Spatial and temporal inhibition of FGFR2b ligands reveals continuous requirements and novel targets in mouse inner ear morphogenesis

Abstract

Morphogenesis of the inner ear epithelium requires coordinated deployment of several signaling pathways and disruptions cause abnormalities of hearing and/or balance. The FGFR2b ligands, FGF3 and FGF10, are expressed throughout otic development and are required individually for normal morphogenesis, but their prior and redundant roles in otic placode induction complicates investigation of subsequent combinatorial functions in morphogenesis. To interrogate these roles and identify new effectors of FGF3 and FGF10 signaling at the earliest stages of otic morphogenesis, we used conditional gene ablation after otic placode induction and temporal inhibition of signaling with a secreted, dominant-negative FGFR2b ectodomain. We show that both ligands are required continuously after otocyst formation for maintenance of the otic ganglion and patterning and proliferation of the epithelium, leading to normal morphogenesis of both the cochlear and vestibular domains. Furthermore, the first genomewide identification of proximal targets of FGFR2b signaling in the early otocyst reveals novel candidate genes for inner ear development and function.

New developments in the biology of fibroblast growth factors

“…Genetic approaches to manipulate FGF signaling in vivo use mice in which ligands (FGF2, FGF7, FGF9, FGF10, and FGF18) or a constitutively activated FGFR1 can be induced to activate cell non‐autonomous or cell autonomous FGF signaling, respectively (Cilvik et al, 2013; Clark et al, 2001; Koo et al, 2018; Tichelaar et al, 2000; Volckaert et al, 2017; White et al, 2006; Whitsett et al, 2002). Similarly, induced expression of dominant‐negative FGFR1 or FGFR2b or the FGFR downstream pathway inhibitor SPRY4, allow cell non‐autonomous or cell autonomous, respectively, suppression of FGF signaling (Eckenstein et al, 2006; Parsa et al, 2008; Perl et al, 2003; Urness et al, 2018). In zebrafish, a transgenic allele encoding a dominant‐negative Fgfr1a with a fluorescent tag (fgfr1‐dn‐cargo) placed under combined Cre/lox and heat shock control allows spatiotemporal perturbation of FGF signaling (Kirchgeorg et al, 2018).…”

“…Mice lacking Fgf10 have a shortened and narrower cochlear duct, as well as defects in non‐sensory regions of the inner ear (Munnamalai & Fekete, 2020; Urness et al, 2015). Inhibition of FGF3 and FGF10 at the otic placode stage with a soluble FGFR2b transgene show that both ligands are required after otocyst formation for maintenance of otic neuroblasts and for patterning and proliferation of the epithelium of both the cochlear and vestibular domains (Urness et al, 2018).…”

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish

“…Another candidate factor for the upregulation of PAX2 is FGF signalling. FGF10 is expressed in the ventromedial part of the otocyst, which is a PAX2-positive area, and hindbrain-derived FGF3 is important for the development of the cochlea [ 43 ]. Therefore, to investigate the possible roles of FGF3 and FGF10 in otic placode induction, we started to culture Day 14 cells in the presence of FGF3/FGF10 as well as LDN and CHIR.…”

Inner Ear Anomalies in Children With Apert Syndrome: A Radiological and Audiological Analysis

“…Mouse model studies have demonstrated the importance of FGFR2b ligands FGF3 and FGF10 in the development of the inner ear including otocyst patterning and vestibular and cochlea morphogenesis. 10 The authors demonstrate various morphological variations of the inner ear structure brought about by altering FGF3 and FGF10 expression. 10 We hypothesize that the inner ear appearances in our patient population arise from a similar embryological mechanism.…”

“…10 The authors demonstrate various morphological variations of the inner ear structure brought about by altering FGF3 and FGF10 expression. 10 We hypothesize that the inner ear appearances in our patient population arise from a similar embryological mechanism.…”