DOCSLIB.ORG

Hedgehog–FGF Signaling Axis Patterns Anterior Mesoderm During Gastrulation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Hedgehog–FGF Signaling Axis Patterns Anterior Mesoderm During Gastrulation Hedgehog–FGF signaling axis patterns anterior mesoderm during gastrulation Alexander Guzzettaa,b,c, Mervenaz Koskaa,b,c, Megan Rowtona,b,c, Kaelan R. Sullivand,e,f, Jessica Jacobs-Lia,b,c, Junghun Kweona,b,c, Hunter Hidalgoa,b,c, Heather Eckartg, Andrew D. Hoffmanna,b,c, Rebecca Backg, Stephanie Lozanog, Anne M. Moond,e,f,1, Anindita Basug,h,1, Michael Bressani,1, Sebastian Pottc,1, and Ivan P. Moskowitza,b,c,1,2 aDepartment of Pediatrics, University of Chicago, Chicago, IL 60637; bDepartment of Pathology, University of Chicago, Chicago, IL 60637; cDepartment of Human Genetics, University of Chicago, Chicago, IL 60637; dDepartment of Molecular and Functional Genomics, Geisinger Clinic, Weis Center for Research, Danville, PA 17822; eDepartment of Pediatrics, University of Utah, Salt Lake City, UT 84112; fDepartment of Human Genetics, University of Utah, Salt Lake City, UT 84112; gDepartment of Medicine, University of Chicago, Chicago, IL 60637; hCenter for Nanoscale Materials, Argonne National Laboratory, Lemont, IL 60439; and iDepartment of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC 27599 Edited by Janet Rossant, Hospital for Sick Children, University of Toronto, Toronto, ON, Canada, and approved May 1, 2020 (received for review August 20, 2019) The mechanisms used by embryos to pattern tissues across their and truncation of GLI2/3 proteins to repressive isoforms GLI2R/ axes has fascinated developmental biologists since the founding 3R, repressing Hh target gene transcription (13, 14). Hh signaling of embryology. Here, using single-cell technology, we interro- participates in the patterning of a diverse array of structures in- gate complex patterning defects and define a Hedgehog (Hh)– cluding the Drosophila wing disk (15), cnidarian pharyngeal mus- fibroblast growth factor (FGF) signaling axis required for anterior culature (16), tetrapod forelimb (17), vertebrate central nervous mesoderm lineage development during gastrulation. Single-cell system (18), and heart (19–21), although it has not been impli- transcriptome analysis of Hh-deficient mesoderm revealed selec- cated in patterning the early A-P axis in vertebrates. tive deficits in anterior mesoderm populations, culminating in Removal of all Hh signaling through germline deletion of Smo defects to anterior embryonic structures, including the pharyn- in mice revealed an essential role for the Hh pathway in early − − geal arches, heart, and anterior somites. Transcriptional profiling mammalian development (22). Smo / embryos exhibit cardiac of Hh-deficient mesoderm during gastrulation revealed disrup- and somitic defects, absence of the anterior dorsal aorta, and tions to both transcriptional patterning of the mesoderm and failure to establish left/right (L-R) axis asymmetry (22, 23). FGF signaling for mesoderm migration. Mesoderm-specific Fgf4/ Fgf8 double-mutants recapitulated anterior mesoderm defects Furthermore, treatment of zebrafish embryos with a small-molecule and Hh-dependent GLI transcription factors modulated enhancers Smo antagonist, cyclopamine, during early but not late gastrulation at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addi- Significance tion of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic Anterior–posterior axis patterning is a fascinating but poorly node and executed by FGF signals in nascent mesoderm to control understood developmental process. Single-cell sequencing anterior mesoderm patterning. was utilized to interrogate patterning abnormalities in a mesoderm-specific Hedgehog (Hh) pathway mutant, which FGF signaling | mesoderm | embryonic node | Hedgehog signaling | revealed selective anterior mesoderm defects. We found that single-cell sequencing Hh signaling was required for fibroblast growth factor (FGF) pathway activity in nascent mesoderm during gastrulation. ne of the first challenges of metazoan development is the Mouse genetics, cell biology, and genomic experiments in- Ogeneration and distribution of mesoderm across the dicate that FGF signaling acts downstream of Hh signaling in anterior–posterior (A-P) axis during gastrulation (1–4). Pre- nascent mesoderm for anterior mesoderm morphogenesis. This sumptive mesoderm migrates through the primitive streak and is work demonstrates the utility of applying single-cell technol- patterned during migration toward the anterior embryonic pole ogies to resolve complex developmental phenotypes. Here, it (1, 3–7). A-P patterning of the mesoderm is determined by se- revealed a role for Hh signaling from the node in organizing creted signals from the node and primitive streak. These signals anterior mesoderm morphogenesis, independent of its role include members of the nodal, fibroblast growth factor (FGF), in left–right determination, by promoting FGF signaling in and Wnt pathways, all of which are required for the patterning gastrulating mesoderm. of the anterior mesoderm (6, 8). While the requirement for these pathways in gastrulation has been historically defined Author contributions: A.G., M.R., M.B., A.B., and I.P.M. designed research; A.G., M.K., M.R., K.R.S., J.J.-L., J.K., H.H., H.E., A.D.H., R.B., S.L., A.M.M., M.B., and S.P. performed by loss-of-function assays, the multicomponent cross-talk be- research; H.E., A.M.M., A.B., and M.B. contributed new reagents/analytic tools; A.G., M.K., tween signaling axes that dictates A-P patterns remain largely M.R., and S.P. analyzed data; and A.G., M.K., A.M.M., M.B., S.P., and I.P.M. wrote unknown. the paper. The Hedgehog (Hh) signaling pathway is required for the The authors declare no competing interest. morphogenesis of organs derived from all three germ layers in This article is a PNAS Direct Submission. most metazoans (9, 10). Hh signaling was first described in a Published under the PNAS license. classic forward genetic screen for genes that determine A-P Data deposition: Both bulk and single-cell RNA-seq data sets have been deposited in the segment polarity during early Drosophila melanogaster develop- Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession ment (11). Hh pathway activation in mammals is initiated by the nos. GSE147868 and GSE149335). binding of Hh ligands (SHH, IHH, or DHH) to PTCH1, re- 1A.M.M., A.B., M.B., S.P., and I.P.M. contributed equally to this work. lieving its inhibition on Smoothened (SMO). Activated SMO 2To whom correspondence may be addressed. Email: [email protected]. then promotes nuclear translocation of full-length GLI2/3 tran- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ scription factors (TFs) that drive Hh target gene transcription (12, doi:10.1073/pnas.1914167117/-/DCSupplemental. 13). Conversely, the absence of Hh signaling triggers proteolysis First published June 19, 2020. 15712–15723 | PNAS | July 7, 2020 | vol. 117 | no. 27 www.pnas.org/cgi/doi/10.1073/pnas.1914167117 Downloaded by guest on September 29, 2021 Mesp1Cre/+; Wt Smo-/- R26 tdTomato/tdTomato A B R26tdTomato/+ Cre/+ Mesp1 SSC-A X Mesp1Cre/+; tdTomato-A Mesp1+/+;R26Gli3R/+Mesp1Cre/+;R26Gli3R/+ R26Gli3R/Gli3R R26 Gli3R/+ SSC-A YFP-A C mesenchyme 5 10 lateral plate mesoderm cardiac 0 allantois 5 P 2 pharyngeal mesoderm -5 UMA UMAP 2 UMAP somite embryonic mesoderm 0 cranial primitive blood mesenchyme -10 yolk sac endothelium intermediate hepatic stellate cells mesoderm -10 -5 0 5 10 0 5 10 15 UMAP 1 UMAP 1 D E Wt Wt 10 Mut 2744 Mut 0.4 1875 5 0.2 action of cells r DEVELOPMENTAL BIOLOGY UMAP 2 UMAP F 751 454 187 124 168 79 420 467 65 358 246 0 418 316 0.0 265 cardiac somite cranial 0 5 10 15 allantois lateral plate pharyngeal UMAP 1 mesenchyme mesoderm mesoderm intermediatemesoderm Cluster mesenchyme FGaSHF pSHF CM Isl1 250 Wnt2 250 Isl1 200 200 Mef2c 150 150 Fgf10 100 100 10 Fgf8 10 50 50 Nkx2-6 0 0 Dusp14 Wnt2 P 2 Tgfb2 5 5 Gata4 UMAP 2 UMAP Tbx5 UMA Sfrp5 Sfrp1 0 0 Myh7 2 Myh6 1 Myl3 0 Ttn 0 5 10 15 0 5 10 15 Nkx2-5 −1 UMAP 1 UMAP 1 Tnnt2 −2 Mef2cAHF-Cre;R26R c/+;Smo +/+ Mef2cAHF-Cre;R26R c/+;Smo -/- HI Wt 187 Ht 0.6 Mut V SHF 353 SHF 0.4 At E9.5 236 0.2 66 Fraction of cells 0.0 aSHF pSHF Cluster Fig. 1. scRNA-seq identifies Hh-dependent selective defects to anterior mesoderm progenitor populations. (A) Whole-mount left lateral views of control and − − Hh-mutant embryos comparing defects observed between Smo / mutants (Upper Right) and Mesp1Cre/+;R26Gli3R-IRES-Venus/+ (Mesp1Cre-Gli3R) mutants (Lower Right). (Scale bars, 200 μm.) (B) Experimental schema for collecting mutant Mesp1Cre-Gli3R and control Mesp1Cre/+;R26tdTomato/+ (Mesp1Cre-tdTomato) Mesp1Cre-labeled cells by FACS for scRNA-seq. (C, Left) UMAP plots for all Mesp1Cre-tdTomato and Mesp1Cre-Gli3R cells used in this study including both extraembryonic (gray and light blue) and embryonic (dark blue) mesoderm lineages. (Right) UMAP for embryonic mesoderm color-coded by cell lineage. (D) UMAP for embryonic mesoderm color-coded for either Mesp1Cre-tdTomato (blue) or Mesp1Cre-Gli3R (orange) genotypes. (E) Represents the proportion of Mesp1Cre-tdTomato (blue) and Mesp1Cre-Gli3R (orange) cells in each annotated lineage from D, where the number linked to each column represents absolute cell counts. Mut, mutant. (F) Heatmap of marker gene expression for aSHF (red), pSHF (green), and cardiomyocytes (blue) where each column represents asinglecell.(G) Expression of aSHF and pSHF markers Isl1 and Wnt2, respectively, superimposed on UMAP plots of C.(H) Proportion of Mesp1Cre-tdTomato (blue) and Mesp1Cre-Gli3R (orange) cells across aSHF, pSHF, and cardiomyocyte (CM) populations. (I) Genetic fate map for aSHF-specific Cre (Mef2cAHF-Cre)witha Cre-dependent lacZ reporter (R26R)inSmo+/+ and Smo−/− backgrounds. Images represent the posterior margin of the aSHF at low and high power in the Upper and Lower, respectively. The arrow points to dorsal mesenchyme marker by Mef2cAHFCre, which is absent in the Smo−/− background.
Load more

Recommended publications
  • Loss of the PTCH1 Tumor Suppressor Defines a New
    Banerjee et al. J Transl Med (2019) 17:246 https://doi.org/10.1186/s12967-019-1995-z Journal of Translational Medicine RESEARCH Open Access Loss of the PTCH1 tumor suppressor defnes a new subset of plexiform fbromyxoma Sudeep Banerjee1,2, Christopher L. Corless3, Markku M. Miettinen4, Sangkyu Noh1, Rowan Ustoy1, Jessica L. Davis3, Chih‑Min Tang1, Mayra Yebra1, Adam M. Burgoyne5 and Jason K. Sicklick1* Abstract Background: Plexiform fbromyxoma (PF) is a rare gastric tumor often confused with gastrointestinal stromal tumor. These so‑called “benign” tumors often present with upper GI bleeding and gastric outlet obstruction. It was recently demonstrated that approximately one‑third of PF have activation of the GLI1 oncogene, a transcription factor in the hedgehog (Hh) pathway, via a MALAT1‑GLI1 fusion protein or GLI1 up‑regulation. Despite this discovery, the biology of most PFs remains unknown. Methods: Next generation sequencing (NGS) was performed on formalin‑fxed parafn‑embedded (FFPE) samples of PF specimens collected from three institutions (UCSD, NCI and OHSU). Fresh frozen tissue from one tumor was utilized for in vitro assays, including quantitative RT‑PCR and cell viability assays following drug treatment. Results: Eight patients with PF were identifed and 5 patients’ tumors were analyzed by NGS. An index case had a mono‑allelic PTCH1 deletion of exons 15–24 and a second case, identifed in a validation cohort, also had a PTCH1 gene loss associated with a suspected long‑range chromosome 9 deletion. Building on the role of Hh signaling in PF, PTCH1, a tumor suppressor protein, functions upstream of GLI1. Loss of PTCH1 induces GLI1 activation and down‑ stream gene transcription.
    [Show full text]
  • The Role of Gli3 in Inflammation
    University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Winter 2020 THE ROLE OF GLI3 IN INFLAMMATION Stephan Josef Matissek University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Matissek, Stephan Josef, "THE ROLE OF GLI3 IN INFLAMMATION" (2020). Doctoral Dissertations. 2552. https://scholars.unh.edu/dissertation/2552 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. THE ROLE OF GLI3 IN INFLAMMATION BY STEPHAN JOSEF MATISSEK B.S. in Pharmaceutical Biotechnology, Biberach University of Applied Sciences, Germany, 2014 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In Biochemistry December 2020 This dissertation was examined and approved in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Biochemistry by: Dissertation Director, Sherine F. Elsawa, Associate Professor Linda S. Yasui, Associate Professor, Northern Illinois University Paul Tsang, Professor Xuanmao Chen, Assistant Professor Don Wojchowski, Professor On October 14th, 2020 ii ACKNOWLEDGEMENTS First, I want to express my absolute gratitude to my advisor Dr. Sherine Elsawa. Without her help, incredible scientific knowledge and amazing guidance I would not have been able to achieve what I did. It was her encouragement and believe in me that made me overcome any scientific struggles and strengthened my self-esteem as a human being and as a scientist.
    [Show full text]
  • FGF Signaling Network in the Gastrointestinal Tract (Review)
    163-168 1/6/06 16:12 Page 163 INTERNATIONAL JOURNAL OF ONCOLOGY 29: 163-168, 2006 163 FGF signaling network in the gastrointestinal tract (Review) MASUKO KATOH1 and MASARU KATOH2 1M&M Medical BioInformatics, Hongo 113-0033; 2Genetics and Cell Biology Section, National Cancer Center Research Institute, Tokyo 104-0045, Japan Received March 29, 2006; Accepted May 2, 2006 Abstract. Fibroblast growth factor (FGF) signals are trans- Contents duced through FGF receptors (FGFRs) and FRS2/FRS3- SHP2 (PTPN11)-GRB2 docking protein complex to SOS- 1. Introduction RAS-RAF-MAPKK-MAPK signaling cascade and GAB1/ 2. FGF family GAB2-PI3K-PDK-AKT/aPKC signaling cascade. The RAS~ 3. Regulation of FGF signaling by WNT MAPK signaling cascade is implicated in cell growth and 4. FGF signaling network in the stomach differentiation, the PI3K~AKT signaling cascade in cell 5. FGF signaling network in the colon survival and cell fate determination, and the PI3K~aPKC 6. Clinical application of FGF signaling cascade in cell polarity control. FGF18, FGF20 and 7. Clinical application of FGF signaling inhibitors SPRY4 are potent targets of the canonical WNT signaling 8. Perspectives pathway in the gastrointestinal tract. SPRY4 is the FGF signaling inhibitor functioning as negative feedback apparatus for the WNT/FGF-dependent epithelial proliferation. 1. Introduction Recombinant FGF7 and FGF20 proteins are applicable for treatment of chemotherapy/radiation-induced mucosal injury, Fibroblast growth factor (FGF) family proteins play key roles while recombinant FGF2 protein and FGF4 expression vector in growth and survival of stem cells during embryogenesis, are applicable for therapeutic angiogenesis. Helicobacter tissues regeneration, and carcinogenesis (1-4).
    [Show full text]
  • The Biology of Hepatocellular Carcinoma: Implications for Genomic and Immune Therapies Galina Khemlina1,4*, Sadakatsu Ikeda2,3 and Razelle Kurzrock2
    Khemlina et al. Molecular Cancer (2017) 16:149 DOI 10.1186/s12943-017-0712-x REVIEW Open Access The biology of Hepatocellular carcinoma: implications for genomic and immune therapies Galina Khemlina1,4*, Sadakatsu Ikeda2,3 and Razelle Kurzrock2 Abstract Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is a leading cause of cancer-related death worldwide. It is highly refractory to most systemic therapies. Recently, significant progress has been made in uncovering genomic alterations in HCC, including potentially targetable aberrations. The most common molecular anomalies in this malignancy are mutations in the TERT promoter, TP53, CTNNB1, AXIN1, ARID1A, CDKN2A and CCND1 genes. PTEN loss at the protein level is also frequent. Genomic portfolios stratify by risk factors as follows: (i) CTNNB1 with alcoholic cirrhosis; and (ii) TP53 with hepatitis B virus-induced cirrhosis. Activating mutations in CTNNB1 and inactivating mutations in AXIN1 both activate WNT signaling. Alterations in this pathway, as well as in TP53 and the cell cycle machinery, and in the PI3K/Akt/mTor axis (the latter activated in the presence of PTEN loss), as well as aberrant angiogenesis and epigenetic anomalies, appear to be major events in HCC. Many of these abnormalities may be pharmacologically tractable. Immunotherapy with checkpoint inhibitors is also emerging as an important treatment option. Indeed, 82% of patients express PD-L1 (immunohistochemistry) and response rates to anti-PD-1 treatment are about 19%, and include about 5% complete remissions as well as durable benefit in some patients. Biomarker-matched trials are still limited in this disease, and many of the genomic alterations in HCC remain challenging to target.
    [Show full text]
  • Targeting FXR and FGF19 to Treat Metabolic Diseases—Lessons
    1720 Diabetes Volume 67, September 2018 Targeting FXR and FGF19 to Treat Metabolic Diseases— Lessons Learned From Bariatric Surgery Nadejda Bozadjieva,1 Kristy M. Heppner,2 and Randy J. Seeley1 Diabetes 2018;67:1720–1728 | https://doi.org/10.2337/dbi17-0007 Bariatric surgery procedures, such as Roux-en-Y gastric diabetes (T2D) (1). Clinical data demonstrate that patients bypass (RYGB) and vertical sleeve gastrectomy (VSG), who have undergone RYGB or VSG experience increased are the most effective interventions available for sus- satiety and major glycemic improvements prior to significant tained weight loss and improved glucose metabolism. weight loss, suggesting that metabolic changes as result Bariatric surgery alters the enterohepatic bile acid cir- of these surgeries are essential to the weight loss and glycemic culation, resulting in increased plasma bile levels as well benefits (2). Therefore, it is important to identify the medi- as altered bile acid composition. While it remains unclear ators that play a role in promoting the benefits of these why both VSG and RYGB can alter bile acids, it is possible surgeries with the goal of improving current surgical that these changes are important mediators of the approaches and developing less invasive therapies that effects of surgery. Moreover, a molecular target of bile harness these effects. acid synthesis, the bile acid–activated transcription fac- The effectiveness of bariatric surgery to reduce body tor FXR, is essential for the positive effects of VSG on weight and improve glucose metabolism highlights the weight loss and glycemic control. This Perspective examines the relationship and sequence of events be- important role the gastrointestinal tract plays in regulat- tween altered bile acid levels and composition, FXR ing a wide range of metabolic processes.
    [Show full text]
  • The Roles of Fgfs in the Early Development of Vertebrate Limbs
    Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW The roles of FGFs in the early development of vertebrate limbs Gail R. Martin1 Department of Anatomy and Program in Developmental Biology, School of Medicine, University of California at San Francisco, San Francisco, California 94143–0452 USA ‘‘Fibroblast growth factor’’ (FGF) was first identified 25 tion of two closely related proteins—acidic FGF and ba- years ago as a mitogenic activity in pituitary extracts sic FGF (now designated FGF1 and FGF2, respectively). (Armelin 1973; Gospodarowicz 1974). This modest ob- With the advent of gene isolation techniques it became servation subsequently led to the identification of a large apparent that the Fgf1 and Fgf2 genes are members of a family of proteins that affect cell proliferation, differen- large family, now known to be comprised of at least 17 tiation, survival, and motility (for review, see Basilico genes, Fgf1–Fgf17, in mammals (see Coulier et al. 1997; and Moscatelli 1992; Baird 1994). Recently, evidence has McWhirter et al. 1997; Hoshikawa et al. 1998; Miyake been accumulating that specific members of the FGF 1998). At least five of these genes are expressed in the family function as key intercellular signaling molecules developing limb (see Table 1). The proteins encoded by in embryogenesis (for review, see Goldfarb 1996). Indeed, the 17 different FGF genes range from 155 to 268 amino it may be no exaggeration to say that, in conjunction acid residues in length, and each contains a conserved with the members of a small number of other signaling ‘‘core’’ sequence of ∼120 amino acids that confers a com- molecule families [including WNT (Parr and McMahon mon tertiary structure and the ability to bind heparin or 1994), Hedgehog (HH) (Hammerschmidt et al.
    [Show full text]
  • Hedgehog Interacting Protein (HHIP) Represses Airway Remodeling And
    www.nature.com/scientificreports OPEN Hedgehog interacting protein (HHIP) represses airway remodeling and metabolic reprogramming in COPD‑derived airway smooth muscle cells Yan Li1,2,7*, Li Zhang2,3, Francesca Polverino4, Feng Guo2, Yuan Hao2, Taotao Lao5, Shuang Xu2, Lijia Li2, Betty Pham2, Caroline A. Owen6 & Xiaobo Zhou2,6* Although HHIP locus has been consistently associated with the susceptibility to COPD including airway remodeling and emphysema in genome‑wide association studies, the molecular mechanism underlying this genetic association remains incompletely understood. By utilizing Hhip+/- mice and primary human airway smooth muscle cells (ASMCs), here we aim to determine whether HHIP haploinsufciency increases airway smooth muscle mass by reprogramming glucose metabolism, thus contributing to airway remodeling in COPD pathogenesis. The mRNA levels of HHIP were compared in normal and COPD‑derived ASMCs. Mitochondrial oxygen consumption rate and lactate levels in the medium were measured in COPD‑derived ASMCs with or without HHIP overexpression as readouts of glucose oxidative phosphorylation and aerobic glycolysis rates. The proliferation rate was measured in healthy and COPD‑derived ASMCs treated with or without 2‑DG. Smooth muscle mass around airways was measured by immunofuorescence staining for α‑smooth muscle actin (α‑SMA) in lung sections from Hhip+/- mice and their wild type littermates, Hhip+/+ mice. Airway remodeling was assessed in Hhip+/- and Hhip+/- mice exposed to 6 months of cigarette smoke. Our results show HHIP inhibited aerobic glycolysis and represses cell proliferation in COPD‑derived ASMCs. Notably, knockdown of HHIP in normal ASMCs increased PKM2 activity. Importantly, Hhip+/- mice demonstrated increased airway remodeling and increased intensity of α‑SMA staining around airways compared to Hhip+/+ mice.
    [Show full text]
  • And Fibroblast Growth Factor Receptor Interactions Usin
    www.nature.com/scientificreports OPEN Characterization of clostridium botulinum neurotoxin serotype A (BoNT/A) and fbroblast growth factor receptor interactions using novel receptor dimerization assay Nicholas G. James1, Shiazah Malik2, Bethany J. Sanstrum1, Catherine Rhéaume2, Ron S. Broide2, David M. Jameson1, Amy Brideau‑Andersen2 & Birgitte S. Jacky2* Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an efective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specifc binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand‑receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rHC/A‑induced dimerization of fuorescently tagged FGFR subtypes (FGFR1‑ 3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization. Botulinum neurotoxin type A (BoNT/A) is a 150 kDa metalloenzyme belonging to the family of neurotoxins produced by Clostridium botulinum.
    [Show full text]
  • Markers of Liver Regeneration—The Role of Growth Factors and Cytokines
    Hoffmann et al. BMC Surgery (2020) 20:31 https://doi.org/10.1186/s12893-019-0664-8 RESEARCH ARTICLE Open Access Markers of liver regeneration—the role of growth factors and cytokines: a systematic review Katrin Hoffmann*†, Alexander Johannes Nagel†, Kazukata Tanabe, Juri Fuchs, Karolin Dehlke, Omid Ghamarnejad, Anastasia Lemekhova and Arianeb Mehrabi Abstract Background: Post-hepatectomy liver failure contributes significantly to postoperative mortality after liver resection. The prediction of the individual risk for liver failure is challenging. This review aimed to provide an overview of cytokine and growth factor triggered signaling pathways involved in liver regeneration after resection. Methods: MEDLINE and Cochrane databases were searched without language restrictions for articles from the time of inception of the databases till March 2019. All studies with comparative data on the effect of cytokines and growth factors on liver regeneration in animals and humans were included. Results: Overall 3.353 articles comprising 40 studies involving 1.498 patients and 101 animal studies were identified and met the inclusion criteria. All included trials on humans were retrospective cohort/observational studies. There was substantial heterogeneity across all included studies with respect to the analyzed cytokines and growth factors and the described endpoints. Conclusion: High-level evidence on serial measurements of growth factors and cytokines in blood samples used to predict liver regeneration after resection is still lacking. To address
    [Show full text]
  • In Vitro Differentiation of Human Umbilical Cord Blood Mesenchymal
    Alexandria Journal of Medicine (2017) 53, 167–173 HOSTED BY Alexandria University Faculty of Medicine Alexandria Journal of Medicine http://www.elsevier.com/locate/ajme In vitro differentiation of human umbilical cord blood mesenchymal stem cells into functioning hepatocytes May H. Hasan a, Kamal G. Botros a, Mona A. El-Shahat a,*, Hussein A. Abdallah b, Mohamed A. Sobh c a Anatomy and Embryology Department, Faculty of Medicine, Mansoura University, Egypt b Medical Biochemistry Department, Faculty of Medicine, Mansoura University, Egypt c Experimental Biology, Urology & Nephrology Center, Mansoura University, Egypt Received 29 January 2016; revised 6 May 2016; accepted 14 May 2016 Available online 5 August 2016 KEYWORDS Abstract Mesenchymal stem cells (MSCs) were isolated by gradient density centrifugation from Umbilical cord blood; umbilical cord blood. Spindle-shaped adherent cells were permitted to grow to 70% confluence Mesenchymal stem cells; in primary culture media which was reached by day 12. Induction of differentiation started by cul- Culture; turing cells with differentiation medium containing FGF-4 and HGF. Under hepatogenic condi- Hepatocytes; tions few cuboidal cells appeared in culture on day 7. From day 21 to day 28, most of cells HGF; became small and round. The control negative cells cultured in serum free media showed FGF-4 fibroblast-like morphology. Urea production and protein secretion by the differentiated hepatocyte-like cells were detected on day 21 and increased on day 28. Protein was significantly increased in comparison with control by day 28. The cells became positive for AFP at day 7 and positive cells could still be detected at days 21 and 28.
    [Show full text]
  • Fgf15 Neurons of the Dorsomedial Hypothalamus Control Glucagon Secretion and Hepatic Gluconeogenesis
    Diabetes Volume 70, July 2021 1443 Fgf15 Neurons of the Dorsomedial Hypothalamus Control Glucagon Secretion and Hepatic Gluconeogenesis Alexandre Picard, Salima Metref, David Tarussio, Wanda Dolci, Xavier Berney, Sophie Croizier, Gwena€el Labouebe, and Bernard Thorens Diabetes 2021;70:1443–1457 | https://doi.org/10.2337/db20-1121 The counterregulatory response to hypoglycemia is an between the brain and these peripheral tissues is ensured, essential survival function. It is controlled by an inte- in large part, by the autonomic nervous system. This is grated network of glucose-responsive neurons, which activated in response to changes in the concentration of trigger endogenous glucose production to restore nor- circulating hormones such as insulin, leptin, or ghrelin moglycemia. The complexity of this glucoregulatory and of nutrients such as glucose and lipids. Glucose-re- network is, however, only partly characterized. In a ge- sponsive neurons, which increase their firing activity in netic screen of a panel of recombinant inbred mice we response to hyperglycemia (glucose-excited [GE] neurons) METABOLISM fi previously identi ed Fgf15, expressed in neurons of the or to hypoglycemia (glucose-inhibited [GI] neurons) (1–3), dorsomedial hypothalamus (DMH), as a negative regula- are thought to couple fluctuations in blood glucose con- tor of glucagon secretion. Here, we report on the gener- centrations to the regulation of sympathetic or parasym- ation of Fgf15CretdTomato mice and their use to further pathetic nerve activity. characterize these neurons. We show that they were A major glucoregulatory role of the central nervous sys- glutamatergic and comprised glucose-inhibited and tem is to maintain glycemic levels at a minimum value of glucose-excited neurons.
    [Show full text]
  • Highly Conserved C-Terminal Region of Indian Hedgehog N-Fragment Contributes to Its Auto-Processing and Multimer Formation
    biomolecules Article Highly Conserved C-Terminal Region of Indian Hedgehog N-Fragment Contributes to Its Auto-Processing and Multimer Formation Xiaoqing Wang 1,2 , Hao Liu 3 , Yanfang Liu 2, Gefei Han 2, Yushu Wang 2 , Haifeng Chen 3,*, Lin He 2,* and Gang Ma 1,2,* 1 Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] 2 Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] (Y.L.); [email protected] (G.H.); [email protected] (Y.W.) 3 State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] * Correspondence: [email protected] (H.C.); [email protected] (L.H.); [email protected] (G.M.) Abstract: Hedgehog (HH) is a highly conserved secretory signalling protein family mainly involved in embryonic development, homeostasis, and tumorigenesis. HH is generally synthesised as a precursor, which subsequently undergoes autoproteolytic cleavage to generate an amino-terminal fragment (HH-N), mediating signalling, and a carboxyl-terminal fragment (HH-C), catalysing the Citation: Wang, X.; Liu, H.; Liu, Y.; auto-processing reaction. The N-terminal region of HH-N is required for HH multimer formation Han, G.; Wang, Y.; Chen, H.; He, L.; to promote signal transduction, whilst the functions of the C-terminal region of HH-N remain Ma, G.
    [Show full text]