The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Dual role for Sox2 in specification of sensory competence and regulation of Atoh1 function

The formation of inner ear sensory epithelia is believed to occur in two steps, initial specification of sensory competent (prosensory) regions followed by determination of specific cell‐types, such as hair cells (HCs) and supporting cells. However, studies in which the HC determination factor Atoh1 was ectopically expressed in nonprosensory regions indicated that expression of Atoh1 alone is sufficient to induce HC formation suggesting that prosensory formation may not be a prerequisite for HC development. To test this hypothesis, interactions between Sox2 and Atoh1, which are required for prosensory and HC formation respectively, were examined. Forced expression of Atoh1 in nonprosensory cells resulted in transient expression of Sox2 prior to HC formation, suggesting that expression of Sox2 is required for formation of ectopic HCs. Moreover, Atoh1 overexpression failed to induce HC formation in Sox2 mutants, confirming that Sox2 is required for prosensory competence. To determine whether expression of Sox2 alone is sufficient to induce prosensory identity, Sox2 was transiently activated in a manner that mimicked endogenous expression. Following transient Sox2 activation, nonprosensory cells developed as HCs, a result that was never observed in response to persistent expression of Sox2. These results, suggest a dual role for Sox2 in inner ear formation. Initially, Sox2 is required to specify prosensory competence, but subsequent down‐regulation of Sox2 must occur to allow Atoh1 expression, most likely through a direct interaction with the Atoh1 promoter. These results implicate Sox2‐mediated changes in prosensory cells as an essential step in their ability to develop as HCs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 3–13, 2017     

Sox2 and Fgf interact with Atoh1 to promote sensory competence throughout the zebrafish inner ear

Atoh1 is required for differentiation of sensory hair cells in the vertebrate inner ear. Moreover, misexpression of Atoh1 is sufficient to establish ectopic sensory epithelia, making Atoh1 a good candidate for gene therapy to restore hearing. However, competence to form sensory epithelia appears to be limited to discrete regions of the inner ear. To better understand the developmental factors influencing sensory-competence, we examined the effects of misexpressing atoh1a in zebrafish embryos under various developmental conditions. Activation of a heat shock-inducible transgene, hs:atoh1a, resulted in ectopic expression of early markers of sensory development within 2 h, and mature hair cells marked by brn3c:GFP began to accumulate 9 h after heat shock. The ability of atoh1a to induce ectopic sensory epithelia was maximal when activated during placodal or early otic vesicle stages but declined rapidly thereafter. At no stage was atoh1a sufficient to induce sensory development in dorsal or lateral non-sensory regions of the otic vesicle. However, co-misexpression of atoh1a with fgf3, fgf8 or sox2, genes normally acting in the same gene network with atoh1a, stimulated sensory development in all regions of the otic vesicle. Thus, expression of fgf3, fgf8 or sox2 strongly enhances competence to respond to Atoh1.     

<Emphasis Type="Italic">Lmx1a</Emphasis> is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a ^dr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a “cochlear-gravistatic” endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants. This work was supported by grants from the NCRR/COBRE (P20 RR 018788; D.H.N.) and NIH (RO1 DC 005590; B.F.). Parts of this investigation were conducted in a facility constructed with support of a Research Facilities Improvement Program Grant from the National Center for Research Resources, National Institutes of Health. We acknowledge the use of the confocal microscope facility of the NCCB, supported by EPSCoR EPS-0346476 (CFD 47.076), and of the University of Nebraska microarray facility, supported by NCRR/COBRE.     

Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea

Background

At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation.

Methodology/Principal Findings

We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells.

Conclusions/Significance

Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 might actually be mediated in large part by Neurod1. We suggest that Neurod1 is regulated by both Neurog1 and Atoh1 and provides a negative feedback for either gene. Through this and other feedback, Neurod1 suppresses alternate fates of neurons to differentiate as hair cells and regulates hair cell subtypes.     

Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity

Background

The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.

Methodology/Principal Findings

We have investigated the role of Prox1 in the developing mouse ear taking advantage of available standard and conditional Prox1 mutant mouse strains using Tg(Pax2-Cre) and Tg(Nes-Cre). A severe reduction in the size of the canal cristae but not of other vestibular organs or the cochlea was identified in the E18.5 Prox1^Flox/Flox; Tg(Pax2-Cre) mutant ear. In these mutant embryos, hair cell differentiated; however, their distribution pattern was slightly disorganized in the cochlea where the growth of type II nerve fibers to outer hair cells along Prox1 expressing supporting cells was severely disrupted. In the case of Nestin-Cre, we found that newborn Prox1^Flox/Flox; Tg(Nestin-Cre) exhibit only a disorganized innervation of outer hair cells despite apparently normal cellular differentiation of the organ of Corti, suggesting a cell-autonomous function of Prox1 in neurons.

Conclusions/Significance

These results identify a dual role of Prox1 during inner ear development; growth of the canal cristae and fiber guidance of Type II fibers along supporting cells in the cochlea.     

Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear

Sox2 has been variously implicated in maintenance of pluripotent stem cells or, alternatively, early stages of cell differentiation, depending on context. In the developing inner ear, Sox2 initially marks all cells in the nascent sensory epithelium and, in mouse, is required for sensory epithelium formation. Sox2 is eventually downregulated in hair cells but is maintained in support cells, the functional significance of which is unknown. Here we describe regulation and function of sox2 in the zebrafish inner ear. Expression of sox2 begins after the onset of sensory epithelium development and is regulated by Atoh1a/b, Fgf and Notch. Knockdown of sox2 does not prevent hair cell production, but the rate of accumulation is reduced due to sporadic death of differentiated hair cells. We next tested the capacity for hair cell regeneration following laser ablation of mature brn3c:gfp-labeled hair cells. In control embryos, regeneration of lost hair cells begins by 12 h post-ablation and involves transdifferentiation of support cells rather than asymmetric cell division. In contrast, regeneration does not occur in sox2-depleted embryos. These data show that zebrafish sox2 is required for hair cell survival, as well as for transdifferentiation of support cells into hair cells during regeneration.     

Effects of DAPT and Atoh1 overexpression on hair cell production and hair bundle orientation in cultured Organ of Corti from neonatal rats

Background

In mammals, hair cells do not undergo spontaneous regeneration when they are damaged and result in permanent hearing loss. Previous studies in cultured Organ of Corti dissected from neonatal animals have shown that both DAPT (r-secretase inhibitor in the Notch signal pathway) treatment and Atoh1 overexpression can induce supernumerary hair cells. The effects of simultaneous DAPT treatment and Atoh1 over expression in the cells of cultured Organ of Corti from neonatal rats are still obscure.

Principal Findings

In this study, we set out to investigate the interaction of DAPT treatment and Atoh1 overexpression as well as culture time and the location of basilar fragment isolated form neonatal rat inner ear. Our results showed that DAPT treatment induced more hair cells in the apical turn, while Atoh1 overexpression induced more extra hair cells in the middle turn of the cultured Organ of Corti. When used together, their effects are additive but not synergistic. In addition, the induction of supernumerary hair cells by both DAPT and Atoh1 overexpression is dependent on the treatment time and the location of the cochlear tissue. Moreover, DAPT treatment causes dramatic changes in the orientation of the stereociliary bundles of hair cells, whereas Atoh1 overexpression didn''t induce drastic change of the polarity of stereociliary bundles.

Conclusions/Significance

Taken together, these results suggest that DAPT treatment are much more potent in inducing supernumerary hair cells than Atoh1 overexpression and that the new hair cells mainly come from the trans-differentiation of supporting cells around hair cells. The orientation change of stereociliary bundle of hair cells may be attributed to the insertion of the newly formed hair cells. The immature hair bundles on the newly formed hair cells may also contribute to the overall chaos of the stereociliary bundle of the sensory epithelia.     

Activated Notch Causes Deafness by Promoting a Supporting Cell Phenotype in Developing Auditory Hair Cells

Purpose

To determine whether activated Notch can promote a supporting cell fate during sensory cell differentiation in the inner ear.

Methods

An activated form of the Notch1 receptor (NICD) was expressed in early differentiating hair cells using a Gfi1-Cre mouse allele. To determine the effects of activated Notch on developing hair cells, Gfi1-NICD animals and their littermate controls were assessed at 5 weeks for hearing by measuring auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). The differentiation of NICD-expressing hair cells was assessed at postnatal day (P) 6, 11 and 20, using histological and molecular markers for hair cells, as well as supporting cells/progenitor cells. We also examined whether the effects of Notch were mediated by SOX2, a gene expressed in supporting cells and a likely downstream target of Notch, by crossing an inducible form of SOX2 to the Gfi1-Cre.

Results

Activation of Notch1 in developing auditory hair cells causes profound deafness. The NICD-expressing hair cells switch off a number of hair cell markers and lose their characteristic morphology. Instead, NICD-expressing hair cells adopt a morphology resembling supporting cells and upregulate a number of supporting cell markers. These effects do not appear to be mediated by SOX2, because although expression of SOX2 caused some hearing impairment, the SOX2-expressing hair cells did not downregulate hair cell markers nor exhibit a supporting cell-like phenotype.

Conclusions

Our data show that Notch signaling inhibits hair cell differentiation and promotes a supporting cell-like phenotype, and that these effects are unlikely to be mediated by SOX2.