Fbxo2 mouse and embryonic stem cell reporter lines delineate in vitro-generated inner ear sensory epithelia cells and enable otic lineage selection and Cre-recombination

Abstract

While the mouse has been a productive model for inner ear studies, a lack of highly specific genes and tools has presented challenges. The absence of definitive otic lineage markers and tools is limiting in vitro studies of otic development, where innate cellular heterogeneity and disorganization increase the reliance on lineage-specific markers. To address this challenge in mice and embryonic stem (ES) cells, we targeted the lineage-specific otic gene Fbxo2 with a multicistronic reporter cassette (Venus/Hygro/CreER = VHC). In otic organoids derived from ES cells, Fbxo2 specifically delineates otic progenitors and inner ear sensory epithelia. In mice, Venus expression and CreER activity reveal a cochlear developmental gradient, label the prosensory lineage, show enrichment in a subset of type I vestibular hair cells, and expose strong expression in adult cerebellar granule cells. We provide a toolbox of multiple spectrally distinct reporter combinations for studies that require use of fluorescent reporters, hygromycin selection, and conditional Cre-mediated recombination.

Application of Mesenchymal Stem Cell Therapy and Inner Ear Regeneration for Hearing Loss: A Review

“…Several factors must be considered when establishing an organoid. Pax2 [52] or Fbxo2 [53] can induce organoid formation to model otic induction, which is a pivotal developmental event when the preplacodal tissue adopts otic fate.…”

Deafness-in-a-dish: modeling hereditary deafness with inner ear organoids

“…PSC-derived inner ear organoids, in contrast, comprise not only hair cell-and support cell-like cells, but also neuron-like cells and distinct regions of PAX8 + non-sensory otic-like epithelium and TFAP2A + /SLUG + periotic-like mesenchyme (Bouchard et al 2010;Koehler et al 2013Koehler et al , 2017. To date, all publications describing the successful generation of PSC-derived inner ear organoids have built upon the foundations of the stepwise induction protocol described by Koehler et al (2013) (see DeJonge et al 2016;Hartman et al 2018;Koehler et al 2017;Liu et al 2016;Perny et al 2017;Schaefer et al 2018;Tang et al 2019). PSC-derived aggregates are treated with BMP-4 and the TGF-beta inhibitor SB-431542, which promote the specification of non-neural ectoderm, while simultaneously inhibiting the formation of mesendoderm.…”

“…Expressed in mouse HC-like cells by day 17 (Hartman et al 2018) Expressed in mouse HCs as early as E13.5 (Xiang et al 1998)…”

“…Expressed in mouse otic-like vesicles at day 14, restricted to HC-like cells by day 15 (DeJonge et al 2016;Hartman et al 2018;Koehler et al 2013;Liu et al 2016;Perny et al 2017;Schaefer et al 2018;Tang et al 2019); expressed in human HC-like cells by day 60 (Koehler et al 2017) Expressed in mouse HCs as early as E13.5 (Xiang et al 1998) Usher syndrome type 1B…”

Inner ear organoids: new tools to understand neurosensory cell development, degeneration and regeneration

“…It is less clear what drives the differences in outcomes in these protocols (Koehler et al, 2017). The generation of reporter lines to track lineage differentiation would lead to improved protocols, with higher efficiency and yield (Hartman et al, 2018;Koehler et al, 2017;Schaefer et al, 2018). The rapidly evolving field of gene editing will surely lead to further implementation of these tools (Nie and Hashino, 2017).…”