Judith D. Speck

Growth factors in combination, but not individually, rescue rd mouse photoreceptors in organ culture.

The rd mouse retina is an animal model for human retinal dystrophy in which the rod photoreceptors undergo apoptosis during the first 4 weeks in vivo or in organ culture. We have examined the effect of different families of trophic factors on the survival of rd mouse photoreceptors in organ culture. Retinas were harvested from rd mice at postnatal day 2 and grown in organ culture for 27 days in vitro (DIV) in DMEM with 10% fetal calf serum. Ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), glial cell line-derived neurotrophic factor (GDNF), neurturin, and persephon were added individually or in combination to the medium at a dose of 50 ng/ml or less. CNTF + BDNF in combination resulted in photoreceptor survival comparable to wild-type retinas after 27 DIV. CNTF + FGF2 or CNTF + GDNF produced a partial prevention of photoreceptor death. Photoreceptor degeneration was not blocked by any of the trophic factors added individually. A significant increase in photoreceptor survival was seen with forskolin added to CNTF, but not to BDNF, FGF2, or GDNF. These results demonstrate that trophic factors promote photoreceptor survival through a synergistic interaction. Increased understanding of receptor interactions and signaling pathways may lead to a potential therapeutic role for combinatorial trophic factors in treatment of photoreceptor dystrophies.

Cochlear and retinal degeneration in the tubby mouse.

A number of autosomal recessive syndromes feature both sensorineural hearing loss and retinal degeneration. The mouse mutant tubby also combines hearing loss with progressive retinal degeneration, and thus may constitute a useful model of one form of human sensorineural deafness/retinal dystrophic syndrome. It has not been directly demonstrated that the hearing loss in this mouse involves the cochlea, however. We have examined the cochleas of adult tubby mice using light microscopy. The tubby cochlea shows pronounced degeneration of the organ of Corti and loss of afferent neurons in the base, with relative sparing of the apex. Our findings support the tubby mouse as a model of human sensorineural deafness/retinal dystrophic syndrome. Possible human counterparts include Usher’s, Alstrom’s, and Bardet–Biedl syndromes.

A reliable method for organ culture of neonatal mouse retina with long-term survival.

Organ culture systems of the central nervous system have proven to be useful tools for the study of development, differentiation, and degeneration. Some studies have been limited by the inability to maintain the cultures over an extended period. Here we describe an organ culture technique for the mouse retina. This method uses commercially available supplies and reproducible procedures to maintain healthy retinas with normal architecture for 4 weeks in vitro. The system is amenable to quantitative analysis. It can be used with both normal and retinal degeneration (rd) retinas to study of the role of various factors in photoreceptor degeneration in retinal cell fate determination and development.

Large Scale Gene Expression Profiles of Regenerating Inner Ear Sensory Epithelia

Loss of inner ear sensory hair cells (HC) is a leading cause of human hearing loss and balance disorders. Unlike mammals, many lower vertebrates can regenerate these cells. We used cross-species microarrays to examine this process in the avian inner ear. Specifically, changes in expression of over 1700 transcription factor (TF) genes were investigated in hair cells of auditory and vestibular organs following treatment with two different damaging agents and regeneration in vitro. Multiple components of seven distinct known signaling pathways were clearly identifiable: TGFβ, PAX, NOTCH, WNT, NFKappaB, INSULIN/IGF1 and AP1. Numerous components of apoptotic and cell cycle control pathways were differentially expressed, including p27KIP and TFs that regulate its expression. A comparison of expression trends across tissues and treatments revealed identical patterns of expression that occurred at identical times during regenerative proliferation. Network analysis of the patterns of gene expression in this large dataset also revealed the additional presence of many components (and possible network interactions) of estrogen receptor signaling, circadian rhythm genes and parts of the polycomb complex (among others). Equal numbers of differentially expressed genes were identified that have not yet been placed into any known pathway. Specific time points and tissues also exhibited interesting differences: For example, 45 zinc finger genes were specifically up-regulated at later stages of cochlear regeneration. These results are the first of their kind and should provide the starting point for more detailed investigations of the role of these many pathways in HC recovery, and for a description of their possible interactions.

Progression of Cochlear and Retinal Degeneration in the tubby (rd5) Mouse

Mice homozygous for a defect of the tub (rd5) gene exhibit cochlear and retinal degeneration combined with obesity, and resemble certain human autosomal recessive sensory deficit syndromes. To establish the progressive nature of sensory cell loss associated with the tub gene, and to differentiate tub-related losses from those associated with the C57 background on which tub arose, we evaluated cochleas and retinas from tub/tub, tub/+, and +/+ mice, aged 2 weeks to 1 year by light and electron microscopy. Cochleas from mice of all three genotypes show progressive inner (IHC) and outer hair cell (OHC) loss. Relative to tub/+ and +/+ animals, however, tub homozygotes show accelerated OHC loss, affecting the extreme cochlear base (hook region) by 1 month, and the apex by 6 months. IHC loss in tub/tub animals is accelerated in the basal half of the cochlea, affecting the hook region by 6 months. Spiral ganglion cell losses were observed only in tub/tub mice, and only in the cochlear base. Retinas of tub/tub mice are abnormal at maturity, exhibiting shortened photoreceptor outer segments by 2 weeks, and progressive photoreceptor loss thereafter. Because the tub mutation causes degeneration of sensory cells in the ear and eye but has no other neurological effects, tubby mice hold unique promise for the study of human syndromic sensory loss.

Dopamine Has a Critical Role in Photoreceptor Degeneration in the rd Mouse

Photoreceptors receive paracrine input from dopaminergic interplexiform cells. Rod photoreceptors in the rd mouse degenerate rapidly due to a specific gene defect. We investigated the effects of dopamine on rd mouse photoreceptors in retinal organ culture. Retinas were harvested from rd or wild-type mice at postnatal day 2 and grown in organ culture for 27 days. When antagonists for either D(1)- or D(2)-family dopamine receptors were added to the media, photoreceptor degeneration was blocked. Furthermore, when dopamine was depleted by the addition of 6-hydroxydopamine and pargyline, photoreceptor survival appeared comparable to wild-type retinal cultures. The addition of a dopamine agonist induced photoreceptor degeneration in dopamine-depleted rd organ cultures. In all cases, photoreceptors maintained robust staining of opsin. These results demonstrate that dopamine antagonists or dopamine depletion blocks photoreceptor degeneration and that dopamine is necessary for photoreceptor degeneration in the rd mouse retinal organ culture model, indicating that dopamine antagonists may represent a therapeutic strategy in retinal degenerative disease.

Epigenetic Influences on Sensory Regeneration: Histone Deacetylases Regulate Supporting Cell Proliferation in the Avian Utricle

The sensory hair cells of the cochlea and vestibular organs are essential for normal hearing and balance function. The mammalian ear possesses a very limited ability to regenerate hair cells and their loss can lead to permanent sensory impairment. In contrast, hair cells in the avian ear are quickly regenerated after acoustic trauma or ototoxic injury. The very different regenerative abilities of the avian vs. mammalian ear can be attributed to differences in injury-evoked expression of genes that either promote or inhibit the production of new hair cells. Gene expression is regulated both by the binding of cis-regulatory molecules to promoter regions as well as through structural modifications of chromatin (e.g., methylation and acetylation). This study examined effects of histone deacetylases (HDACs), whose main function is to modify histone acetylation, on the regulation of regenerative proliferation in the chick utricle. Cultures of regenerating utricles and dissociated cells from the utricular sensory epithelia were treated with the HDAC inhibitors valproic acid, trichostatin A, sodium butyrate, and MS-275. All of these molecules prevent the enzymatic removal of acetyl groups from histones, thus maintaining nuclear chromatin in a “relaxed” (open) configuration. Treatment with all inhibitors resulted in comparable decreases in supporting cell proliferation. We also observed that treatment with the HDAC1-, 2-, and 3-specific inhibitor MS-275 was sufficient to reduce proliferation and that two class I HDACs—HDAC1 and HDAC2—were expressed in the sensory epithelium of the utricle. These results suggest that inhibition of specific type I HDACs is sufficient to prevent cell cycle entry in supporting cells. Notably, treatment with HDAC inhibitors did not affect the differentiation of replacement hair cells. We conclude that histone deacetylation is a positive regulator of regenerative proliferation but is not critical for avian hair cell differentiation.

Expression of GATA3 and tenascin in the avian vestibular maculae: Normative patterns and changes during sensory regeneration

Sensory receptors in the vestibular organs of birds can regenerate after ototoxic injury. Notably, this regenerative process leads to the restoration of the correct patterning of hair cell phenotype and afferent innervation within the repaired sensory epithelium. The molecular signals that specify cell phenotype and regulate neuronal guidance during sensory regeneration are not known, but they are likely to be similar to the signals that direct these processes during embryonic development. The present study examined the recovery of hair cell phenotype during regeneration in the avian utricle, a vestibular organ that detects linear acceleration and head orientation. First, we show that Type I hair cells in the avian vestibular maculae are immunoreactive for the extracellular matrix molecule tenascin and that treatment with the ototoxic antibiotic streptomycin results in a nearly complete elimination of tenascin immunoreactivity. Cells that express tenascin begin to recover after about 2 weeks and are then contacted by calyx terminals of vestibular neurons. In addition, our previous work had shown that the zinc finger transcription factor GATA3 is uniquely expressed within the striolar reversal zone of the utricle (Hawkins et al. [ 2003 ] Hum Mol Genet 12:1261–1272), and we show here that this regionalized expression of GATA3 is maintained after severe hair cell lesions and after transplantation of the sensory epithelium onto a chemically defined substrate. In contrast, the expression of three other supporting cell markers—α‐ and β‐tectorin and SCA—is reduced following ototoxic injury. These observations suggest that GATA3 expression may maintain positional information in the maculae during sensory regeneration. J. Comp. Neurol. 500:646–657, 2007.

An RNA interference-based screen of transcription factor genes identifies pathways necessary for sensory regeneration in the avian inner ear.

Sensory hair cells of the inner ear are the mechanoelectric transducers of sound and head motion. In mammals, damage to sensory hair cells leads to hearing or balance deficits. Nonmammalian vertebrates such as birds can regenerate hair cells after injury. In a previous study, we characterized transcription factor gene expression during chicken hair cell regeneration. In those studies, a laser microbeam or ototoxic antibiotics were used to damage the sensory epithelia (SE). The current study focused on 27 genes that were upregulated in regenerating SEs compared to untreated SEs in the previous study. Those genes were knocked down by siRNA to determine their requirement for supporting cell proliferation and to measure resulting changes in the larger network of gene expression. We identified 11 genes necessary for proliferation and also identified novel interactive relationships between many of them. Defined components of the WNT, PAX, and AP1 pathways were shown to be required for supporting cell proliferation. These pathways intersect on WNT4, which is also necessary for proliferation. Among the required genes, the CCAAT enhancer binding protein, CEBPG, acts downstream of Jun Kinase and JUND in the AP1 pathway. The WNT coreceptor LRP5 acts downstream of CEBPG, as does the transcription factor BTAF1. Both of these genes are also necessary for supporting cell proliferation. This is the first large-scale screen of its type and suggests an important intersection between the AP1 pathway, the PAX pathway, and WNT signaling in the regulation of supporting cell proliferation during inner ear hair cell regeneration.

Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells.

Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.