The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

Chromatin remodelers and lineage-specific factors interact to target enhancers to establish proneurosensory fate within otic ectoderm.

Specification of Sox2+ proneurosensory progenitors within otic ectoderm is a prerequisite for the production of sensory cells and neurons for hearing. However, the underlying molecular mechanisms driving this lineage specification remain unknown. Here, we show that the Brg1-based SWI/SNF chromatin-remodeling complex interacts with the neurosensory-specific transcriptional regulators Eya1/Six1 to induce Sox2 expression and promote proneurosensory-lineage specification. Ablation of the ATPase-subunit Brg1 or both Eya1/Six1 results in loss of Sox2 expression and lack of neurosensory identity, leading to abnormal apoptosis within the otic ectoderm. Brg1 binds to two of three distal 3' Sox2 enhancers occupied by Six1, and Brg1-binding to these regions depends on Eya1-Six1 activity. We demonstrate that the activity of these Sox2 enhancers in otic neurosensory cells specifically depends on binding to Six1. Furthermore, genome-wide and transcriptome profiling indicate that Brg1 may suppress apoptotic factor Map3k5 to inhibit apoptosis. Together, our findings reveal an essential role for Brg1, its downstream pathways, and their interactions with Six1/Eya1 in promoting proneurosensory fate induction in the otic ectoderm and subsequent neuronal lineage commitment and survival of otic cells.

Dynamic changes in cis-regulatory occupancy by Six1 and its cooperative interactions with distinct cofactors drive lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to cis-regulatory elements changes dramatically at cell-state transitions. Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1's downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance.

BACKGROUND: Eya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown. METHODS: We engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo. RESULTS: Eya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death-inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor-specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor-specific expression in response to Six2 activity. CONCLUSIONS: Our results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

Eya2 expression during mouse embryonic development revealed by Eya2lacZ knockin reporter and homozygous mice show mild hearing loss.

BACKGROUND: Eya2 expression during mouse development has been studied by in situ hybridization and it has been shown to be involved skeletal muscle development and limb formation. Here, we generated Eya2 knockout (Eya2- ) and a lacZ knockin reporter (Eya2lacZ ) mice and performed a detailed expression analysis for Eya2lacZ at different developmental stages to trace Eya2lacZ -positive cells in Eya2-null mice. We describe that Eya2 is not only expressed in cranial sensory and dorsal root ganglia, retina and olfactory epithelium, and somites as previously reported, but also Eya2 is specifically detected in other organs during mouse development. RESULTS: We found that Eya2 is expressed in ocular and trochlear motor neurons. In the inner ear, Eya2lacZ is specifically expressed in differentiating hair cells in both vestibular and cochlear sensory epithelia of the inner ear and Eya2-/- or Eya2lacZ/lacZ mice displayed mild hearing loss. Furthermore, we detected Eya2 expression during both salivary gland and thymus development and Eya2-null mice had a smaller thymus. CONCLUSIONS: As Eya2 is coexpressed with other members of the Eya family genes, these results together highlight that Eya2 as a potential regulator may act synergistically with other Eya genes to regulate the differentiation of the inner ear sensory hair cells and the formation of the salivary gland and thymus.

The Eya1 Phosphatase Mediates Shh-Driven Symmetric Cell Division of Cerebellar Granule Cell Precursors.

During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.

Six1 is essential for differentiation and patterning of the mammalian auditory sensory epithelium.

The organ of Corti in the cochlea is a two-cell layered epithelium: one cell layer of mechanosensory hair cells that align into one row of inner and three rows of outer hair cells interdigitated with one cell layer of underlying supporting cells along the entire length of the cochlear spiral. These two types of epithelial cells are derived from common precursors in the four- to five-cell layered primordium and acquire functionally important shapes during terminal differentiation through the thinning process and convergent extension. Here, we have examined the role of Six1 in the establishment of the auditory sensory epithelium. Our data show that prior to terminal differentiation of the precursor cells, deletion of Six1 leads to formation of only a few hair cells and defective patterning of the sensory epithelium. Previous studies have suggested that downregulation of Sox2 expression in differentiating hair cells must occur after Atoh1 mRNA activation in order to allow Atoh1 protein accumulation due to antagonistic effects between Atoh1 and Sox2. Our analysis indicates that downregulation of Sox2 in the differentiating hair cells depends on Six1 activity. Furthermore, we found that Six1 is required for the maintenance of Fgf8 expression and dynamic distribution of N-cadherin and E-cadherin in the organ of Corti during differentiation. Together, our analyses uncover essential roles of Six1 in hair cell differentiation and formation of the organ of Corti in the mammalian cochlea.

Eya-six are necessary for survival of nephrogenic cord progenitors and inducing nephric duct development before ureteric bud formation.

BACKGROUND: Specification of the metanephric mesenchyme is a central step of kidney development as this mesenchyme promotes nephric duct induction to form a ureteric bud near its caudal end. Before ureteric bud formation, the caudal nephric duct swells to form a pseudostratified epithelial domain that later emerges as the tip of the bud. However, the signals that promote the formation of the transient epithelial domain remain unclear. Here, we investigated the early roles of the mesenchymal factor Six family and its cofactor Eya on the initial induction of nephric duct development. RESULTS: The nephrogenic progenitor population is initially present but significantly reduced in mice lacking both Six1 and Six4 and undertakes an abnormal cell death pathway to be completely eliminated by ∼E10.5-E11.0, similar to that observed in Eya1(-/-) embryos. Consequently, the nephric duct fails to be induced to undergo normal proliferation to pseudostratify and form the ureteric bud in Six1(-/-) ;Six4(-/-) or Eya1(-/-) embryos. CONCLUSIONS: Our data support a model where Eya-Six may form a complex to regulate nephron progenitor cell development before metanephric specification and are critical mesenchymal factors for inducing nephric duct development.

Tbx18 is essential for normal development of vasculature network and glomerular mesangium in the mammalian kidney.

Tbx18 has been shown to be essential for ureteral development. However, it remains unclear whether it plays a direct role in kidney development. Here we addressed this by focusing on examining the pattern and contribution of Tbx18+ cells in the kidney and its role in kidney vascular development. Expression studies and genetic lineage tracing revealed that Tbx18 is expressed in renal capsule, vascular smooth muscle cells and pericytes and glomerular mesangial cells in the kidney and that Tbx18-expressing progenitors contribute to these cell types. Examination of Tbx18(-/-) kidneys revealed large reduction in vasculature density and dilation of glomerular capillary loops. While SMA+ cells were reduced in the mutant, PDGFRß+ cells were seen in early capillary loop renal corpuscles in the mutant, but fewer than in the controls, and further development of the mesangium failed. Analysis of kidney explants cultured from E12.5 excluded the possibility that the defects observed in the mutant were caused by ureter obstruction. Reduced proliferation in glomerular tuft and increased apoptosis in perivascular mesenchyme were observed in Tbx18(-/-) kidneys. Thus, our analyses have identified a novel role of Tbx18 in kidney vasculature development.

EYA1 and SIX1 drive the neuronal developmental program in cooperation with the SWI/SNF chromatin-remodeling complex and SOX2 in the mammalian inner ear.

Inner ear neurogenesis depends upon the function of the proneural basic helix-loop-helix (bHLH) transcription factors NEUROG1 and NEUROD1. However, the transcriptional regulation of these factors is unknown. Here, using loss- and gain-of-function models, we show that EYA1 and SIX1 are crucial otic neuronal determination factors upstream of NEUROG1 and NEUROD1. Overexpression of both Eya1 and Six1 is sufficient to convert non-neuronal epithelial cells within the otocyst and cochlea as well as the 3T3 fibroblast cells into neurons. Strikingly, all the ectopic neurons express not only Neurog1 and Neurod1 but also mature neuronal markers such as neurofilament, indicating that Eya1 and Six1 function upstream of, and in the same pathway as, Neurog1 and Neurod1 to not only induce neuronal fate but also regulate their differentiation. We demonstrate that EYA1 and SIX1 interact directly with the SWI/SNF chromatin-remodeling subunits BRG1 and BAF170 to drive neurogenesis cooperatively in 3T3 cells and cochlear nonsensory epithelial cells, and that SOX2 cooperates with these factors to mediate neuronal differentiation. Importantly, we show that the ATPase BRG1 activity is required for not only EYA1- and SIX1-induced ectopic neurogenesis but also normal neurogenesis in the otocyst. These findings indicate that EYA1 and SIX1 are key transcription factors in initiating the neuronal developmental program, probably by recruiting and interacting with the SWI/SNF chromatin-remodeling complex to specifically mediate Neurog1 and Neurod1 transcription.

SIX1 acts synergistically with TBX18 in mediating ureteral smooth muscle formation.

Dysfunction of the ureter often leads to urine flow impairment from the kidney to the bladder, causing dilation of the ureter and/or renal pelvis. Six1 is a crucial regulator of renal development: mutations in human SIX1 cause branchio-oto-renal (BOR) syndrome and Six1(-/-) mice exhibit renal agenesis, although the ureter is present. It remains unclear whether Six1 plays a role in regulating ureter morphogenesis. We demonstrate here that Six1 is differentially expressed during ureter morphogenesis. It was expressed in undifferentiated smooth muscle (SM) progenitors, but was downregulated in differentiating SM cells (SMCs) and had disappeared by E18.5. In Six1(-/-) mice, the ureteral mesenchymal precursors failed to condense and differentiate into normal SMCs and showed increased cell death, indicating that Six1 is required for the maintenance and normal differentiation of SM progenitors. A delay in SMC differentiation was observed in Six1(-/-) ureters. A lack of Six1 in the ureter led to hydroureter and hydronephrosis without anatomical obstruction when kidney formation was rescued in Six1(-/-) embryos by specifically expressing Six1 in the metanephric mesenchyme, but not the ureter, under control of the Eya1 promoter. We show that Six1 and Tbx18 genetically interact to synergistically regulate SMC development and ureter function and that their gene products form a complex in cultured cells and in the developing ureter. Two missense mutations in SIX1 from BOR patients reduced or abolished SIX1-TBX18 complex formation. These findings uncover an essential role for Six1 in establishing a functionally normal ureter and provide new insights into the molecular basis of urinary tract malformations in BOR patients.

The EYA-SO/SIX complex in development and disease.

Eyes absent (EYA) and Sine oculis (SO/SIX) proteins function as transcriptional activation complexes and play essential roles in organogenesis during embryonic development in regulating cell proliferation and survival and coordination of particular differentiation programs. Mutations of the Eya and So/Six genes cause profound developmental defects in organisms as diverse as flies, frogs, fish, mice, and humans. EYA proteins also possess an intrinsic phosphatase activity, which is essential for normal development. Here, we review crucial roles of EYA and SO/SIX in development and disease in mice and humans.

The role of Eya1 and Eya2 in the taste system of mice from embryonic stage to adulthood.

Members of the Eya family, which are a class of transcription factors with phosphatase activity, are widely expressed in cranial sensory organs during development. However, it is unclear whether these genes are expressed in the taste system during development and whether they play any role in specifying taste cell fate. In this study, we report that Eya1 is not expressed during embryonic tongue development but that Eya1-expressing progenitors in somites or pharyngeal endoderm give rise to tongue musculature or taste organs, respectively. In the Eya1-deficient tongues, these progenitors do not proliferate properly, resulting in a smaller tongue at birth, impaired growth of taste papillae, and disrupted expression of Six1 in the papillary epithelium. On the other hand, Eya2 is specifically expressed in endoderm-derived circumvallate and foliate papillae located on the posterior tongue during development. In adult tongues, Eya1 is predominantly expressed in IP3R3-positive taste cells in the taste buds of the circumvallate and foliate papillae, while Eya2 is persistently expressed in these papillae at higher levels in some epithelial progenitors and at lower levels in some taste cells. We found that conditional knockout of Eya1 in the third week or Eya2 knockout reduced Pou2f3+, Six1+ and IP3R3+ taste cells. Our data define for the first time the expression patterns of Eya1 and Eya2 during the development and maintenance of the mouse taste system and suggest that Eya1 and Eya2 may act together to promote lineage commitment of taste cell subtypes.

Smarca4 deficiency induces Pttg1 oncogene upregulation and hyperproliferation of tubular and interstitial cells during kidney development.

Kidney formation and nephrogenesis are controlled by precise spatiotemporal gene expression programs, which are coordinately regulated by cell-cycle, cell type-specific transcription factors and epigenetic/chromatin regulators. However, the roles of epigenetic/chromatin regulators in kidney development and disease remain poorly understood. In this study, we investigated the impact of deleting the chromatin remodeling factor Smarca4 (Brg1), a human Wilms tumor-associated gene, in Wnt4-expressing cells. Smarca4 deficiency led to severe tubular defects and a shortened medulla. Through unbiased single-cell RNA sequencing analyses, we identified multiple types of Wnt4 Cre-labeled interstitial cells, along with nephron-related cells. Smarca4 deficiency increased interstitial cells but markedly reduced tubular cells, resulting in cells with mixed identity and elevated expression of cell-cycle regulators and genes associated with extracellular matrix and epithelial-to-mesenchymal transition/fibrosis. We found that Smarca4 loss induced a significant upregulation of the oncogene Pttg1 and hyperproliferation of Wnt4 Cre-labeled cells. These changes in the cellular state could hinder the cellular transition into characteristic tubular structures, eventually leading to fibrosis. In conclusion, our findings shed light on novel cell types and genes associated with Wnt4 Cre-labeled cells and highlight the critical role of Smarca4 in regulating tubular cell differentiation and the expression of the cancer-causing gene Pttg1 in the kidney. These findings may provide valuable insights into potential therapeutic strategies for renal cell carcinoma resulting from SMARCA4 deficiency.

Six1 regulates Grem1 expression in the metanephric mesenchyme to initiate branching morphogenesis.

Urinary tract morphogenesis requires subdivision of the ureteric bud (UB) into the intra-renal collecting system and the extra-renal ureter, by responding to signals in its surrounding mesenchyme. BMP4 is a mesenchymal regulator promoting ureter development, while GREM1 is necessary to negatively regulate BMP4 activity to induce UB branching. However, the mechanisms that regulate the GREM1-BMP4 signaling are unknown. Previous studies have shown that Six1-deficient mice lack kidneys, but form ureters. Here, we show that the tip cells of Six1(-/-) UB fail to form an ampulla for branching. Instead, the UB elongates within Tbx18- and Bmp4-expressing mesenchyme. We find that the expression of Grem1 in the metanephric mesenchyme (MM) is Six1-dependent. Treatment of Six1(-/-) kidney rudiments with GREM1 protein restores ampulla formation and branching morphogenesis. Furthermore, we demonstrate that genetic reduction of BMP4 levels in Six1(-/-) (Six1(-/-); Bmp4(+/-)) embryos restores urinary tract morphogenesis and kidney formation. This study uncovers an essential function for Six1 in the MM as an upstream regulator of Grem1 in initiating branching morphogenesis.

Six1 and Six2 of the Sine Oculis Homeobox Subfamily are Not Functionally Interchangeable in Mouse Nephron Formation.

The vertebrate Six1 and Six2 arose by gene duplication from the Drosophila sine oculis and have since diverged in their developmental expression patterns. Both genes are expressed in nephron progenitors of human fetal kidneys, and mutations in SIX1 or SIX2 cause branchio-oto-renal syndrome or renal hypodysplasia respectively. Since ∼80% of SIX1 target sites are shared by SIX2, it is speculated that SIX1 and SIX2 may be functionally interchangeable by targeting common downstream genes. In contrast, in mouse kidneys, Six1 expression in the metanephric mesenchyme lineage overlaps with Six2 only transiently, while Six2 expression is maintained in the nephron progenitors throughout development. This non-overlapping expression between Six1 and Six2 in mouse nephron progenitors promoted us to examine if Six1 can replace Six2. Surprisingly, forced expression of Six1 failed to rescue Six2-deficient kidney phenotype. We found that Six1 mediated Eya1 nuclear translocation and inhibited premature epithelialization of the progenitors but failed to rescue the proliferation defects and cell death caused by Six2-knockout. Genome-wide binding analyses showed that Six1 selectively occupied a small subset of Six2 target sites, but many Six2-bound loci crucial to the renewal and differentiation of nephron progenitors lacked Six1 occupancy. Altogether, these data indicate that Six1 cannot substitute Six2 to drive nephrogenesis in mouse kidneys, thus demonstrating that the difference in physiological roles of Six1 and Six2 in kidney development stems from both transcriptional regulations of the genes and divergent biochemical properties of the proteins.

SIX1/EYA1 are novel liver damage biomarkers in chronic hepatitis B and other liver diseases.

BACKGROUND: This study aimed to investigate the clinicopathological significance of sine oculis homeobox homolog 1 (SIX1) and eyes absent 1 (EYA1) in patients with chronic hepatitis B (CHB) and other liver diseases. METHODS: SIX1 and EYA1 levels were detected in human serum and liver tissues by enzyme linked immunosorbent assay (ELISA) and immunofluorescent staining method, respectively. RESULTS: The serum SIX1 and EYA1 levels in 313 CHB patients were 7.24±0.11 and 25.21±0.51 ng/mL, respectively, and these values were significantly higher than those in 33 healthy controls (2.84±0.15 and 13.11±1.01 ng/mL, respectively; P<0.05). Serum SIX1 and EYA1 levels were also markedly increased in patients with numerous other liver diseases, including liver fibrosis, hepatocellular carcinoma, fatty liver disease, alcoholic liver disease, fulminant hepatic failure, autoimmune liver disease, and hepatitis C, compared to the healthy controls (P<0.05). Dynamic observation of these proteins over time in 35 selected CHB patients revealed that SIX1 and EYA1 serum levels increased over an interval. Immunofluorescent staining revealed that both SIX1 and EYA1 were only expressed in hepatic stellate cells (HSCs), and their increased expression was evident in CHB liver tissue. CONCLUSIONS: SIX1 and EYA1 are novel biomarkers of liver damage in patients of CHB and other liver diseases, with potential clinical utility.

Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4.

Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1(-/-) embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1(-/-) OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.

Eya1 gene dosage critically affects the development of sensory epithelia in the mammalian inner ear.

Haploinsufficiency of the transcription co-activator EYA1 causes branchio-oto-renal syndrome, congenital birth defects that account for as many as 2% of profoundly deaf children; however, the underlying cause for its dosage requirement and its specific role in sensory cell development of the inner ear are unknown. Here, an allelic series of Eya1 were generated to study the basis of Eya1 dosage requirements for sensory organ development. Our results show different threshold requirements for the level of Eya1 in different regions of the inner ear. Short and disorganized hair cell sterocilia was observed in wild-type/null heterozygous or hypomorphic/hypomorphic homozygous cochleae. Patterning and gene-marker analyses indicate that in Eya1 hypomorphic/null heterozygous mice, a reduction of Eya1 expression to 21% of normal level causes an absence of cochlear and vestibular sensory formation. Eya1 is initially expressed in the progenitors throughout the epithelium of all six sensory regions, and later on during sensory cell differentiation, its expression becomes restricted to the differentiating hair cells. We provide genetic evidence that Eya1 activity, in a concentration-dependent manner, plays a key role in the regulation of genes known to be important for sensory development. Furthermore, we show that Eya1 co-localizes with Sox2 in the sensory progenitors and both proteins physically interact. Together, our results indicate that Eya1 appears to be upstream of very early events during the sensory organ development, hair cell differentiation and inner-ear patterning. These results also provide a molecular mechanism for understanding how hypomorphic levels of EYA1 cause inner-ear defects in humans.

Using Sox2 to alleviate the hallmarks of age-related hearing loss.

Age-related hearing loss (ARHL) is the most prevalent sensory deficit. ARHL reduces the quality of life of the growing population, setting seniors up for the enhanced mental decline. The size of the needy population, the structural deficit, and a likely research strategy for effective treatment of chronic neurosensory hearing in the elderly are needed. Although there has been profound advancement in auditory regenerative research, there remain multiple challenges to restore hearing loss. Thus, additional investigations are required, using novel tools. We propose how the (1) flat epithelium, remaining after the organ of Corti has deteriorated, can be converted to the repaired-sensory epithelium, using Sox2. This will include (2) developing an artificial gene regulatory network transmitted by (3) large viral vectors to the flat epithelium to stimulate remnants of the organ of Corti to restore hair cells. We hope to unite with our proposal toward the common goal, eventually restoring a functional human hearing organ by transforming the flat epithelial cells left after the organ of Corti loss.