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Hedgehog–FGF Signaling Axis Patterns Anterior Mesoderm During Gastrulation

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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- DEVELOPMENTAL BIOLOGY 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 www.pnas.org/cgi/doi/10.1073/pnas.1914167117 PNAS Latest Articles | 1of12 Downloaded by guest on September 27, 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 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.
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