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      RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

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      PLoS Genetics
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          Abstract

          During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.

          Author Summary

          The vertebrate inner ear is a complex three-dimensional structure with hearing and balance functions. To form a functional ear in the embryo, it is crucial that the right cells develop at the right time and in the right place. These cells include the sensory hair cells that detect sound and movement, neurons that relay sensory information to the brain, and structural cells. We have investigated patterning and maintenance events in the developing ear of the zebrafish embryo. We show that two signalling pathways, FGF and Retinoic Acid (RA), act in an antagonistic manner to regulate the numbers of sensory hair cells that develop, together with the expression of a key gene, otx1b, required for the development of structural cells. However, the two signalling pathways act in concert to regulate the emergence of neuronal cells. Our data also indicate that FGF and RA signalling form a feedback loop, placing them at the heart of the regulatory network that ensures correct patterning is maintained in the ear. Both FGF and RA signalling are employed to generate hair cells and neurons for replacement therapies to treat hearing loss. Understanding the roles of FGF and RA signalling underpins the development of such therapies.

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          Most cited references60

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          nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.

          We report the isolation and identification of a new mutation affecting pigment cell fate in the zebrafish neural crest. Homozygous nacre (nac(w2)) mutants lack melanophores throughout development but have increased numbers of iridophores. The non-crest-derived retinal pigment epithelium is normal, suggesting that the mutation does not affect pigment synthesis per se. Expression of early melanoblast markers is absent in nacre mutants and transplant experiments suggested a cell-autonomous function in melanophores. We show that nac(w2) is a mutation in a zebrafish gene encoding a basic helix-loop-helix/leucine zipper transcription factor related to microphthalmia (Mitf), a gene known to be required for development of eye and crest pigment cells in the mouse. Transient expression of the wild-type nacre gene restored melanophore development in nacre(-/-) embryos. Furthermore, misexpression of nacre induced the formation of ectopic melanized cells and caused defects in eye development in wild-type and mutant embryos. These results demonstrate that melanophore development in fish and mammals shares a dependence on the nacre/Mitf transcription factor, but that proper development of the retinal pigment epithelium in the fish is not nacre-dependent, suggesting an evolutionary divergence in the function of this gene.
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            Evolution of the Fgf and Fgfr gene families.

            Fibroblast growth factors (Fgfs) and Fgf receptors (Fgfrs) comprise a signaling system that is conserved throughout metazoan evolution. Twenty-two Fgfs and four Fgfrs have been identified in humans and mice. During evolution, the Fgf family appears to have expanded in two phases. In the first phase, during early metazoan evolution, Fgfs expanded from two or three to six genes by gene duplication. In the second phase, during the evolution of early vertebrates, the Fgf family expanded by two large-scale gen(om)e duplications. By contrast, the Fgfr family has expanded only in the second phase. However, the acquisition of alternative splicing by Fgfrs has increased their functional diversity. The mechanisms that regulate alternative splicing have been conserved since the divergences of echinoderms and vertebrates. The expansion of the Fgf and Fgfr gene families has enabled this signaling system to acquire functional diversity and, therefore, an almost ubiquitous involvement in developmental and physiological processes.
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              Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension.

              Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.
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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, USA )
                1553-7390
                1553-7404
                December 2014
                4 December 2014
                : 10
                : 12
                : e1004858
                Affiliations
                [1]MRC Centre for Developmental and Biomedical Genetics, Bateson Centre and Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom
                University of Pennsylvania School of Medicine, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: ECM. Performed the experiments: ECM. Analyzed the data: ECM TTW. Wrote the paper: ECM TTW.

                Article
                PGENETICS-D-14-00316
                10.1371/journal.pgen.1004858
                4256275
                25473832
                851753f8-8f75-4da3-ab12-22a6aa328425
                Copyright @ 2014

                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.

                History
                : 5 February 2014
                : 29 October 2014
                Page count
                Pages: 18
                Funding
                This work was funded by an EU Marie Curie Intra European Fellowship (275978) to ECM and a BBSRC Project Grant (BB/J003050) to TTW. The MRC CDBG zebrafish aquaria were supported by the MRC (G0700091). Imaging facilities (GR077544AIA) and the Sanger Institute Zebrafish Mutation Resource (077047/Z/05/Z) were sponsored by the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Research and Analysis Methods
                Model Organisms
                Biology and Life Sciences
                Developmental Biology
                Genetics
                Neuroscience

                Genetics
                Genetics

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