Transcriptomic, proteomic, and metabolomic landscape of positional memory in the caudal fin of zebrafish.

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.

Wnt/ß-catenin signaling promotes regeneration after adult zebrafish spinal cord injury.

Unlike mammals, zebrafish can regenerate their injured spinal cord and regain control of caudal tissues. It was recently shown that Wnt/ß-catenin signaling is necessary for spinal cord regeneration in the larval zebrafish. However, the molecular mechanisms of regeneration may or may not be conserved between larval and adult zebrafish. To test this, we assessed the role of Wnt/ß-catenin signaling after spinal cord injury in the adult zebrafish. We show that Wnt/ß-catenin signaling is increased after spinal cord injury in the adult zebrafish. Moreover, overexpression of Dkk1b inhibited Wnt/ß-catenin signaling in the regenerating spinal cord of adult zebrafish. Dkk1b overexpression also inhibited locomotor recovery, axon regeneration, and glial bridge formation in the injured spinal cord. Thus, our data illustrate a conserved role for Wnt/ß-catenin signaling in adult and larval zebrafish spinal cord regeneration.

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.

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.

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).

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.

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.

Effects of 3,3'-Iminodipropionitrile on Hair Cell Numbers in Cristae of CBA/CaJ and C57BL/6J Mice.

This study examines absolute hair cell numbers in the cristae of C57BL/6J mice and CBA/CaJ mice from weaning to adulthood as well as the dose required for 3,3'-iminodiproprionitrile (IDPN)-injury of the cristae in C57BL/6J mice and CBA/CaJ mice, the two mouse strains most commonly used by inner ear researchers. In cristae of CBA/CaJ and C57BL/6J mice, no loss of hair cells was observed up to 24 weeks. In both strains, dose-dependent loss of hair cells was observed 7 days after IDPN treatment of 2-month-old mice (IC50 = 16.1 mmol/kg in C57BL/6J mice vs. 25.21 mmol/kg in CBA/CaJ mice). Four-month-old C57BL/6J mice exposed to IDPN developed dose-dependent vestibular dysfunction as indicated by increased activity and circling behavior in open field tests and by failure to swim 7 days after treatment. IDPN-hair cell injury in C57BL/6J mice and CBA/CaJ mice represents a fast and predictable experimental model for the study of vestibular degeneration and a platform for the testing of vestibular therapies.