RESEARCH ARTICLE FGF signaling refines Wnt gradients to regulate the patterning of taste papillae Michaela Prochazkova 1,2 , Teemu J. Ha ̈ kkinen 3 , Jan Prochazka 1,2 , Frantisek Spoutil 2 , Andrew H. Jheon 1 , Youngwook Ahn 4 , Robb Krumlauf 4,5 , Jukka Jernvall 3, * and Ophir D. Klein 1,6, * ABSTRACT The patterning of repeated structures is a major theme in developmental biology, and the inter-relationship between spacing and size of such structures is an unresolved issue. Fungiform papillae are repeated epithelial structures that house taste buds on the anterior tongue. Here, we report that FGF signaling is a crucial regulator of fungiform papillae development. We found that mesenchymal FGF10 controls the size of the papillary area, while overall patterning remains unchanged. Our results show that FGF signaling negatively affects the extent of canonical Wnt signaling, which is the main activation pathway during fungiform papillae development; however, this effect does not occur at the level of gene transcription. Rather, our experimental data, together with computational modeling, indicate that FGF10 modulates the range of Wnt effects, likely via induction of Sostdc1 expression. We suggest that modification of the reach of Wnt signaling could be due to local changes in morphogen diffusion, representing a novel mechanism in this tissue context, and we propose that this phenomenon might be involved in a broader array of mammalian developmental processes. KEY WORDS: FGF, Wnt, Tongue, Taste papilla INTRODUCTION Taste is one of the fundamental senses in vertebrates and is crucial for discrimination between nutritious substances and potentially toxic ones. The basic taste structures of mammals consist of clusters of neuroepithelial receptor cells called taste buds. Although taste buds can be found at various locations within the oral cavity, including on the palate or epiglottis, most reside on the dorsal surface of the tongue. Taste buds are housed in highly organized structures called taste papillae. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found on the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Among members of a given species, there is high intra-individual variability in the number of taste buds and in the number of taste cells within the taste bud in fungiform papillae; this is often connected to the terms ‘supertaster’ or ‘nontaster’ in humans (Bartoshuk et al., 1994; Miller and Reedy, 1990). Therefore, the number and size of the fungiform papillae are important for taste quality, and understanding the process of fungiform papillae patterning is required for determining how tastes are perceived. The general rules of patterning of functional structures such as taste papillae or hair follicles are central aspects of developmental biology. Over the past few decades, research using rodent models has shown that the patterning and distribution of mature fungiform papillae is driven by specific cellular and molecular mechanisms that occur during prenatal tongue development (Chaudhari and Roper, 2010; Kapsimali and Barlow, 2013). This process starts at embryonic day (E) 12.5 in mouse by formation of epithelial thickenings called taste placodes. From E13.5 to E14.5, the placodes evaginate, forming raised structures called papillae. Around birth, taste bud cells differentiate within the mature papillae (Kaufman, 1992; Mistretta and Liu, 2006). Canonical Wnt/β-catenin signaling has been identified as a positive effector of fungiform papillae development. In vitro treatment with LiCl, an activator of Wnt signaling via GSK3 inhibition, makes papillae both larger and more numerous, whereas overexpression of the Wnt antagonist Dkk1 or inactivation of β-catenin cause a severe decrease or complete elimination of papillae (Liu et al., 2007). The Wnt ligand WNT10B has been proposed to play a major role in placode formation, and the fungiform papillae are reduced but not completely eliminated in Wnt10b-null mice (Iwatsuki et al., 2007). Another Wnt ligand reported to be expressed in tongue epithelium during the fungiform patterning period is WNT10A (Liu et al., 2007), and mutations in Wnt10a cause marked reduction of fungiform papillae in humans (Adaimy et al., 2007). Whereas canonical Wnt signaling is known to induce the development of fungiform papillae, multiple pathways have been shown to inhibit their formation. One of the best studied of these is the SHH pathway. Shh expression lies downstream of Wnt signaling and was described to function in a negative-feedback loop with Wnt (Iwatsuki et al., 2007). SHH has both long- and short-range inhibitory effects on taste papillae, as blocking of SHH activity by cyclopamine, jervine or the anti-SHH 5E1 antibody leads to both larger and more numerous papillae (Hall et al., 2003; Iwatsuki et al., 2007; Liu et al., 2004). Despite these inhibitory effects during development, Shh expression also serves as a reliable and widely used marker of the fungiform placodes (Iwatsuki et al., 2007; Liu et al., 2004, 2013). SHH also plays an important role in the differentiation of taste cells. Lineage tracing experiments revealed that cells of taste placodes expressing Shh are taste cell progenitors, but do not contribute to the rest of the mature papilla epithelium, which forms later (Thirumangalathu et al., 2009). After E16 in rat Received 19 December 2016; Accepted 28 April 2017 1 Department of Orofacial Sciences and Program in Craniofacial Biology, University of California San Francisco, San Francisco, CA 94143, USA. 2 Institute of Molecular Genetics of the CAS, v. v. i., Czech Centre for Phenogenomics and Laboratory of Transgenic Models of Diseases, Division BIOCEV, Prumyslova 595, Vestec 252 42, Czech Republic. 3 Developmental Biology Program, Institute of Biotechnology, University of Helsinki, PO Box 56, Helsinki FIN-00014, Finland. 4 Stowers Institute for Medical Research, Kansas City, MO 64110, USA. 5 Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA. 6 Department of Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, CA 94143, USA. *Authors for correspondence ( jernvall@fastmail.fm; ophir.klein@ucsf.edu) M.P., 0000-0002-3773-8995; J.J., 0000-0001-6575-8486; O.D.K., 0000-0002- 6254-7082 2212 © 2017. Published by The Company of Biologists Ltd | Development (2017) 144, 2212-2221 doi:10.1242/dev.148080 DEVELOPMENT