Retinoic Acid Signaling Mediates Hair Cell Regeneration by Repressing p27kip and sox2 in Supporting Cells.

During development, otic sensory progenitors give rise to hair cells and supporting cells. In mammalian adults, differentiated and quiescent sensory cells are unable to generate new hair cells when these are lost due to various insults, leading to irreversible hearing loss. Retinoic acid (RA) has strong regenerative capacity in several organs, but its role in hair cell regeneration is unknown. Here, we use genetic and pharmacological inhibition to show that the RA pathway is required for hair cell regeneration in zebrafish. When regeneration is induced by laser ablation in the inner ear or by neomycin treatment in the lateral line, we observe rapid activation of several components of the RA pathway, with dynamics that position RA signaling upstream of other signaling pathways. We demonstrate that blockade of the RA pathway impairs cell proliferation of supporting cells in the inner ear and lateral line. Moreover, in neuromast, RA pathway regulates the transcription of p27(kip) and sox2 in supporting cells but not fgf3. Finally, genetic cell-lineage tracing using Kaede photoconversion demonstrates that de novo hair cells derive from FGF-active supporting cells. Our findings reveal that RA has a pivotal role in zebrafish hair cell regeneration by inducing supporting cell proliferation, and shed light on the underlying transcriptional mechanisms involved. This signaling pathway might be a promising approach for hearing recovery. SIGNIFICANCE STATEMENT: Hair cells are the specialized mechanosensory cells of the inner ear that capture auditory and balance sensory input. Hair cells die after acoustic trauma, ototoxic drugs or aging diseases, leading to progressive hearing loss. Mammals, in contrast to zebrafish, lack the ability to regenerate hair cells. Here, we find that retinoic acid (RA) pathway is required for hair cell regeneration in vivo in the zebrafish inner ear and lateral line. RA pathway is activated very early upon hair cell loss, promotes cell proliferation of progenitor cells, and regulates two key genes, p27(kip) and sox2. Our results position RA as an essential signal for hair cell regeneration with relevance in future regenerative strategies in mammals.

Intravitreal Administration of rhNGF Enhances Regenerative Processes in a Zebrafish Model of Retinal Degeneration.

Nerve growth factor (NGF) is the best characterized neurotrophin, and it is known to play an important role in ocular homeostasis. Here, we demonstrated the expression of NGF receptors in adult zebrafish retina and optimized a light-induced retina degeneration (LID) zebrafish model that mimics human cone-rod disorders, demonstrating that intravitreal (IV) administration of rhNGF can boost zebrafish retinal regeneration in this model. Adult zebrafish retinae exposed to 60 h of light irradiation (60 h LID) displayed evident reduction of outer nuclear layer (ONL) thickness and cell number with presence of apoptotic cells. Retinal histologic evaluation at different time points showed that IV therapeutic injection of rhNGF resulted in an increase of ONL thickness and cell number at late time points after damage (14 and 21 days post injury), ultimately accelerating retinal tissue recovery by driving retinal cell proliferation. At a molecular level, rhNGF activated the ERK1/2 pathway and enhanced the regenerative potential of Müller glia gfap- and vim-expressing cells by stimulating at early time points the expression of the photoreceptor regeneration factor Drgal1-L2. Our results demonstrate the highly conserved nature of NGF canonical pathway in zebrafish and thus support the use of zebrafish models for testing new compounds with potential retinal regenerative properties. Moreover, the pro-regenerative effects of IV-injected NGF that we observed pave the way to further studies aimed at evaluating its effects also in mammals, in order to expedite the development of novel rhNGF-based therapeutic approaches for ophthalmological disorders.

CRISPR Meets Zebrafish: Accelerating the Discovery of New Therapeutic Targets.

Bringing a new drug to the market costs an average of US$2.6 billion and takes more than 10 years from discovery to regulatory approval. Despite the need to reduce cost and time to increase productivity, pharma companies tend to crowd their efforts in the same indications and drug targets. This results in the commercialization of drugs that share the same mechanism of action (MoA) and, in many cases, equivalent efficacies among them-an outcome that helps neither patients nor the balance sheet of the companies trying to bring therapeutics to the same patient population. Indeed, the discovery of new therapeutic targets, based on a deeper understanding of the disease biology, would likely provide more innovative MoAs and potentially greater drug efficacies. It would also bring better chances for identifying appropriate treatments according to the patient's genetic stratification. Nowadays, we count with an enormous amount of unprocessed information on potential disease targets that could be extracted from omics data obtained from patient samples. In addition, hundreds of pharmacological and genetic screenings have been performed to identify innovative drug targets. Traditionally, rodents have been the animal models of choice to perform functional genomic studies. The high experimental cost, combined with the low throughput provided by those models, however, is a bottleneck for discovering and validating novel genetic disease associations. To overcome these limitations, we propose that zebrafish, in conjunction with the use of CRISPR/Cas9 genome-editing tools, could streamline functional genomic processes to bring biologically relevant knowledge on innovative disease targets in a shorter time frame.

Loss of the sphingolipid desaturase DEGS1 causes hypomyelinating leukodystrophy.

Sphingolipid imbalance is the culprit in a variety of neurological diseases, some affecting the myelin sheath. We have used whole-exome sequencing in patients with undetermined leukoencephalopathies to uncover the endoplasmic reticulum lipid desaturase DEGS1 as the causative gene in 19 patients from 13 unrelated families. Shared features among the cases include severe motor arrest, early nystagmus, dystonia, spasticity, and profound failure to thrive. MRI showed hypomyelination, thinning of the corpus callosum, and progressive thalamic and cerebellar atrophy, suggesting a critical role of DEGS1 in myelin development and maintenance. This enzyme converts dihydroceramide (DhCer) into ceramide (Cer) in the final step of the de novo biosynthesis pathway. We detected a marked increase of the substrate DhCer and DhCer/Cer ratios in patients' fibroblasts and muscle. Further, we used a knockdown approach for disease modeling in Danio rerio, followed by a preclinical test with the first-line treatment for multiple sclerosis, fingolimod (FTY720, Gilenya). The enzymatic inhibition of Cer synthase by fingolimod, 1 step prior to DEGS1 in the pathway, reduced the critical DhCer/Cer imbalance and the severe locomotor disability, increasing the number of myelinating oligodendrocytes in a zebrafish model. These proof-of-concept results pave the way to clinical translation.

Mitotic cell rounding and epithelial thinning regulate lumen growth and shape.

Many organ functions rely on epithelial cavities with particular shapes. Morphogenetic anomalies in these cavities lead to kidney, brain or inner ear diseases. Despite their relevance, the mechanisms regulating lumen dimensions are poorly understood. Here, we perform live imaging of zebrafish inner ear development and quantitatively analyse the dynamics of lumen growth in 3D. Using genetic, chemical and mechanical interferences, we identify two new morphogenetic mechanisms underlying anisotropic lumen growth. The first mechanism involves thinning of the epithelium as the cells change their shape and lose fluids in concert with expansion of the cavity, suggesting an intra-organ fluid redistribution process. In the second mechanism, revealed by laser microsurgery experiments, mitotic rounding cells apicobasally contract the epithelium and mechanically contribute to expansion of the lumen. Since these mechanisms are axis specific, they not only regulate lumen growth but also the shape of the cavity.