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 otic neuroblasts and for patterning and proliferation of the epithelium, leading to normal morphogenesis of both the cochlear and vestibular domains. Furthermore, the first genome-wide identification of proximal targets of FGFR2b signaling in the early otocyst reveals novel candidate genes for inner ear development and function.

Slc26a9P2ACre, a new CRE driver to regulate gene expression in the otic placode lineage and other FGFR2b-dependent epithelia

“…6C,C'), and was similar to all of 3 Fgf10 -/or 4 Fgf10;Tg(Pax2-cre) CKO cochleae examined previously ( Fig. 6G and Urness et al, 2015;Urness et al, 2018). Another cochlea had a shortened Reissner's membrane and lacked the outer sulcus ( Fig.…”

“…Fgf10 is required for inner ear morphogenesis, but germline null or conditional mutants generated with Tg(Pax2-cre) do not survive past birth (Min et al, 1998;Sekine et al, 1999;Urness et al, 2018). To assess postnatal Fgf10 inner ear phenotypes we generated Fgf10 conditional mutants using Slc26a9 P2ACre and observed motor behavior and measured ABR thresholds.…”

“…To evaluate cochlear development in Fgf10;Slc26a9 P2ACre CKOs relative to previous studies of Fgf10 -/and Fgf10;Tg(Pax2-cre) CKOs (Urness et al, 2018;Urness et al, 2015), we examined histologic cross sections of CKO and littermate control cochleae at E18.5 ( Fig. 6B-G).…”

“…To assess the utility of Slc26a9 P2ACre for activating gene expression, we crossed Rosa26 lslrtTA/lslrtTA to Slc26a9 P2ACre/+ ;Tg(dnFgfr2b)/0 mice and treated pregnant/lactating females from E17.5-P3 with DOX. The cross generated all 4 expected genotypes, of which only one (Slc26a9 P2ACre/+ ;Rosa26 lslrtTA/+ ;Tg(dnFgfr2b)/0) was expected to express dnFGFR2b, a ligand trap that inhibits signaling by FGF3 and FGF10 in the inner ear (Urness et al, 2018). Offspring were tested at 3-5 weeks for ABR thresholds.…”

“…The utility of this strain for postnatal studies of otic genes, however, can be limited when modulating the expression of a given gene-of-interest in an extraotic portion of the CRE lineage affects viability. For example, we studied embryonic inner ear development following inactivation of Fgf10 in the Tg(Pax2-cre) lineage (Urness et al, 2018), but could not analyze such conditional knockout (CKO) mice postnatally because of P0 lethality, which could be caused by loss of Fgf10 in the developing kidney (Michos et al, 2010), a component of the Tg(Pax2-cre) lineage (Ohyama and Groves, 2004). Similarly, we have been unable to study postnatal Fgf8;Tg(Pax2-cre) CKO mice due to P0 lethality (unpublished data) suspected to be caused by the requirement for Fgf8 in the developing mid/hindbrain (Chi et al, 2003), another component of the Tg(Pax2-cre) lineage (Ohyama and Groves, 2004) or in the kidney (Perantoni et al, 2005).…”