut . Mutatio hindgut  largely  hindgut . We concfgf8 and fgf10, we tested if a temporal FGF GOF would phenocopy Tbx5 LOF  and pulmonary endoderm genes (shh and nkx2-1), whilst decreasing aSHF markers   , similarnd tbx1) . Moreovend tbx1) . Interesuires RA . Combineshh and dhh transcription in Xenopus foregut endoderm explants where the tbx5/aldh1a2+ lpm, the source of endogenous RA, had been removed , which is located more than 800 kb from Shh, within an intron of the Rnf32 gene  in part by RA treatment  Tbx5-RA and FGF-Cyp form mutually antagonistic modules, with the Tbx5-RA loop promoting pSHF/CPP identity and suppressing aSHF fate, and Cyp-mediated RA degradation refining the spatial domain of RA activity; and (3) Direct RXR/RAR activation of the MACS1 enhancer at the shh locus, which provides a mechanism underlying the cell-non-autonomous activation of endodermal Hh ligand expression by Tbx5/Aldh1a2-dependent RA signaling in the pSHF. Reception of Hh signaling in the pSHF mesoderm activates Gli TFs, which cooperate with Tbx5 to directly activate wnt2/2b transcription; Wnt2/2b then induce pulmonary fate in the foregut endoderm  and in mice . Adult transgenic X. laevis and X. tropicalis Wnt/B-catenin reporter (VlemxXla.Tg(WntREs:dEGFP), NXR_0064; and VlemxXtr.Tg(WntREs:dEGFP), NXR_1094), and adult transgenic X. laevis nkx2-5:GFP (MohuXla.Tg.(nkx2-5:GFP), NXR_0030) frogs were purchased from the National Xenopus Resource (RRID:SCR_013713). Ovulation, in-vitro fertilization and natural mating, embryo de-jellying, and microinjection were performed as described (Xenopus GR-Tbx5 (Addgene 117248), Xenopus Tbx5 (Addgene 117247) (Xenopus dominant-negative RARa (TBX5 (Horizon Discovery OHS5894-202500411) was gateway sub-cloned from its entry vector pENTR223 into the expression vector pCSf107mT-Gateway-3′myc (Addgene 67617) using clonase (ThermoFisher 11791020) according to manufacturer’s instructions. Linearized plasmid templates were used to make mRNA for injection using the Ambion mMessage mMachine SP6 RNA Synthesis Kit (ThermoFisher AM1340). Total amounts of injected mRNA were as follows: GR-Tbx5 RNA, 125 pg; dN-RARa, 200 pg; and human TBX5-myc, 100 pg. Previously validated translation-blocking MOs against Tbx5 . MOs were purchased from GeneTools  and were as follows: Tbx5-MO1: 5′-TTA GGA AAG TGT CTC TGG TGT TGC C-3′; a negative control Tbx5 mismatch MO1 with three nucleotides mutated: 5′-TCA GTA AAG TAT CTC TGG TGT TGC C-3′; Tbx5-MO2: 5′-CAT AAG CCT CCT CTG TGT CCG CCA T-3′; Tbx5 3 bp mismatch MO2: 5′-TAT CAG ACT CCT CTG TGT CCG CCA T-3′ ; Aldh1a2-MO: 5′-GCA TCT CTA TTT TAC TGG AAG TCAT-3′; Cyp26a1 MO: 5′-TAG TGA GCA GAG TAT ACA GAT CCA T-3′; and Cyp26c1 MO: 5′-TAC AAG ATG TTC CTC CTT GAG ATC A-3′.WT adult escribed . Plasmid 117247) , and Xenive RARa  were prenst Tbx5 , Aldh1a2nst Tbx5 , Cyp26a1nst Tbx5 , and Cypnst Tbx5  were injX. trop tbx5 exon 5 (DNA-binding domain) that causes frameshift mutations was synthesized in-vitro as previously described . In all experiments, corresponding amounts of vehicle controls (DMSO or 0.2% fatty-acid free BSA) were used.For Gastrula or CP-foregut explants (containing both endoderm and lpm) were micro-dissected in 1× MBS +50 μg/ml gentamycin sulfate ±10 U/ml dispase  and were cultured in 0.5× MBS +0.2% fatty acid free BSA (Fisher BP9704)+50 μg/ml gent with the following concentrations of factors: 1 μM dexamethasone ; 1 μM cycloheximide ; 25 nM all-trans RA (Sigma R2625); 100 ng/ml WNT2B (R&D Systems 3900-WN-025); 1 μM DEAB (Sigma D86256); 100 ng/ml FGF8b (R&D Systems 423-F8-025); and 0.5 μM ketoconazole (Tocris 1103). In CHX experiments, explants were treated for 2 hr in CHX prior to DEX+CHX treatment for 6 hr.Xenopus RT-qPCR primer sequences are listed in odc, was determined using the 2−ΔΔCt method. Graphs display the average 2−ΔΔCt value ± standard deviation. Statistical significance (p<0.05) was determined using parametric two-tailed paired t-test, relative to uninjected, untreated explants. Each black dot in the RT-qPCR graphs represents an independent biological replicate containing four explants. Heat map of Xenopus RT-qPCR gene expression was generated using Morpheus software (https://software.broadinstitute.org/morpheus/) and shows the average 2−ΔΔCt value from three biological replicates for each condition.Xenopus explants were dissected from embryos of 2–3 separate fertilization/injection experiments, frozen on dry ice in 200 μl of TRIzol (ThermoFisher 15596018), and stored at –80°C. RNA was extracted using TRIzol and purified using the Direct-zol RNA miniprep plus kit (ZymoResearch R2070); 500 ng RNA was used in cDNA synthesis reactions using Superscript Vilo Mastermix (ThermoFisher 11755050), all according to the manufacturer’s instructions. qPCR reactions were carried out using PowerUp Mastermix (ThermoFisher A25742) on ABI StepOnePlus or QuantStudio3 machines. Xenopus embryos was performed as described (In-situ hybridization of escribed  with minEmbryos were fixed in 100 mM HEPES (pH 7.5), 100 mM NaCl, 2.7% methanol-free formaldehyde for 2 hr at room temperature, dehydrated directly into Dent’s post-fixative (80% Methanol/20% DMSO), washed five times in Dent’s, and stored in Dent’s at −20°C for at least 48 hr. Embryos were serially rehydrated  into PBS +0.1% TritonX-100 (PBSTr). Embryos were then cut in a transverse plane through the pharynx and posterior to the liver to create a foregut sample using a fine razor blade on a 2% agarose-coated dish in PBSTr. Foreguts were subjected to antigen retrieval in 1× R-universal epitope recovery buffer (Electron Microscopy Sciences 62719-10) for 1 hr at 60–65°C, washed 2× 10 min in PBSTr, blocked for 1–2 hr in PBSTr +10% normal donkey serum (Jackson ImmunoResearch 017-000-001) + 11% DMSO at room temperature, and incubated overnight at 4°C in this blocking solution+primary antibodies: chicken anti-GFP , mouse anti-Sox2 , rabbit anti-Aldh1a2 , and goat anti-Tbx5 . Secondary antibodies were donkey anti-chicken 488, donkey anti-rabbit Cy3, and donkey anti-mouse Cy5, donkey anti-goat 405 . After extensive washing in PBSTr, samples were incubated overnight at 4°C in PBSTr +0.2% DMSO+secondary antibodies. Samples were again extensively washed in PBSTr, dehydrated into 100% methanol, washed five times in 100% methanol, cleared, and imaged in Murray’s Clear  on a metal slide with glass coverslip bottom using a Nikon A1R confocal microscope to obtain optical sections.Xenopus tropicalis v9.1 genome on Xenbase.org; RRID:SCR_003280 , X. trop aldh1a2 enh1 , X.trop shh MACS1, and human SHH MACS1  enhancers, as well as their respective mutant forms, were commercially synthesized  and cloned into the pGL4.23 firefly luc2/miniP vector (Promega E8411). For enh1 enhancer assays, embryos were co-injected with 5 pg of pRL-TK:renilla luciferase plasmid (Promega E2241) + 50 pg of the pGL4.23 luc2/miniP enhancer:luciferase plasmid and the following amounts of MOs or mRNAs into each dorsal marginal zone (dmz) region of 4–8 cell embryos: 3.5 ng of tbx5-MO or 3 bp mismatch-MO; 62.5 pg GR-Tbx5 RNA; 50 pg Xenopus Tbx5 RNA; 50 pg human TBX5-myc RNA. For hindgut mesendoderm injections, the luciferase reporters were injected into the ventral-posterior marginal zone at the 4–8 cell stage±Tbx5 RNA. For analysis of Shh MACS1 enhancer activity in endoderm, C1 (foregut) or C4 (hindgut) blastomeres were injected at the 16- or 32-cell stage with 5 pg pRL-TK+50 pg MACS1:luc±100 pg dnRARa RNA.The R_003280  was usedg and then 25 µl of the clear supernatant lysate was used separately in firefly (Biotium #30085-1) and renilla (Biotium 300821) luciferase assays according to the manufacturer’s instructions. Relative luciferase activity was determined by normalizing firefly to renilla levels for each sample. Graph show the average relative luciferase activity ± standard deviation with dots showing values of biological replicates. Statistical significance was determined by parametric two-tailed paired t-test, *p<0.05.Each biological replicate contained a pool of five embryos, obtained from 2 to 3 separate fertilization/injection experiments which were frozen on dry ice in a minimal volume of 0.1× MBS and stored at –80°C. To assay luciferase activity samples were lysed in 100 µl of 100 mM TRIS-Cl pH 7.5, centrifuged for 10 min at ~13,000×Xenopus transgenic plasmids were constructed using the pI-SceI-d2EGFP plasmid backbone (Addgene 32674). First, a fragment containing the mouse or X. trop enh1 enhancers upstream of a minimal TATA box promoter . Columns in the +/+Tbx5 WT N=5, −/Tbx5− N=2), and each column replicate contained n=4 pooled CP dissected regions.RNA-seq of the micro-dissected E9.5 WT and Gapdh and RT-qPCR primers are listed in Shh probe was provided by Elizabeth Grove (University of Chicago). Immunofluorescence of WT CD-1 (Charles River) or Shh:GFP (B6.129×1(Cg)-Shhtm6Amc/J; Jax Labs Stock Number #008466) mouse embryos was performed as described  mouse E9.5 pSHF/CPP regions was performed as described , cDNA geescribed  using mohttp://www.adobe.com) and reconstructed with AMIRA . Manual review of each image in the stack was performed and corrections were made when necessary. LabelFields for gene expression and tissue were generated from the same series of sections using separate CastField and LabelVoxel modules. The SurfaceGen module was used to generate surfaces from these LabelFields. Gene expression models for two different genes were initially aligned using the Landmark (two sets) module, and a minimum of three landmarks were used to align the separate models. These landmarks were located using the pharyngeal endoderm and ventral edge of the SHF. Final alignments were fine-tuned manually using the Transform editor.Reconstructions of whole-mount in-situ hybridizations were generated using previously published methods . In brieAldh1a2, Tbx1, Tbx5, and Shh was generated using the single cells Spatial Mouse Atlas . Images are centered around the cardiothoracic region with cardiomyocytes highlighted.Digital in-situ hybridization for Tbx5OE-mESC line was previously generated , .RNA-seq of the micro-dissected E9.5 WT and ogists), . Differe−/Tbx5− embryos compared to the single-cell data sets by GSEA  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Acceptance summary:Your study provides critical data on the early events regulated by mesoderm-endoderm signaling required for lung development. These studies are important for assessment of early foregut development and help to define causal cell types in genetic diseases of lung formation.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Edward Morrisey as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Tien Peng (Reviewer #1).Thank you for submitting your article "TBX5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) Please address the issues raised by the reviewers on the specificity of the pharmacological inhibitors through the use of more specific techniques such as morpholinos. Also clearly state the limitations of such pharmacological inhibitors.2) Address the requests of additional histological and gene expression analysis with new data where noted by the reviewers.3) The manuscript needs to be revised to better link the current studies with previous work and to highlight to the uninitiated the importance of your study.4) The manuscript also needs additional revision to temper some of the claims as mentioned by the reviewers.Reviewer #1 (Recommendations for the authors):1. In Figure 1H,I, labeling of major anatomic landmarks would be helpful to delineate the domain of Aldh1a2 expression. Furthermore, it would be nice to highlight the relationship of the Shh expression domain alongside Aldh1a2 in Figure 1I as their complement anatomic relationship sets up the hypothesis studied in this manuscript.2. In Figure 2a-c, it is clear that while there are some overlaps between Tbx5 and Aldh1a2, there are clearly domains of Aldh1a2 expression that are Tbx5-negative. It would be easier to visualize the overlap of the two domains in Figure 2b by coloring Tbx5 and Aldh1a2 green and red respectively with some quantification of the overlap. Furthermore, it is not clear how the reduction in Aldh1a2 is quantified. Finally, some discussion on what is driving Aldh1a2 expression in cells that are not Tbx5+.3. In Figure 3A, a set of data shows that RA inhibition by DEAB can phenocopy RNA targeting of Tbx5, however, this is not really specific to the expression of Aldh1a2 per say. What is the phenotype of using MO to target Aldh1a2 in these set of experiments?4. In Figure 4G, it would be nice to colocalize the expression of the GFP with in situ of Adh1a2 to see where the enhancer-driven GFP overlap with actual gene expression.5. In Figure 5, I would raise a similar point as Comment 3, which is that the use of ketoconazole as a Cyp inhibitor is not as specific as MO targeting Cyp26a1. Would targeting of Cyp26a1 block the effect of FGF8 directly. This would provide stronger mechanistic proof for the model described.6. In Figure 6E, can RA administration directly increase the reporter activity driven by the MACS1 enhancer? This would better link the Shh enhancer described to an RA response.Reviewer #2 (Recommendations for the authors):Prior to publication, a few points need to be considered.1. Figure 1H. It will be nice to zoom in the foregut region in addition to showing the whole embryo. Although it is not necessary, it will be informative if the authors can show the in situ hybridization of Fgf10, Shh in TBX5 mutants using the similar projection method.2. Fgf10 is known  to be localized in the future lung budding sites. In between the budding sites Fgf10 is not expressed. Is Tbx5 expressed in this region? It will be informative if authors can also show the projection image of Tbx5 as other genes in H.3. Figure 1I, Can the authors label the projection images, e.g. heart field, lung budding site.4. Does TBX5 deletion/kd induce stronger foregut phenotypes in frog than mouse? It appears that trachea-esophageal separation is abnormal in frog models . Can the authors provide more details?5. Does addition of exogenous Tbx5 RNA or RA in the rescue experiments affect the development of other organs? The presence of any phenotypes can further confirm that the extra Tbx5 or RA works in addition to the qPCR data.Reviewer #3 (Recommendations for the authors):Phrasing throughout the manuscript should be cautious. For example, differentially expressed genes are not necessarily Tbx5-dependent as worded on page 8 second paragraph.Listing Tbx5 regulation of shh signaling as a key finding is an overstatement.Can the authors demonstrate loss of Aldh1a2 expression in Tbx5null embryos?The authors should mention the role of Aldh2a2 in retinoic acid processing so that the reader can understand how it functions to regulate RA signaling. The only description present is RA-producing enzyme.The Wnt GFP reporter data in figure 2 B and C could be moved to supplemental data as it is more distracting than informative.A co-stain for lateral plate mesoderm would be beneficial in figure 2 B and C.For comparison purposes and because the readout is qualitative Figure 3 should be organized in a manner such that DEAB is next to tbx5 MO and 25 nM RA is next to mismatch MO. TBX5 RNA could go to the far right.Limitation of drug inhibition should be stated. There are likely many more developmental processes disrupted by addition of RA inhibitor at the NF20 stage.Authors might consider highlighting what new data they have added to the illustration in figure 7.Figure legend 3 should be revised to reflect the panels in the figure.Several of the effects observed in Figure 3 A-J appear to span beyond the cardiopulmonary region of the embryo. The author should recognized this in the interpretation of their data.The diagram in 3L is based primarily on one time point examination. It may be prudent to remove this figure.Wording in figure 4A legend has mixed verb tenses.The statement that Tbx5 activates and represses certain genes goes beyond the data demonstrated in Figure 4A.The authors should try to separate the description of the results from their discussion of the results. For example, p 12 the entire first full paragraph is more discussion than results. Essential revisions:1) Please address the issues raised by the reviewers on the specificity of the pharmacological inhibitors through the use of more specific techniques such as morpholinos. Also clearly state the limitations of such pharmacological inhibitors.We have repeated the Aldh1a2 and Cyp loss of function experiments with morpholinos that completely support the inhibitor studies. This data is presented in new Figure 3—figure supplement 1 and Figure 5—figure supplement 1. We have also clearly stated the limitations and advantages of pharmacological inhibitors.2) Address the requests of additional histological and gene expression analysis with new data where noted by the reviewers.We have added new analysis gene expression analysis in both sections and wholemount as requested and better annotated the anatomical structures in the images. These new data are presented in a revised Figure 1 D-F and in a revised Figure 1—figure supplement 1F-H.2) The manuscript needs to be revised to better link the current studies with previous work and to highlight to the uninitiated the importance of your study.We have revised the introduction and discussion to better link our results to previous work and to better highlight the importance of the work and the significance of the advance.3) The manuscript also needs additional revision to temper some of the claims as mentioned by the reviewers.We have revised the text to temper some of the claims as suggested by the reviewers.Reviewer #1 (Recommendations for the authors):1. In Figure 1H,I, labeling of major anatomic landmarks would be helpful to delineate the domain of Aldh1a2 expression. Furthermore, it would be nice to highlight the relationship of the Shh expression domain alongside Aldh1a2 in Figure 1I as their complement anatomic relationship sets up the hypothesis studied in this manuscript.Aldh1a2 and Tbx5 in the pSHF, Tbx1 in the aSHF and endodermal Shh . We also added better labels of anatomical structures on the 3D in situ reconstructions as requested. Finally, we also present 3D maximum projections of confocal images showing the co-expression domain Tbx5-Ald1ha2 in the Xenopus pSHF/foregut lpm .In retrospect, we agree that the relative expression domains were not as clear as they should have been. While the each of these gene expression patterns has been documented before in previous publications, their direct comparison is key for our hypothesis. Therefore, we performed additional 3D whole mount confocal imaging of the E9.5 mouse embryo simultaneously staining Tbx5, Aldh1a2, Shh-GFP and Nkx2-1 , which clearly shows the co-expression of Tbx5 and Aldh1a2 in the pSHF adjacent to the Shh/Nk2-1 expressing pulmonary endoderm. In addition, we used anew online single cells expression resource from the Marioni lab , to generate a “digital” in situs hybridization of sagittal section through the cardiothoracic region of a E9.5 mouse showing the overlapping expression domains of 2. In Figure 2a-c, it is clear that while there are some overlaps between Tbx5 and Aldh1a2, there are clearly domains of Aldh1a2 expression that are Tbx5-negative. It would be easier to visualize the overlap of the two domains in Figure 2b by coloring Tbx5 and Aldh1a2 green and red respectively with some quantification of the overlap. Furthermore, it is not clear how the reduction in Aldh1a2 is quantified. Finally, some discussion on what is driving Aldh1a2 expression in cells that are not Tbx5+.As requested, we revised the immunostaining in Figure 2B with Tbx5 in green and Aldh1a2 in red. This better shows the overlapping expression domains in yellow, which we quantified by generating a 3D volume with Imaris imaging software . We also quantified the reduction in Aldh1a2 protein levels based on the average immunostaining pixel intensity in the lpm in both the Tbx5-MO and tbx5-CRISPR mutants .Yes there are some Aldh1a2 expression domains that do not overlap with Tbx5 including the somite and kidney mesenchyme (figures ??), which must be regulated by different enhancers independent of Tbx5. While studying these other enhancers and expression domains is interesting, this are not really relevant to the present and so we only briefly mention this in the revised discussion.3. In Figure 3A, an set of data shows that RA inhibition by DEAB can phenocopy RNA targeting of Tbx5, however, this is not really specific to the expression of Aldh1a2 per say. What is the phenotype of using MO to target Aldh1a2 in these set of experiments?We agree that this is an important point given the potential off target effects of inhibitors. As requested, we performed Aldh1a2-MO knockdowns with targeted microinjection to the foregut lpm. Immunostaining confirmed loss of Aldh1a2 protein, and resulted in gene expression changes largely identical to the DEAB treatment. Moreover, addition of exogenous RA rescued the Aldh1a2-MO phenotype. Since Aldh1a2 has earlier functions in the paraxial mesoderm, if the targeting was not efficient the embryos can have other defects, which is why we initially used the DEAB where we can control the timing of RA inhibition. We address the limitations and advantages of inhibitors in the revised manuscript. This new data presented in Figure 3—figure supplement 1A-E further strengthens our conclusions.4. In Figure 4G, it would be nice to colocalize the expression of the GFP with in situ of Adh1a2 to see where the enhancer-driven GFP overlap with actual gene expression.As requested, we performed co-staining of the enh1-GFP transgenes with endogenous Aldh1a2, which shows a specific overlap in the Tbx5+ foregut lpm, but not in the somites . Again this is consistent with other Tbx5-independent enhancers regulating Aldh1a2 in other tissues.5. In Figure 5, I would raise a similar point as Comment 3, which is that the use of ketoconazole as a Cyp inhibitor is not as specific as MO targeting Cyp26a1. Would targeting of Cyp26a1 block the effect of FGF8 directly. This would provide stronger mechanistic proof for the model described.shh, tbx5, wnt2b and nkx2-1 expression and elevating their expression levels in untreated foregut explants . This new data provides a stronger mechanistic proof that FGF8 restricts RA-dependent cardiopulmonary development in the anterior foregut domain by regulating expression of Cyp26 RA-degrading enzymes. We thank the reviewer for the suggestion.To address this, we performed MO knockdown of redundant Cyp26a1 and Cyp26a1, both of which are direct FGF targets. RT-PCR analysis of dissected foregut tissue shows that the cyp261a/c-MO phenocopies the Cyp-inhibitors ketoconazole, rescuing FGF8’s inhibition of 6. In Figure 6E, can RA administration directly increase the reporter activity driven by the MACS1 enhancer? This would better link the Shh enhancer described to an RA response.Xenopus MACS1 enhancers can be activate by RA in isolated foregut endoderm and that this activation is dependent on RXR/RAR DNA-binding sites .We have added new luciferase assay data that demonstrate that both the human and Reviewer #2 (Recommendations for the authors):Prior to publication, a few points need to be considered.1. Figure 1H. It will be nice to zoom in the foregut region in addition to showing the whole embryo. Although it is not necessary, it will be informative if the authors can show the in situ hybridization of Fgf10, Shh in TBX5 mutants using the similar projection method.Aldh1a2 and Tbx5 in the pSHF, Tbx1 in the aSHF and endodermal Shh . We focused on Tbx5, Aldh1a2 and Shh rather than Fgf10 since these are the main focus of the work. As the mouse RNA-seq and RT-PCR as well as the Xenopus data show reduced Fgf10 and Shh expression of in Tbx5 mutants we did not repeat this by in situ in mice, given our time constraints.As suggested, we have improved the imaging of the cardiopulmonary region with additional high magnification 3D whole mount confocal imaging of the E9.5 mouse embryo simultaneously staining Tbx5, Aldh1a2, Shh-GFP and Nkx2-1 , which clearly shows the co-expression of Tbx5 and Aldh1a2 in the pSHF adjacent to the Shh/Nk2-1 expressing pulmonary endoderm. In addition, we used a new online single cells expression resource from the Marioni lab , to generate a “digital” in situs hybridization in sagittal section of the E9.5 mouse cardiothoracic region showing the overlapping expression domains of 2. Fgf10 is known  to be localized in the future lung budding sites. In between the budding sites Fgf10 is not expressed. Is Tbx5 expressed in this region? It will be informative if authors can also show the projection image of Tbx5 as other genes in H.Fgf10, Wnt2, as well as branching morphogenesis. It is unclear whether RA is involved in this role, but we have mentioned the similarity in the discussion.Arora et al., PLOS Genetics, 2012, have already published a description of Tbx5 and Tbx4 expression in the fetal mouse lung, including the distal mesenchyme where they regulate expression of 3. Figure 1I, Can the authors label the projection images, e.g. heart field, lung budding site.In addition to providing better analysis of the gene expression, we have labeled the anatomy on the 3D in situ reconstruction.4. Does TBX5 deletion/kd induce stronger foregut phenotypes in frog than mouse? It appears that trachea-esophageal separation is abnormal in frog models . Can the authors provide more details?Xenopus Tbx5 null mutant phenotypes are very similar. As previously described, both have major defects in cardiac morphogenesis and fail to specify respiratory fate in the ventral foregut . Tbx5-/- null mice die between E9.5 and E10.5 due to cardiac defects, before trachea-esophageal separation at E11.5. Xenopus tadpoles on the other hand are well known to survive for many days with severe heart defects as they can absorb oxygen from the water, which is one reason that we used Xenopus for this study. We now make this clearer in the manuscript and show data on the late tbx5-MO phenotype confirming the previously published hypoplastic atria and vertical lacking trabeculae as well as the failed trachea-esophageal separation .The mouse and 5. Does addition of exogenous Tbx5 RNA or RA in the rescue experiments affect the development of other organs? The presence of any phenotypes can further confirm that the extra Tbx5 or RA works in addition to the qPCR data.We did not look at other organs, but focused our analysis on early heart and lung specification, which is the theme of the paper. We show that addition of Tbx5 or RA can rescue cardiopulmonary development as assessed by immunostaining, in situ hybridization and RT-qPCR. There are publications suggesting that Tbx5 and RA may also interact in other tissue such as the limb, but the logic of the regulatory networks appears to be somewhat different. We address this in the discussion. While beyond the scope of this work, in the future it would be interesting to compare the different developmental contexts.Reviewer #3 (Recommendations for the authors):Phrasing throughout the manuscript should be cautious. For example, differentially expressed genes are not necessarily Tbx5-dependent as worded on page 8 second paragraph.We have revised the phasing to be more accurate and cautious as requested.Listing Tbx5 regulation of shh signaling as a key finding is an overstatement.shh expression via RA is a major finding and one of the key mechanisms linking mesoderm and endoderm development. We have revised how we describe this to be clear that Tbx5 regulates shh expression.We are not sure what the reviewer means here. In our view the finding that Tbx5 indirectly regulates Can the authors demonstrate loss of Aldh1a2 expression in Tbx5null embryos?Xenopus. Give the time constraints, and the other revisions requested did not repeat this experiment with immunostaining or in situ.The RNA-seq and RT-PCR of the Tbx5-null mouse foregut do clearly demonstrate the loss of Aldh1a2, as does the many experiments in The authors should mention the role of Aldh2a2 in retinoic acid processing so that the reader can understand how it functions to regulate RA signaling. The only description present is RA-producing enzyme.Yes, we agree that this would be helpful, and we have added this. Thank you for the suggestion.The Wnt GFP reporter data in figure 2 B and C could be moved to supplemental data as it is more distracting than informative.A co-stain for lateral plate mesoderm would be beneficial in figure 2 B and C.We understand the reviewer perspective, but we have shown the figure to a number of colleagues take a different view and find the Wnt-GFP reporter helpful as this shows the respiratory progenitors. Having said that we take the reviewers point and simplified the description of these data. We also show the panels without the GFP for those readers who find it distracting. To better show the Tbx5-Aldh1a2 co-staining in lateral plate mesoderm we have new immunostaining  showing the overlap in the ventral-lateral foregut lateral plate mesoderm.For comparison purposes and because the readout is qualitative Figure 3 should be organized in a manner such that DEAB is next to tbx5 MO and 25 nM RA is next to mismatch MO. TBX5 RNA could go to the far right.We respectfully prefer to keep the original organization of the columns.Limitation of drug inhibition should be stated. There are likely many more developmental processes disrupted by addition of RA inhibitor at the NF20 stage.We have now clearly stated the limitation of the inhibitor experiments. In addition, we have added new Aldh1a2-MO and Cyp261a/c-MO experiments to complement the DEAB and ketoconazole experiments, which support our model. We also acknowledge that these inhibitors can affect other organ system notably the kidney where RA is known to play a role as published by others. While these to not change the interpretation in the cardiopulmonary tissue we agree that it is important to state this.Authors might consider highlighting what new data they have added to the illustration in figure 7.Thank you for this suggestion we agree that is helpful.Figure legend 3 should be revised to reflect the panels in the figure.Done. Thank you for pointing out this error.Several of the effects observed in Figure 3 A-J appear to span beyond the cardiopulmonary region of the embryo. The author should recognized this in the interpretation of their data.We agree that the Tbx5 depleted embryos have non-cell autonomous defects in other tissues including the kidney primordia, pharynx, head and ectoderm. We interpret this to be due to the disrupted RA and FGF signaling that we have documented. We have modified the text to explain this.The diagram in 3L is based primarily on one time point examination. It may be prudent to remove this figure.We have revised the figure legend of the model to make it clear that we are refereeing to a specific time point. We agree that this is important since Tbx5-RA interactions may be different at different times.Wording in figure 4A legend has mixed verb tenses.Thank you we have corrected this.The statement that Tbx5 activates and represses certain genes goes beyond the data demonstrated in Figure 4A.We have revise language to state that expression levels increase and decreases in Tbx5 GOF.The authors should try to separate the description of the results from their discussion of the results. For example, p 12 the entire first full paragraph is more discussion than results.We have done this, although we kept some description of previous work where is provides a rational for the experiments.


Network analysis of large-scale neuroimaging data is a particularly challenging computational problem. Here, we adapt a novel analytical tool, the community dynamic inference method (CommDy), for brain imaging data from young and aged mice. CommDy, which was inspired by social network theory, has been successfully used in other domains in biology; this report represents its first use in neuroscience. We used CommDy to investigate aging-related changes in network metrics in the auditory and motor cortices by using flavoprotein autofluorescence imaging in brain slices and in vivo. We observed that auditory cortical networks in slices taken from aged brains were highly fragmented compared to networks observed in young animals. CommDy network metrics were then used to build a random-forests classifier based on NMDA receptor blockade data, which successfully reproduced the aging findings, suggesting that the excitatory cortical connections may be altered during aging. A similar aging-related decline in network connectivity was also observed in spontaneous activity in the awake motor cortex, suggesting that the findings in the auditory cortex reflect general mechanisms during aging. These data suggest that CommDy provides a new dynamic network analytical tool to study the brain and that aging is associated with fragmentation of intracortical networks. In this work, we introduce a novel technique  which is based on social network theory, to analyze neural data. We use CommDy to analyze imaging data from the cerebral cortex of awake mice and mouse brain slices. In each case, data were obtained from young and aged mice. Using CommDy, we found that in both types of data, aging is associated with a fragmentation of cortical circuitry. Specifically, the strength of intracortical connectivity diminishes with aging, leaving fragmented cortical networks. Thus, CommDy is a novel tool now available to the neuroscience community, and has revealed aging-related pathology of cortical networks. Normal aging is associated with a gradual loss of cognitive function . The mecNetwork analysis tools are emerging approaches to understand functional connectivity of the brain . UnfortuPrevious investigators have suggested that complex dynamic networks, be they populations of neurons, large ecosystems, or social networks, share common underlying organizational motifs . TherefoIn the current report, we adapt CommDy for the analysis of Two datasets were examined in this study: brain slice imaging data from the auditory cortex during Prior to applying the CommDy technique, there are several data preprocessing steps required to transform raw brain imaging data into CommDy input, including using the time series of each pixel to construct a simple network representation, and then static community detection in each time step. After that, CommDy was applied to infer dynamic communities. “Static” networks refer to temporal networks with multiple snapshots, and “dynamic” networks refer to those networks linked over time. To create the network representation, pixels were used as nodes and a weighted, thresholded network of pixel value correlations across an empirically determined number of time steps (the “window size”) was then generated. The correlation threshold was determined empirically, and correlation values above the threshold were used to build the correlation matrix.To determine the appropriate time window and correlation thresholds for CommDy analysis applied to flavoprotein autofluorescence imaging data for slice data, the analysis window size was systematically changed from 25 frames (352 ms) to 200 frames  and the resulting spatial distributions of the average By sliding the correlation window one step or frame each time following each correlation extraction, and doing this over the entire time line, a time series of correlation networks was obtained. CommDy can take the resulting dynamic network as the input directly or, for more efficient processing, it takes any grouping of the time steps as input. In each time step of these time series, we applied the Louvain static community inference method  to find −3, which is lower than previous reports  change over time, we use the CommDy method . In CommCommDy can be used to quantitatively describe network activity in terms of the node and structural network metrics based on network theory . These mSD] edges per active node, mean in aged animals = 149.1 ± 162.4 [SD] edges per active node, p = 0.005, Mann–Whitney). These data suggest that the average connectivity between nodes is higher in younger animals than in aging animals and cannot be accounted for by any differences in the magnitude in the response of the young versus aged animals.Paroxysmal depolarizations were imaged in brain slices taken from five young  and five aged  animals . Initially, static analysis was performed on auditory cortical networks in young and aging mice. To do this, the average degree of each node was computed for each pixel as the average number of edges at each node during the 100-frame window surrounding the peak of the paroxysmal depolarization. These values were then averaged across paroxysmal depolarizations within each animal, normalized to the overall magnitude of the response, and the resulting grand average for each group of animals is shown in Average degree of each node does not provide insights into the changing associations between nodes. We therefore performed dynamic analysis using CommDy, which is built upon static communities detected by the Louvain algorithm independently in each time step. Dynamic analysis of paroxysmal depolarizations in young and aged animals using CommDy revealed that network activity differed between these two groups. Young animals showed distinct patterns of activity across the auditory cortex during paroxysmal depolarizations. See SD] for young vs. 0.058 ± 0.046 [SD] for aged; p = 0.0001) and normalized community size . The differences in homing, a measure of network cohesion, trended toward significance but did not survive correction for multiple comparisons . See CommDy network metrics were compared between young and aged mice. To ensure that differences in network measures were not due to changes in the absolute magnitude of activation, individual group and community sizes were normalized by the total number of nodes in each activation. Using a mixed-effects model, age was found to be a significant predictor for both normalized group size  and compared to those with low ABR thresholds . Similar to separation of groups based on age (n = 5 per group), using a mixed-effects model, normalized group size, and normalized community size were both larger in the better hearing animals. The values for normalized group size = 0.189 ± 0.070 (SD) for low ABR threshold versus 0.120 ± 0.052 (SD) for high ABR threshold,  for young vs. 0.146 ± 0.052 (SD) for aged , whereas for group size, cortical thickness was the only significant predictor , as has been seen previously . These data suggest that spontaneous flavoprotein autofluorescence signals track with more commonly measured indicators of spontaneous activity, such as Nine young  and nine aged  mice were used for these experiments. Because flavoprotein autofluorescence has not been used previously to measure spontaneous neural activity in vivo, we initially characterized the properties of the signals. It was observed that the majority of the power is in the delta range (approximately 3 Hz) in both young and aged animals . To valieviously , with nop = 0.3, . To deteSD] edges/node vs. 257 ± 476 [SD] edges/node, p = 0.03, Mann–Whitney; To compare the overall degree of connectedness between nodes in aged versus young animals, the degree of each node was computed in young and aged animals. Similar to the findings in brain slices from the auditory cortex, the degree of connectivity, as measured by average number of edges per node, was significantly decreased in the aged compared to the young animals , switching , and normalized community size , visiting , and average stay ; but these did not survive multiple comparisons correction. To determine if the drop in community size with aging is related to diminished intracortical connectivity, analogous to that seen in vitro . Slices containing the auditory cortex were cut using a modification of the method developed by Cruikshank et al.  and incubated at 32°C for 1 hr prior to experimentation. The ACSF used for the experiments use equimolar MgCl2 and CaCl2 and contained 1 μM of the GABAA antagonist The CBA/CaJ strain was used because of its gradual aging-related hearing loss . Five yok et al. , modifiek et al. , see FigAn initial stereotactic surgery under ketamine/xylazine  was done to glue a threaded headbolt onto the skull using OptiBond XTR kit  cement. After at least 3 days to recover, mice were gradually acclimated to an imaging chamber within a soundproof booth. Their headbolts were affixed to a holder, and the body was suspended while under isoflurane (4%) anesthesia. The mice were then allowed to emerge from anesthesia, initially for 5 min, and gradually up to 15–20 min. Awake data were obtained once isoflurane was removed for at least 10 min. Once awake data were obtained, the animal was reanesthetized with ketamine/xylazine  for additional imaging.For the slice work, flavoprotein autofluorescence imaging was done with a fluorescence illuminator (Prior Lumen 200) and a UMNIB Olympus filter cube , Award ID: 1515587. Daniel Adolfo Llano, National Institutes of Health (https://dx.doi.org/10.13039/100000002), Award ID: DC012125. Daniel Adolfo Llano, National Institutes of Health (https://dx.doi.org/10.13039/100000002), Award ID: AG059103. Daniel Adolfo Llano, American Federation for Aging Research (https://dx.doi.org/10.13039/100000965), Award ID: 2011-03147. Daniel Adolfo Llano, Alzheimer's Association (https://dx.doi.org/10.13039/100000957), Award ID: NIRG-12-242848. Daniel Adolfo Llano, Kiwanis Neuroscience Research Foundation.Daniel Adolfo Llano, National Science Foundation (Click here for additional data file.Click here for additional data file.Click here for additional data file.


Knowledge translation (KT) is an important means of improving the health service quality. Most research on the effectiveness of KT strategies has focused on individual strategies, i.e., those directly targeting the modification of allied health professionals’ knowledge, attitudes, and behaviors, for example. In general, these strategies are moderately effective in changing practices (maximum 10% change). Effecting change in organizational contexts  is part of a promising new avenue to service quality improvement through the implementation of evidence-based practices. The objective of this study will be to identify why, how, and under what conditions organizational KT strategies have been shown to be effective or ineffective in changing the (a) knowledge, (b) attitudes, and (c) clinical behaviors of allied health professionals in traumatology settings.This is a realist review protocol involving four iterative steps: (1) Initial theory formulation, (2) search for Evidence search, (3) knowledge extraction and synthesis, and (4) recommendations. We will search electronic databases such as PubMed, Embase, CINHAL, Cochrane Library, and Conference Proceedings Citation Index - Science. The studies included will be those relating to the use of organizational KT strategies in trauma settings, regardless of study designs, published between January 1990 and October 2020, and presenting objective measures that demonstrate change in allied health professionals’ knowledge, attitudes, and clinical behaviors. Two independent reviewers will select, screen, and extract the data related to all relevant sources in order to refine or refute the context-mechanism-outcome (CMO) configurations developed in the initial theory and identify new CMO configurations.Using a systematic and rigorous method, this review will help guide decision-makers and researchers in choosing the best organizational strategies to optimize the implementation of evidence-based practices.PROSPERO CRD42020216105The online version contains supplementary material available at 10.1186/s13643-021-01793-4. Knowledge translation (KT)  is an imA promising new avenue for supporting service quality improvement through the translation of evidence-based practices targets changes in organizational contexts  –11. IncrA realist review is required to assess the different action mechanisms of organizational KT strategies  and to facilitate the choice of effective strategies that will enable effective and sustainable implementation of evidence-based practice for allied health professionals in traumatology settings. Realist synthesis (or review) is a scientific literature review approach that provides an explanatory analysis of how theories work (or fail to work) in different contexts . It is uWe will conduct a realist review . We repoThe research team consists of seven co-investigators: three academic researchers , two knowledge users , one research coordinator (JD), and one postdoctoral fellow (KL). The project involves a multidisciplinary team of experts in knowledge synthesis (AL), neurotrauma rehabilitation (MEL), management in traumatology settings , and KT . Knowledge users are decision-makers representing health and social services settings in Quebec City. The postdoctoral fellow is working on developing expertise in knowledge synthesis and KT. All team members will contribute to the different stages of this project through document revision, reflection, and decision-making with regard to the methodological approach and the various analyses. Monthly written follow-up is planned, as well as two face-to-face meetings at the beginning of the project and one at the end of the project. Other meetings (by telephone or in-person) may be convened as needed in the course of the project.The realist review consists of four main stages: (1) formulation of initial theories, (2) search for evidence, (3) knowledge extraction and synthesis, and (4) recommendations .In terms specific to the realist review, we will put forward initial theories to explain the context-mechanism-outcome (CMO) system. From a methodological point of view, researchers begin by extracting from the literature the main ideas that relate to a class of interventions . These initial theories explain how and why a class of interventions  work and generate the outcome(s) of interest .We will use the Consolidated Framework for Implementation Research (CFIR)  to guideBased on CFIR and team members’ own clinical and organizational experience, we will develop initial theories explaining why, how, and in what context organizational KT strategies can work. Specifically, we will hold a 1.5-h, audio-recorded, group discussion to develop a first draft theory answering the following question: how (context and mechanism) an organizational KT strategy (intervention) has been shown to be effective or ineffective in changing the (a) knowledge, (b) attitudes, and (c) clinical behaviors of allied health professionals (outcomes). We will make an initial schematic representation based on the discussion and refine it through revision of the audio-recorded material. This representation will then be tested against data from studies included in the review.The search will be conducted in the MEDLINE, CINAHL, Embase, Cochrane, Ergonomic Abstracts, and Web of Science Core Collection databases and may be extended to other databases of a more social or sociological nature as required (planned date coverage between January 1990 and October 2020). It should be noted that, depending on the realist review methodology, subsequent iterative research may be conducted to generate additional data on a particular aspect of the research question.Two knowledge management specialists will perform the primary article search in the various databases. Subsequently, the research team will use three different mechanisms: (1) the list of cited references will be reviewed to extract articles of interest, 2) the “find similar articles” function of the databases will be used to check if other articles of interest are related to the primary articles, and (3) the authors who have published more than two articles deemed relevant will be contacted by e-mail to request a list of relevant references, published or not, on our subject. All articles found during the secondary search will in turn be put through the three secondary search mechanisms to complete the search. Finally, Grey Matters, a practical tool for searching health-related grey literature , will be the “finhttps://cihr-irsc.gc.ca/e/29529.html):We will consider French- and English-language studies relating to organizational KT strategies published. The definition of KT used for the purpose of article selection  is the one provided by the Canadian Institutes of Health Research  in traumatology settings. For the outcomes, we will include all objective measures that demonstrate a change in the clinical knowledge, attitudes, and behaviors of allied health professionals.First (by title and abstract) and second (full-text review) screenings will be carried out independently by two reviewers  using Covidence . We willStudy characteristics: title, first author, publication year, country, participants , study objectives, trauma setting , and outcomes (what specific changes have been measured in the (a) knowledge, (b) attitudes, and (c) clinical behaviors)Intervention: type of organizational KT strategiesProgram theory: refined or refuted CMO configurations developed in the initial theory and new CMO configurationsMethodology: data collection method and type of analysisWe will carry out a thorough reading of the selected articles followed by the extraction of relevant data. The extraction will be also performed in parallel by two research team members  using a standard Covidence  extractiThe extraction form will be piloted with five studies. Here again, data extraction can lead to questions about the focus of the synthesis and, consequently, to iterative reflection on the research question and selection of articles. These questions and reflections will also be referred to the research team.Two independent analysts will examine the selected articles and indicate to what extent they correspond to each CMO configurations developed in the original theory. They will also identify any new CMOs that may emerge from the studies. Results will then be compare in a cross-tabulation matrix. A consensus will have to be reached before results are presented to the rest of the team. Data synthesis will take into account the circumstances in which these organizational KT strategies were used, including the characteristics of the targeted evidence-based practices, as well as internal, external, and personal contexts of individuals involved, in addition to any other information that supports the understanding of the context. Based on the emerging results of the studies, the research team will be able to validate (confirm or refute) the initial theories on the effectiveness of organizational KT strategies through group discussion. The team will perform a comparative analysis between the initial theories and the emerging results of the review as well as a search for both contradictory and consensual results , 26. SpeUsing a systematic and rigorous method, this review will help guide decision-makers and researchers in choosing the best organizational strategies to optimize the implementation of evidence-based practices in the trauma field.Researchers have explained some of the challenges that are specific to realist reviews. Among other things, making the distinction between mechanism and context can be difficult –34. RobeAdditional file 1. PRISMA-P populated checklist.Additional file 2. Draft search strategy for MEDLINE.


The Covid-19 pandemic is affecting the entire world population. During the first spread, most governments have implemented quarantine and strict social distancing procedures. Similar measures during recent pandemics resulted in an increase in post-traumatic stress, anxiety and depression symptoms. The development of novel interventions to mitigate the mental health burden are of utmost importance.In this rapid review, we aimed to provide a systematic overview of the literature with regard to associations between physical activity (PA) and depression and anxiety during the COVID-19 pandemic.We searched major databases  and preprint servers , for relevant papers up to 25/07/2020.We included observational studies with cross-sectional and longitudinal designs. To qualify for inclusion in the review, studies must have tested the association of PA with depression or anxiety, using linear or logistic regressions. Depression and anxiety must have been assessed using validated rating scales.Effect sizes were represented by fully adjusted standardized betas and odds ratios (OR) alongside 95% confidence intervals (CI). In case standardized effects could not be obtained, unstandardized effects were presented and indicated.We identified a total of 21 observational studies , including information of 42,293  participants from five continents. The early evidence suggests that people who performed PA on a regular basis with higher volume and frequency and kept the PA routines stable, showed less symptoms of depression and anxiety. For instance, those reporting a higher total time spent in moderate to vigorous PA had 12–32% lower chances of presenting depressive symptoms and 15–34% of presenting anxiety.Performing PA during Covid-19 is associated with less depression and anxiety. To maintain PA routines during Covid-19, specific volitional and motivational skills might be paramount to overcome Covid-19 specific barriers. Particularly, web-based technologies could be an accessible way to increase motivation and volition for PA and maintain daily PA routines. With 106,125,682 confirmed cases all over the world , COVID-19 is a global public health emergency. COVID-19 is characterized by a fast human-to-human transmission through droplet or close contact. Given the lack of appropriate treatments and vaccines during the early stage of the pandemic, many countries implemented procedures recommended by the World Health Organization (WHO) .Quarantine and social distancing measures had already been successfully enforced during earlier pandemics, such as the 2003 outbreak of SARS and the 2014 outbreak of Ebola , 6. ThesNotably psychiatric disorders result in a considerable burden of disease, accounting for 6.7% of overall disability-adjusted life years  and beinIn this rapid review, we sought for observational studies examining the associations of PA and depression and anxiety during the COVID-19 pandemic. Inclusion criteria were: (1) observational studies in any population, including cross-sectional and longitudinal designs. Longitudinal studies could be either prospective or retrospective; (2) studies have tested the association of PA with depression or anxiety, using linear or logistic regressions; (3) depression and anxiety were assessed using validated screening or diagnostic tools. We excluded opinion pieces, systematic reviews, and studies addressing other viruses.We searched the electronic databases PubMed, EMBASE, SPORTDiscus, and Web of Science using the following strategy:  AND (coronavirus OR sars-cov-2 OR COVID* OR severe acute respiratory syndrome OR pandemic) AND . Preprints were searched in MedRxiv, SportRxiv, and SciELO Preprints using the following strategy: “ AND (coronavirus OR sars-cov-2 OR COVID* OR severe acute respiratory syndrome OR pandemic)”. Additional hand searches were performed on COVID-19 online repositories on ResearchGate and Google Scholar. Searches were made by an experienced reviewer (FS) on 29th July, 2020. Study selection was conducted in three steps: (1) duplicates removal; (2) screening at the title and abstract level; and (3) assessment based on full-text. The selection was made by one reviewer (FS). Data extraction of selected studies was then performed by three researchers . Data extracted were: author and year, country of the included sample, study design, sample size, age group of the sample included, when possible, mean or range of age sample, % of women, instrument/question used to assess PA levels, instruments used to assess depression and anxiety, publication type and statistical outcomes (regression standardized beta coefficients and odd’s ratios). If they were indicated in the report, fully adjusted coefficients and odd’s ratios were extracted. As studies included in this review used very heterogeneous statistical approaches, a meta-analysis could not be conducted. Instead, we summarized the evidence and presented effect sizes [betas and odds ratios (OR)] with confidence intervals and indicated significant associations between PA and depression or anxiety, separately  were peer-reviewed, seven studies were published as preprints. A summary of studies is provided in Table Data form a total of 42,293 (median = 68% of women) participants were included. Only 1 study was exclusively composed by older adults (over 50 years), 4 were in children, adolescents, or young adults, while 13 studies were in adults (over 18). Only 7 studies used validated measures to assess PA levels. A wide range of scales to measure depression or anxiety were used, the most used scales being the Beck Depression and Anxiety inventory and the DASS-21. Most studies (Results are summarized and presented in Table The risk of bias of individual studies is presented in Table The present study is, to the best of our knowledge, the first study to summarize the evidence on the associations of PA with depression and anxiety during the COVID-19 pandemic. The majority of studies included in the present review showed that those who performed PA on a regular basis with higher volume and frequency and kept the PA routines stable, showed less symptoms of depression and anxiety. There was consistent evidence that those who could not keep their PA routine stable during the pandemic showed more depression and anxiety symptoms , 41–43. Indeed, the observed reduction in PA behavior during COVID-19 specific conditions is highly expected. For example, due to social distancing, exercising in a group setting was limited or completely prohibited. However, high social support is associated with more engagement in PA . Indeed,A web-based tool, e.g., a smartphone application could be a low-threshold and cost-effective option to train, supervise, apply, and adopt such BCTs, especially in terms of COVID-19. First empirical evidence showed preliminary efficacy of apps in promoting PA. Users of such apps are more likely to meet recommendations on PA than non-users –59. HoweMost of the studies included in this review used cross-sectional research designs. A causal nature of these associations, therefore, remains unclear. There are notably differences in effect sizes which point at a high heterogeneity of the effects. Several studies further showed methodological shortcomings, e.g., not reporting the participation rate, including less than 50% of eligible participants, no validated tools to assess PA and failure to report standardized coefficients. Heterogeneity in research designs and statistical analyses hindered meta-analytic approaches, which would have provided a more sophisticated overall effect estimate. Finally, several included studies were published as preprints and are currently in review processes for final publications. It is, therefore, planned to update this review in the future.This rapid review shows promising evidence that higher volume and frequency of PA and the keeping of regular PA habits during the Covid-19 pandemic are associated with less symptoms of depression and anxiety. For instance, those reporting a higher total time spent in moderate to vigorous PA had 12–32% lower chances of presenting depressive symptoms and 15–34% of presenting anxiety. Thus, the promotion of PA habits and routines might be a cost-effective and comprehensive worldwide applicable strategy to overcome the severe gap between people in need and people receiving mental health care, especially in low-income countries with even non-existing mental health supplies. Particularly, web-based technologies, could be an easily accessible way to increase motivation and volition for PA and maintain daily PA routines even under pandemic-specific barriers. However, only very few apps or websites have been tested in RCTs with high methodological standards , 61. Thu


Pituitary metastases are rare, often deriving from lung or breast cancer owing to the upper vena cava proximity. Pituitary metastases can manifest with signs and symptoms of pituitary tumors, consequent to mass effect  and/or hormonal alterations . Immune checkpoint inhibitors burst immunity against tumors, significantly increasing patients’ survival, but their autoimmune side effects frequently involve the skin, the gastrointestinal tract, and the endocrine glands .A 77-year-old Caucasian man had undergone trans-nasal sphenoidal surgery for a nonsecreting pituitary macroadenoma in 2001, without remnant or endocrine deficits. In 2016, he was operated for a shoulder melanoma. In February 2018, imaging evaluation demonstrated metastases in lung, liver, and femur. Therefore, treatment with pembrolizumab (anti-programmed death 1) was scheduled in May 2018, but, before starting this therapy, a brain computed tomography performed for a sudden loss of consciousness detected a sellar mass of 17 × 12 mm, which extended to the pituitary stalk and compressed the optic chiasma. Focused magnetic resonance imaging confirmed the size and characteristics of the lesion, while emergency evaluation of the hormonal profile demonstrated an impairment of adrenal and thyroid function. The pituitary lesion demonstrated a remarkable shrinkage (8 × 6 mm), which was confirmed by subsequent imaging evaluations.This is the first case reporting on effectiveness of immune checkpoint inhibitors in a patient with pituitary metastasis from a melanoma. Pituitary metastases (PMs) are rare, accounting for 1% of all pituitary tumors and 0.4% of all intracranial metastatic tumors, respectively . They moClinically, PMs present with signs/symptoms in about 20% of patients only, occurring in end-stage disease . The mosRegarding PM treatment, surgery by trans-nasal sphenoidal (TNS) approach is often the first-line therapy, mainly considered for the relief of symptoms rather than for survival improvement that otherwise depends on the primary tumor . RadiothImmune checkpoint inhibitors (ICIs) are novel drugs targeting the molecular regulators of immune response to boost it against tumor tissue: anti-cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed death 1 (PD-1), and its ligand (PD-1L) , 13. So Herein, we describe one case of PM that benefited greatly from treatment with an anti-PD1 (pembrolizumab) for advanced melanoma.A 77-year-old Caucasian male was referred to our endocrine unit in June 2018, for evaluation of a suspected PM from melanoma.In 2001, the patient had been diagnosed with a nonsecreting pituitary macroadenoma of 25 mm in maximum diameter with suprasellar extension. He had been treated with TNS surgery in another center, and follow-up MRI studies did not demonstrate any residual tissue, up to 5 years after intervention, and pituitary function was preserved. Unfortunately, these images from the previous patient’s history are not available since he moved from another town.In 2016, he had undergone surgical resection of a melanoma of the right shoulder, and then he was regularly followed up by an oncologist in another hospital, reporting no relapse after surgery.Keytruda, MSD), 2 mg/kg in cycles of four intravenous infusions each with a 3-week interval, was scheduled. Nevertheless, at the beginning of May 2018 before starting this treatment, a sudden onset of nausea and dizziness followed by loss of consciousness required an emergency brain computed tomography (CT) evaluation, which revealed the appearance a sellar mass of about 17 × 12 mm. A focused sellar MRI confirmed the presence of this lesion, isointense with the surrounding parenchyma in T1-weighted sequences and extending from the anterior pituitary towards the peduncle and the optical chiasma , normal heart rate at 72 beats per minute, and slightly high blood pressure (145/95 mmHg). Endocrine evaluation showed mild hyperprolactinemia (prolactin (PRL) 482 µIU/ml, normal range 86–324 µIU/ml) and panhypopituitarism, with an insufficient dose of thyroid hormone and an excess of urinary free cortisol (UFC) indicating corticosteroid overtreatment  and CT scans performed in August 2018 after one cycle of pembrolizumab infusions revealed reduced metabolic activity and morphovolumetric stability of the other known secondary lesions. The head-MRI study performed in September 2018 demonstrated a significant volume decrease (47%) of the pituitary lesion, thus confirming its secondary nature. In fact, only a paramedian left 8 × 6 × 6 mm remnant was described, without alterations in the surrounding structures , hypoadrenalism , sexual dysfunction , or headache or visual field impairment in a patient with neoplasia. DI is a frequent manifestation of PMs (about 50% of cases) since the posterior pituitary is directly perfused by the inferior hypophyseal arteries, lacking a portal system like the anterior lobe and thus being more susceptible to hematogenous spread. On the contrary, anterior pituitary deficiency is reported in 25–45% of cases, mainly manifesting itself with central hypothyroidism and secondary adrenal insufficiency , 5. Howeet al. reporting two cases from the authors’ practice, the lesions were characterized by similar histopathological features, appearing hemorrhagic with an Mib1 proliferation index of 15–20%, and one of them also presented with scattered deposits of melanin and BRAFV600E mutation positivity [There are few reports in the literature regarding pituitary metastases from melanoma. In a recent paper by Mattogno sitivity . PMs frositivity . The tensitivity , 19.plus anti-PD1/PD1L inhibitors) [ICIs are successfully prolonging patients’ survival in the context of advanced solid tumors, but their use is often accompanied with autoimmune endocrine adverse events that are more frequent with the “oldest” anti-CTLA4  than with the newest anti-PD1 and anti-PD1L , with the latter potentially causing more toxicity at the thyroid level . The patibitors) .The case we present is paradigmatic for several reasons. First, the patient had already been treated for a pituitary macroadenoma almost a decade before, with neither evidence of remnant tissue nor hormone deficits for several years post-surgery. Second, the patient had demonstrated signs of pituitary dysfunction  before starting pembrolizumab, consistently with the mass effect exerted by the pituitary lesion. Third, pembrolizumab treatment dramatically led to pituitary mass shrinkage, confirming its secondary nature without any significant endocrine or metabolic side effect.Some evidence of the efficacy exerted by novel anticancer treatments at the pituitary/central nervous system level is emerging in the literature, such as a recent report describing the responsiveness to ICIs by a pituitary adrenocorticotropic hormone (ACTH)-secreting carcinoma . In thisplus pembrolizumab) [V600E, an intriguing therapeutical perspective is represented by BRAF inhibitors  that could add another pharmacological approach to these lesions [PMs can be included in the broader category of brain metastases (BMs), which complicate neoplastic diseases in 8–10% of cases, with the highest cumulative incidence in melanoma . In theslizumab) , 28. Thelizumab) . Besideslizumab) . An emerlizumab) . A recenlizumab) . Of notelizumab) . However lesions .In conclusion, this clinical case reminds us of the possibility of PMs originating not only from breast or lung cancer as in most cases, but also from other tumors like melanoma, thus highlighting the importance of accurate follow-up of cancer patients to identify potential pituitary involvement. It generally occurs at an advanced stage, and the onset is often sudden and dramatic, due to hormonal dysfunction and/or mass effect. Finally, this report, for the first time, sheds light on the role of ICIs in the management of PMs from “ICI-sensitive” malignancies, considering that often surgery is not feasible in such patients. In these cases, targeting the immune checkpoints could significantly prolong the OS rate. Larger studies comparing the role of ICIs, alone or combined with each other or with RT in PMs from “ICI-sensitive” cancers, should be carried on to extend their use in these rare and complicated clinical settings.


Deanship of Scientific Research of King Abdul-Aziz University, D-1025-253-1443, was not included.In the original publication , the funResults section, Paragraph Number 8, where the word “positive” has been corrected to “negative”:A correction has also been made to the The general arithmetic mean value for the axis of negative attitudes was 3.43 out of 5.0, which means that the average was located in the fourth category of the Likert scale. Thus, the respondents mostly selected “agree.” The arithmetic mean value of the degrees of approval ranged between 2.76 and 3.91, which means that the averages were between “agree” and “uncertain”.Paragraph Number 2 from the Results section, where duplicated paragraph was removed:Another correction has also been made to Knowledge assessment was divided into four main sections: nutrition and diabetes; nutrition and obesity; nutrition and heart diseases; and nutrition and other diseases. The study participants’ responses to the questions on the nutrition and diabetes axis showed that correct answers accounted for 55.6% of the questions, and wrong answers accounted for 44.4%, which illustrated the difference in their knowledge about this subject. The question that most of the participants answered correctly (81.0%) was about the complication of prednisone therapy that requires nutritional intervention. Most of the students (76.0%) did not know the correct answer to the question on how many grams of carbohydrate are contained in one serving unit.The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.


This paper presents a Brix sensor based on the differential pressure measurement principle. Two piezoresistive silicon pressure sensors were applied to measure the specific gravity of the liquid, which was used to calculate the Brix level. The pressure sensors were mounted inside custom-built water-tight housings connected together by fixed length metallic tubes containing the power and signal cables. Two designs of the sensor were prepared; one for the basic laboratory testing and validation of the proposed system and the other for a fermentation experiment. For lab tests, a sugar solution with different Brix levels was used and readings from the proposed sensor were compared with a commercially available hydrometer called Tilt. During the fermentation experiments, fermentation was carried out in a 1000 L tank over 7 days and data was recorded and analysed. In the lab experiments, a good linear relationship between the sugar content and the corresponding Brix levels was observed. In the fermentation experiment, the sensor performed as expected but some problems such as residue build up were encountered. Overall, the proposed sensing solution carries a great potential for continuous monitoring of the Brix level in liquids. Due to the usage of low-cost pressure sensors and the interface electronics, the cost of the system is considered suitable for large scale deployment at wineries or juice processing industries. In many industries, such as distillery, juice processing, maple and corn syrup production, the outcome always relies on a solution with sugar  as the major solute. Similarly, in brewing and wine making, the final fermented product is dependent on the starting sugar content of the must and the way it changes during fermentation. Therefore, it is essential to monitor the sugar content in these industries, preferably in a continuous manner . DissolvHydrometer is the most commonly used, hand-held, and economical device to measure SG. However, it includes a manual measurement process that can cause an incorrect reading of the meniscus. In addition, temperature compensation is required in the final reading as its operation is temperature dependent ,12,13. TSeveral in-line or continuous monitoring Brix sensors have also been developed. Among them, vis/NIR spectroscopy is a widely used method, especially in wineries, as it can allow the additional benefit of composition analysis ,18. It iThere are commercially available in-line measurement sensors, such as Liquiphant M vibrating fork, Fermetrol probe, Micro-LDS, and Tilt hydrometer . LiquiphIn this paper, we present a low-cost liquid Brix measurement sensor based on the differential pressure measurement principle. The sensor is intended for continuous measurement of Brix to monitor processes such as wine fermentation, brewing, etc. The use of differential pressure for level  and densP is the pressure, g is the acceleration of gravity with a value of 9.8 m/s2, and h is is the level of the liquid in the container. Equation ∘Bx=Pressure sensor selection is the most important step when designing the proposed Brix sensor. The major criteria considered while selecting the pressure sensor are the pressure range, measurement resolution, output interface, and packaging type. The pressure range is dependent upon the depth at which the sensors are immersed in the testing tank. In the process industry, tank height can vary from 1 m up to 5 m. Hence, a pressure sensor with a range of 0 to 25 psi is desired if the sensors are immersed in the tank for the full depth. However, this is not required for Brix measurement as the sugar content is uniform in the whole tank and measurements at any depth are representative of the solution’s Brix value. This means a pressure sensor with a smaller range can be used in the proposed method of measurement.−2. However, at 0.1 °Bx the density changes to 1000.39 g/L, producing a pressure of 2.8458 psi. The difference of pressures (2.8458–2.8447) is 0.0011.To obtain the accuracy of ± 0.1 °Bx, the preferred resolution of the pressure sensor needs to be 0.001 psi. This is determined by calculating the pressure at a fixed depth for 0 °Bx and 0.1 °Bx. For example, at 2 m depth, the pressure is 2.8447 psi at 0 °B if the density of the liquid is 1000 g/L and the acceleration of gravity is 9.80665 msThe output interface and packaging type of the pressure sensor are important as they relate to how the sensors are interfaced with the measurement electronics and mounted inside the housing. Sensors with analogue or digital interfaces are available and can be used; however, an analogue sensor requires analogue to digital conversion (ADC) and other signal conditioning for interfacing with a microcontroller. The packaging type can be flush mount or port type, but flush mount is preferred as it is less susceptible to residue build up and clogging. Furthermore, the sensor should be suitable for liquid media and temperature-compensated, with an operating temperature range up to 80 °C. Sensor MPRLS0025PA00001A is a port type sensor with digital output. In real life application, residues/ material can be stuck in the port, which can cause fouling of the sensor, and cleaning of the sensor will become challenging. Some additional packaging may be required to avoid this problem. Hence, preference is given to the TE connectivity sensors. The output of the sensor 86-015G-C is analogue voltage, which requires additional signal conditioning circuitry . On the other hand, 86BSD015PG provides digital output directly in response to the applied pressure. Due to the digital signal output, flush mount, and lower cost compared to 86-015G-C, 86BSD015PG is selected.2C port is directly connected to the I2C bus of the Arduino board. The SD card is used to store the sensor’s output and it is connected to the SPI bus of the Arduino board. The block diagram of the measuring circuit is shown in The pressure read from the sensors is processed in the microcontroller to calculate the density and Brix value of the liquid. The data is stored on the SD card in the CSV format. The flow chart in In general, pressure sensors measure stress over a surface area (diaphragm) that is not totally uniform over the measurement range of the sensors and leads to non-linear output. Therefore, least squares regression is selected and implemented in the embedded algorithm to improve the linearity.y represents the theoretical value of the sensor, x is the actual value of the sensor, a is the gradient, and b is an intercept of the linear polynomial.To obtain the best fit of the line, the following linear polynomial is usedThe least squares regression minimizes the error between the each theoretical data and calibration data. Therefore, the partial derivatives of a and b are zero and are expressed as (9)a=NΣxN is the number of points,From the sensors’ actual values, the sensors’ linear polynomials are calculated, as shown in Initially, all six pressure sensors were calibrated against the standard pressure. The results of the calibrations are shown in The experiment was performed under the atmospheric pressure condition. The Brix sensor a was immA commercially available hydrometer Tilt  was alsoAs mentioned earlier, the SG values of the proposed sensor were evaluated with the commercially available Tilt hydrometer’s output. After achieving satisfactory results in the sugar monitoring experiments, fermentation monitoring was performed to observe the sensor functionality. Fermentation was performed in a tank using a sugar solution and yeast. For this purpose, a 1000 L sugar solution was prepared by mixing 200 kg sugar into 1000 L of water. The mixture was circulated overnight with the help of a pump to dissolve sugar. The following morning specific gravity of the solution was measured with the help of a traditional hydrometer, which was 1.0848. The yeast was activated using lukewarm water and added to the sugar solution to start the process of fermentation. The proposed sensor illustrated in During fermentation, the sugar present in the solution was transformed into ethanol due to the presence of yeasts. The process released carbon dioxide as a by-product. The sugar content started to decrease as fermentation progressed. The decrease in sugar level was continuously recorded in terms of the SG level by the proposed sensor. Additionally, every morning the SG of the solution was measured using a hydrometer, a Tilt hydrometer, and the proposed sensor  for compThe curves in During fermentation monitoring, four sensors were used. Each of the sensors were connected 250 mm apart from each other. This allowed us to observe the effect of increasing the distance between two sensors on the Brix measurement. It is clear from Continuous in-line Brix monitoring is important in many industries, but a suitable and low-cost method does not exist. The available sample-based methods for measuring the liquid Brix value are manual, time-consuming, and most importantly cannot be the solution for continuous monitoring. On the other hand, existing in-line measurement methods are expensive or too cumbersome to use.The Brix sensor we have presented is based on the measurement of differential pressure in the liquid. The sensor is fully automatic due to the use of an embedded microcontroller. The Brix sensor uses two pressure sensors for differential pressure measurement. Hence, the key element of the Brix sensor is the linearity and accuracy of the pressure sensors. The least-squares regression model is used to provide the linear compensation of the proposed Brix sensor. The experimental results suggest that the SG and Brix measurement in an open tank is feasible and accurate. It is independent of the liquid level in the tank. The linearization caused an improvement in the accuracy of the SG value. The results of the proposed sensor in sugar monitoring experiment are compared with a commercially available Tilt hydrometer. The proposed sensor shows the SG value more accurately than the Tilt hydrometer. The Brix value of the sensor shows some offset that exists due to the offset present in the pressure sensors.To show the industrial application of the proposed Brix sensor, it was tested in a real fermentation experiment. The sensor successfully monitored the changing levels of Brix over a period of seven days. Multiple pressure sensors were mounted on the mounting rod to study the effect of the distance between the sensors on the resulting Brix values. It was found that the measurements were independent of the distance between the sensors used for the differential pressure calculation.This is significantly important as it allows the proposed sensor to be used as long as it is fully immersed in the liquid.Problems such as residue build up and sensor failure due to liquid ingress were found and need to be remedied for application of the proposed sensor in actual industrial applications. Nevertheless, the sensor has proven to be a successful design and a low-cost method of monitoring Brix in liquid solutions. We assumed a homogeneous temperature in the experimental tank due to its small size. However, in future, we aim to include the temperature compensation and focus on removing the offset with the help of software algorithms. Additionally, the interface electronics will be modified to include IoT features in the sensor.