Phoenix Is Required for Mechanosensory Hair Cell Regeneration in the Zebrafish Lateral Line Martine Behra1, John Bradsher2, Rachid Sougrat3, Viviana Gallardo 4 , Miguel L. Allende4, Shawn M. Burgess1* 1 National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States of America, 2 National Cancer Institute, Bethesda, Maryland, United States of America, 3 National Institute of Child Health and Human Development, Bethesda, Maryland, United States of America, 4 Center for Genomics of the Cell, Facultad de Ciencias, Universidad de Chile, Santiago, Chile Abstract In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear’s neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration. Citation: Behra M, Bradsher J, Sougrat R, Gallardo V, Allende ML, et al. (2009) Phoenix Is Required for Mechanosensory Hair Cell Regeneration in the Zebrafish Lateral Line. PLoS Genet 5(4): e1000455. doi:10.1371/journal.pgen.1000455 Editor: Mary C. Mullins, University of Pennsylvania School of Medicine, United States of America Received September 3, 2008; Accepted March 16, 2009; Published April 17, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This research was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health (SB). MLA was supported by FONDECYT (1070867) and ICM (P06-037F). VG received a CONICYT Fellowship and a travel grant from the Vicerrector´ıa de Asuntos Acade´micos, Departamento de Postgrado y Post´ıtulo. Part of this work was supported by the Intramural Program of Eunice Kennedy Shriver National Institute of Child Health and Human Development. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: burgess@mail.nih.gov Introduction During development of the vertebrate inner ear, a subset of neuroepithelial cells specialize to give rise to hair cells and supporting cells [1–3]. These two cell populations assume distinct and complementary functions. The hair cell is a highly differentiated mechanoreceptor cell, transducing sound waves in the cochlea or acceleration and head movements in the vestibular organ, into electrical signals [4,5]. The supporting cells provide cohesive support [6,7] and have secretory functions [8,9]. More importantly, they have been clearly implicated in the addition of new hair cells, both during normal growth and during restoration of the sensory epithelium after damage in various animal models [10–14]. Thus, among supporting cells, there exists a tissue- specific population of progenitor cells. However, in mammals, this ‘‘stem cell like’’ property is lost shortly after birth in most neuroepithelia of the inner ear [15]. With the exception of some limited regeneration in a sub-region of the vestibular organ [16,17], post natal hair cell loss is permanent and irreversible. Consequently, a large majority of deafness cases in humans are linked to absent or damaged hair cells. To restore the regenerative capacity of supporting cells is an obvious therapeutic aim, but our understanding of the regenerative process is incomplete. Because birds, amphibians, reptiles, and fish have retained the ability to regenerate hair cells [14,18–25] they provide opportunities to find genes involved in the regeneration process and its maintenance. Fish and amphibians have an additional organ related to the inner ear called the lateral line, which is used to detect water currents [26–28]. It is a superficial organ running along each side of the body which consists of stereotypically distributed patches of neuroepithelium, called neuromasts. These are discrete organs made of hair cells that project into the aqueous environment and supporting cells that surround them. In the zebrafish embryo, the lateral line neuromasts first appear in the head approximately 2 days post fertilization (dpf) and in parallel begin to develop along the entire length of the trunk and tail [29]. Hair cells are fully PLoS Genetics | www.plosgenetics.org 1 April 2009 | Volume 5 | Issue 4 | e1000455