Toshinori Hayashi

Fgf20 Is Required for Sensory Epithelial Specification in the Developing Cochlea

Tissue-specific deletion of Fgfr1 results in severe defects in the development of both hair cells and support cells (Pirvola et al., 2002). Despite the importance of Fgfr1 in this early phase of cochlear development, the timing for the requirement for FGF signaling at this stage is not known. Therefore, we investigated the requirement for FGF signaling at early stages of cochlear development using an FGF receptor inhibitor. We find that inhibition of FGF signaling from embryonic day 14 (E14) to E16 has a dramatic effect on the development of the sensory epithelium, causing a severe reduction in hair cells and support cells, similar to that reported for the Fgfr1 deletion. The effects of inhibition of FGF signaling on sensory specification are not explained by increases in cell death or changes in proliferation but lead to a rapid reduction in Pea3 and Erm and a loss of Math1 expression. We also show that a specific FGF, FGF20, is the likely ligand for FGFR1 at this sensory specification phase of cochlear development; Fgf20 is expressed at the right time and place to mediate sensory cell specification, and blocking FGF20 with a specific antibody inhibits hair cell and support cell development in a manner similar to the FGF receptor inhibitor. Our results thus define the period of FGF-dependent sensory cell specification and the ligand that mediates this step in cochlear development.

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea.

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.

Acheate-scute like 1 (Ascl1) is required for normal delta-like (Dll) gene expression and notch signaling during retinal development

Delta gene expression in Drosophila is regulated by proneural basic helix–loop–helix (bHLH) transcription factors, such as acheate‐scute. In vertebrates, multiple Delta‐like and proneural bHLH genes are expressed during neurogenesis, especially in the retina. We recently uncovered a relationship between Acheate‐scute like 1 (Ascl1), Delta‐like genes, and Notch in chick retinal progenitors. Here, we report that mammalian retinal progenitors are also the primary source of Delta‐like genes, likely signaling through Notch among themselves, while differentiating neurons expressed Jagged2. Ascl1 is coexpressed in Delta‐like and Notch active progenitors, and required for normal Delta‐like gene expression and Notch signaling. We also reveal a role for Ascl1 in the regulation of Hes6, a proneurogenic factor that inhibits Notch signaling to promote neural rather than glial differentiation. Thus, these results suggest a molecular mechanism whereby attenuated Notch levels coupled with reduced proneurogenic activity in progenitors leads to increased gliogenesis and decreased neurogenesis in the Ascl1‐deficient retina. Developmental Dynamics 238:2163–2178, 2009.

Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A. Detailed evaluation of inner ear phenotype in the Clrn1 knockout mouse (Clrn1(-/-)) coupled with expression pattern of Clrn1 in the inner ear are presented here. Clrn1 was expressed as early as embryonic day 16.5 in the auditory and vestibular hair cells and associated ganglionic neurons, with its expression being higher in outer hair cells (OHCs) than inner hair cells. Clrn1(-/-) mice showed early onset hearing loss that rapidly progressed to severe levels. Two to three weeks after birth (P14-P21), Clrn1(-/-) mice showed elevated auditory-evoked brainstem response (ABR) thresholds and prolonged peak and interpeak latencies. By P21, approximately 70% of Clrn1(-/-) mice had no detectable ABR and by P30 these mice were deaf. Distortion product otoacoustic emissions were not recordable from Clrn1(-/-) mice. Vestibular function in Clrn1(-/-) mice mirrored the cochlear phenotype, although it deteriorated more gradually than cochlear function. Disorganization of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cell dysfunction and prolonged peak latencies in vestibular and cochlear evoked potentials in Clrn1(-/-) mice strongly indicate that Clrn1 is necessary for hair cell function and associated neural activation.

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.

CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.

Dll3 is expressed in developing hair cells in the mammalian cochlea

Notch mediates the process of lateral inhibition that controls the production of hair cells in the inner ear. Hair cells are known to express Notch ligands Dll1 and Jag2, which signal through Notch1 in adjacent supporting cells. However, recent genetic and pharmacological studies indicate that the level of Notch‐mediated lateral inhibition is greater than can be accounted for by Dll1 and Jag2. Here, we report that another Notch ligand, Dll3, is expressed in developing hair cells, in a pattern that overlaps that of Dll1 and Jag2. We analyzed the cochleae of Dll3pu mutant mice, but did not detect any abnormalities. However, earlier studies have demonstrated that there is functional redundancy among Notch ligands in cochlear development and loss of one ligand can be at least partially compensated for by another. Thus Dll3 may play a role in lateral inhibition similar to that of Dll1 and Jag2. Developmental Dynamics 236:2875–2883, 2007.

Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration.

Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator‐like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single‐cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.

Expression patterns of FGF receptors in the developing mammalian cochlea

Many studies have shown the importance of the fibroblast growth factor (FGF) family of factors in the development of the mammalian cochlea. There are four fibroblast growth factor receptors (FGFR1–4) and all four are expressed in the cochlea during development. While there are examples in the literature of expression patterns of some of the receptors at specific stages of cochlear development there has been no systematic study. We have assembled a full analysis of the patterns of receptor expression during cochlear development for all four Fgfrs using in situ hybridization. We have analyzed the expression patterns from embryonic day 13.5 through postnatal ages. We find that Fgfr1, 2, and 3 are expressed in the epithelium of the cochlear duct and Fgfr4 is limited in its expression to the mesenchyme surrounding the duct. We compare the receptor expression pattern to markers of the sensory domain (p27kip1) and the early hair cells (math1). Developmental Dynamics 239:1019–1026, 2010.