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      Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face

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          Abstract

          The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways − Jagged-Notch, Endothelin1 (Edn1), and Bmp − regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face.

          Author Summary

          The exquisite functions of the vertebrate face require the precise formation of its underlying bones. Remarkably, many of the genes required to shape the facial skeleton are the same from fish to man. In this study, we use the powerful zebrafish system to understand how the skeletal components of the face acquire different shapes during development. To do so, we analyze a series of mutants that disrupt patterning of the facial skeleton, and then assess how the genes affected in these mutants control cell fate in skeletal progenitor cells. From these genetic studies, we found that several pathways converge to control when and where progenitor cells commit to a cartilage fate, thus controlling the size and shape of cartilage templates for the later-arising bones. Our work thus reveals how regulating the timing of when progenitor cells make skeleton helps to shape the bones of the zebrafish face. As mutations in many of the genes studied are implicated in human craniofacial defects, differences in the timing of progenitor cell differentiation may also explain the wonderful diversity of human faces.

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          Most cited references102

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          Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

          A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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            A transcription activator-like effector toolbox for genome engineering.

            Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.
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              A two-color acid-free cartilage and bone stain for zebrafish larvae.

              Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone, which negatively affects bone staining. We have developed an acid-free method to stain cartilage and bone simultaneously in zebrafish larvae. This method has the additional advantage that PCR genotyping of stained specimens is possible.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                8 April 2016
                April 2016
                : 12
                : 4
                Affiliations
                [1 ]Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, University of Southern California Keck School of Medicine, Los Angeles, California, United States of America
                [2 ]Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America
                University of Pennsylvania School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LB AA EZ BB JGC. Performed the experiments: LB AA EZ BB PB JTN JGC. Analyzed the data: LB AA JGC. Wrote the paper: LB AA JGC.

                [¤]

                Current address: Department of Biological Chemistry, HHMI, David Geffen School of Medicine, University of California, Los Angeles, California, United States of America

                Article
                PGENETICS-D-15-02374
                10.1371/journal.pgen.1005967
                4825933
                27058748
                c231a21a-568e-4d19-9d0a-9335d4658ca2
                © 2016 Barske et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 10, Tables: 0, Pages: 33
                Product
                Funding
                This work was supported by the National Institute of Dental and Craniofacial Research ( http://www.nidcr.nih.gov/) (R01 DE018405 to JGC and K99/R00 DE024190 to JTN), as well as by the March of Dimes ( http://www.marchofdimes.org) (JGC), the National Institute on Deafness and Other Communication Disorders ( http://www.nidcd.nih.gov/) (5T32DC009975 fellowship to LB) and the A.P. Giannini Foundation ( http://apgianninifoundation.org/) (postdoctoral fellowship to LB). The bioinformatics software and computing resources used in the analysis were funded by the USC Office of Research and the Norris Medical Library. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Cartilage
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Cartilage
                Biology and Life Sciences
                Developmental Biology
                Embryology
                Embryos
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Notch Signaling
                Research and Analysis Methods
                Model Organisms
                Animal Models
                Zebrafish
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Fishes
                Osteichthyes
                Zebrafish
                Biology and life sciences
                Cell biology
                Signal transduction
                Cell signaling
                BMP signaling
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Membrane Receptor Signaling
                Immune Receptor Signaling
                Physical Sciences
                Physics
                Condensed Matter Physics
                Phase Transitions
                Condensation
                Biology and Life Sciences
                Anatomy
                Head
                Face
                Medicine and Health Sciences
                Anatomy
                Head
                Face
                Custom metadata
                RNAseq files have been deposited in NCBI’s Gene Expression Omnibus and are accessible through the GEO Series accession number GSE72985. All other relevant data are contained within the paper and the Supporting Information files.

                Genetics
                Genetics

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