Onset of taste bud cell renewal starts at birth and coincides with a shift in SHH function.

Embryonic taste bud primordia are specified as taste placodes on the tongue surface and differentiate into the first taste receptor cells (TRCs) at birth. Throughout adult life, TRCs are continually regenerated from epithelial progenitors. Sonic hedgehog (SHH) signaling regulates TRC development and renewal, repressing taste fate embryonically, but promoting TRC differentiation in adults. Here, using mouse models, we show TRC renewal initiates at birth and coincides with onset of SHHs pro-taste function. Using transcriptional profiling to explore molecular regulators of renewal, we identified Foxa1 and Foxa2 as potential SHH target genes in lingual progenitors at birth and show that SHH overexpression in vivo alters FoxA1 and FoxA2 expression relevant to taste buds. We further bioinformatically identify genes relevant to cell adhesion and cell locomotion likely regulated by FOXA1;FOXA2 and show that expression of these candidates is also altered by forced SHH expression. We present a new model where SHH promotes TRC differentiation by regulating changes in epithelial cell adhesion and migration.

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

SOX2 regulation by hedgehog signaling controls adult lingual epithelium homeostasis.

Adult tongue epithelium is continuously renewed from epithelial progenitor cells, a process that requires hedgehog (HH) signaling. In mice, pharmacological inhibition of the HH pathway causes taste bud loss within a few weeks. Previously, we demonstrated that sonic hedgehog (SHH) overexpression in lingual progenitors induces ectopic taste buds with locally increased SOX2 expression, suggesting that taste bud differentiation depends on SOX2 downstream of HH. To test this, we inhibited HH signaling in mice and observed a rapid decline in Sox2 and SOX2-GFP expression in taste epithelium. Upon conditional deletion of Sox2, differentiation of both taste and non-taste epithelial cells was blocked, and progenitor cell number increased. In contrast to basally restricted proliferation in controls, dividing cells were overabundant and spread to suprabasal epithelial layers in mutants. SOX2 loss in progenitors also led non-cell-autonomously to taste cell apoptosis, dramatically shortening taste cell lifespans. Finally, in tongues with conditional Sox2 deletion and SHH overexpression, ectopic and endogenous taste buds were not detectable; instead, progenitor hyperproliferation expanded throughout the lingual epithelium. In summary, we show that SOX2 functions downstream of HH signaling to regulate lingual epithelium homeostasis.

Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.

The integrity of taste buds is intimately dependent on an intact gustatory innervation, yet the molecular nature of this dependency is unknown. Here, we show that differentiation of new taste bud cells, but not progenitor proliferation, is interrupted in mice treated with a hedgehog (Hh) pathway inhibitor (HPI), and that gustatory nerves are a source of sonic hedgehog (Shh) for taste bud renewal. Additionally, epithelial taste precursor cells express Shh transiently, and provide a local supply of Hh ligand that supports taste cell renewal. Taste buds are minimally affected when Shh is lost from either tissue source. However, when both the epithelial and neural supply of Shh are removed, taste buds largely disappear. We conclude Shh supplied by taste nerves and local taste epithelium act in concert to support continued taste bud differentiation. However, although neurally derived Shh is in part responsible for the dependence of taste cell renewal on gustatory innervation, neurotrophic support of taste buds likely involves a complex set of factors.

The RNA-binding protein LIN28B regulates developmental timing in the mammalian cochlea.

Proper tissue development requires strict coordination of proliferation, growth, and differentiation. Strict coordination is particularly important for the auditory sensory epithelium, where deviations from the normal spatial and temporal pattern of auditory progenitor cell (prosensory cell) proliferation and differentiation result in abnormal cellular organization and, thus, auditory dysfunction. The molecular mechanisms involved in the timing and coordination of auditory prosensory proliferation and differentiation are poorly understood. Here we identify the RNA-binding protein LIN28B as a critical regulator of developmental timing in the murine cochlea. We show that Lin28b and its opposing let-7 miRNAs are differentially expressed in the auditory sensory lineage, with Lin28b being highly expressed in undifferentiated prosensory cells and let-7 miRNAs being highly expressed in their progeny-hair cells (HCs) and supporting cells (SCs). Using recently developed transgenic mouse models for LIN28B and let-7g, we demonstrate that prolonged LIN28B expression delays prosensory cell cycle withdrawal and differentiation, resulting in HC and SC patterning and maturation defects. Surprisingly, let-7g overexpression, although capable of inducing premature prosensory cell cycle exit, failed to induce premature HC differentiation, suggesting that LIN28B's functional role in the timing of differentiation uses let-7 independent mechanisms. Finally, we demonstrate that overexpression of LIN28B or let-7g can significantly alter the postnatal production of HCs in response to Notch inhibition; LIN28B has a positive effect on HC production, whereas let-7 antagonizes this process. Together, these results implicate a key role for the LIN28B/let-7 axis in regulating postnatal SC plasticity.

Euglycemic agent-mediated hypothalamic transcriptomic manipulation in the N171-82Q model of Huntington disease is related to their physiological efficacy.

Our aim was to employ novel analytical methods to investigate the therapeutic treatment of the energy regulation dysfunction occurring in a Huntington disease (HD) mouse model. HD is a neurodegenerative disorder that is characterized by progressive motor impairment and cognitive alterations. Changes in neuroendocrine function, body weight, energy metabolism, euglycemia, appetite function, and gut function can also occur. It is likely that the locus of these alterations is the hypothalamus. We determined the effects of three different euglycemic agents on HD progression using standard physiological and transcriptomic signature analyses. N171-82Q HD mice were treated with insulin, Exendin-4, and the newly developed GLP-1-Tf to determine whether these agents could improve energy regulation and delay disease progression. Blood glucose, insulin, metabolic hormone levels, and pancreatic morphology were assessed. Hypothalamic gene transcription, motor coordination, and life span were also determined. The N171-82Q mice exhibited significant alterations in hypothalamic gene transcription signatures and energy metabolism that were ameliorated, to varying degrees, by the different euglycemic agents. Exendin-4 or GLP-1-Tf (but not insulin) treatment also improved pancreatic morphology, motor coordination, and increased life span. Using hypothalamic transcription signature analyses, we found that the physiological efficacy variation of the drugs was evident in the degree of reversal of the hypothalamic HD pathological signature. Euglycemic agents targeting hypothalamic and energy regulation dysfunction in HD could potentially alter disease progression and improve quality of life in HD.