Non-Coding RNAs in Retinal Development

Retinal development is dependent on an accurately functioning network of transcriptional and translational regulators. Among the diverse classes of molecules involved, non-coding RNAs (ncRNAs) play a significant role. Members of this family are present in the cell as transcripts, but are not translated into proteins. MicroRNAs (miRNAs) are small ncRNAs that act as post-transcriptional regulators. During the last decade, they have been implicated in a variety of biological processes, including the development of the nervous system. On the other hand, long-ncRNAs (lncRNAs) represent a different class of ncRNAs that act mainly through processes involving chromatin remodeling and epigenetic mechanisms. The visual system is a prominent model to investigate the molecular mechanisms underlying neurogenesis or circuit formation and function, including the differentiation of retinal progenitor cells to generate the seven principal cell classes in the retina, pathfinding decisions of retinal ganglion cell axons in order to establish the correct connectivity from the eye to the brain proper, and activity-dependent mechanisms for the functionality of visual circuits. Recent findings have associated ncRNAs in several of these processes and uncovered a new level of complexity for the existing regulatory mechanisms. This review summarizes and highlights the impact of ncRNAs during the development of the vertebrate visual system, with a specific focus on the role of miRNAs and a synopsis regarding recent findings on lncRNAs in the retina

Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea

Background: At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation. Methodology/Principal Findings: We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells. Conclusions/Significance: Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 migh

Expression of Neurog1 Instead of Atoh1 Can Partially Rescue Organ of Corti Cell Survival

In the mammalian inner ear neurosensory cell fate depends on three closely related transcription factors, Atoh1 for hair cells and Neurog1 and Neurod1 for neurons. We have previously shown that neuronal cell fate can be altered towards hair cell fate by eliminating Neurod1 mediated repression of Atoh1 expression in neurons. To test whether a similar plasticity is present in hair cell fate commitment, we have generated a knockin (KI) mouse line (Atoh1KINeurog1) in which Atoh1 is replaced by Neurog1. Expression of Neurog1 under Atoh1 promoter control alters the cellular gene expression pattern, differentiation and survival of hair cell precursors in both heterozygous (Atoh1+/KINeurog1) and homozygous (Atoh1KINeurog1/KINeurog1) KI mice. Homozygous KI mice develop patches of organ of Corti precursor cells that express Neurog1, Neurod1, several prosensory genes and neurotrophins. In addition, these patches of cells receive afferent and efferent processes. Some cells among these patches form multiple microvilli but no stereocilia. Importantly, Neurog1 expressing mutants differ from Atoh1 null mutants, as they have intermittent formation of organ of Corti-like patches, opposed to a complete ‘flat epithelium’ in the absence of Atoh1. In heterozygous KI mice co-expression of Atoh1 and Neurog1 results in change in fate and patterning of some hair cells and supporting cells in addition to the abnormal hair cell polarity in the later stages of development. This differs from haploinsufficiency of Atoh1 (Pax2cre; Atoh1f/+), indicating the effect of Neurog1 expression in developing hair cells. Our data suggest that Atoh1KINeurog1 can provide some degree of functional support for survival of organ of Corti cells. In contrast to the previously demonstrated fate plasticity of neurons to differentiate as hair cells, hair cell precursors can be maintained for a limited time by Neurog1 but do not transdifferentiate as neurons

A Cross-Species Analysis of MicroRNAs in the Developing Avian Face

Higher vertebrates use similar genetic tools to derive very different facial features. This diversity is believed to occur through temporal, spatial and species-specific changes in gene expression within cranial neural crest (NC) cells. These contribute to the facial skeleton and contain species-specific information that drives morphological variation. A few signaling molecules and transcription factors are known to play important roles in these processes, but little is known regarding the role of micro-RNAs (miRNAs). We have identified and compared all miRNAs expressed in cranial NC cells from three avian species (chicken, duck, and quail) before and after species-specific facial distinctions occur. We identified 170 differentially expressed miRNAs. These include thirty-five novel chicken orthologs of previously described miRNAs, and six avian-specific miRNAs. Five of these avian-specific miRNAs are conserved over 120 million years of avian evolution, from ratites to galliforms, and their predicted target mRNAs include many components of Wnt signaling. Previous work indicates that mRNA gene expression in NC cells is relatively static during stages when the beak acquires species-specific morphologies. However, miRNA expression is remarkably dynamic within this timeframe, suggesting that the timing of specific developmental transitions is altered in birds with different beak shapes. We evaluated one miRNA:mRNA target pair and found that the cell cycle regulator p27KIP1 is a likely target of miR-222 in frontonasal NC cells, and that the timing of this interaction correlates with the onset of phenotypic variation. Our comparative genomic approach is the first comprehensive analysis of miRNAs in the developing facial primordial, and in species-specific facial development

Loss of Prickle1 leads to aberrant afferent outgrowth in the apical cochlea.

<p>A select population of type II fibers was labeled by dye tracing in <i>Prickle1</i>^{<i>C251X/C251X</i>} mutants and their littermate controls at E18.5 and P0. (A) In the base, both control and <i>Prickle1</i>^{<i>C251X/C251X</i>} mutants formed three rows of type II fibers growing towards the base. (B-F) In the apex of the <i>Prickle1</i>^{<i>C251X/C251X</i>} mutant cochlea, outgrowth of some type II afferents was disrupted. (G and H’) Some afferents were not in the same focal plane as the radial fibers growing towards the hair cells (HCs). (H’) A higher magnification view of (H). Filled triangle, fibers that branched; arrow, fibers growing toward the apex; empty triangle, fibers that grew past HCs.</p

Normalized gene expression in the cochlea from RNA-seq data in multiple studies.

<p>Normalized gene expression in the cochlea from RNA-seq data in multiple studies.</p

Central projections from apical afferents are expanded in the cochlear nuclei in <i>Prickle1</i>^{<i>C251X/C251X</i>} mutants.

<p>Different colors of lipophilic dyes were applied to apex and the base of the cochlear (A, B), and their central projection were analyzed (A’, B’, C-E). (A and B) Overview of the cochlea showing the application of red dye to the base and green dye to the apex in control (A) and <i>Prickle1</i>^{<i>C251X/C251X</i>} mutant (B). After 3 days of diffusion, there was partial overlap of the dye. (A’ and B’) Selective bundles of afferents and olivocochlear efferents (OCE) passed along the vestibular ganglion (VG). Only in <i>Prickle1</i>^{<i>C251X/C251X</i>} mutant (B’), OCE separated into several bundles. In addition, several vestibular ganglion neurons (VG) were labeled. (C-E) Projections to the cochlear nucleus of the control (C) and the <i>Prickle1</i>^{<i>C251X/C251X</i>} mutant (D, E). (C) In controls, afferent bundles from the apex and the base segregated and formed distinct fascicles. (D, E) In the mutant, although afferents from the base projected normally to the dorsal cochlear nucleus complex DCN, afferents from the apex formed collaterals that spread out throughout the DCN and the anterior-ventral cochlear nuclei (AVCN). (E) Higher magnification of the DCN of (D), showing details of apical afferents passing basal turn afferents to branch in the most dorsal aspect of the cochlear nucleus complex. Arrow, afferents innervating vestibular nuclei. Scale bars, 100 μm.</p

A Novel Atoh1 “Self-Terminating” Mouse Model Reveals the Necessity of Proper Atoh1 Level and Duration for Hair Cell Differentiation and Viability

<div><p>Atonal homolog1 (<em>Atoh1</em>) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of <em>Atoh1</em> at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of <em>Atoh1</em> expression required for proper hair cell differentiation and maintenance remain unknown. We generated an <em>Atoh1</em> conditional knockout (CKO) mouse line using <em>Tg(Atoh1-cre)</em>, in which the <em>cre</em> expression is driven by an <em>Atoh1</em> enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of <em>Atoh1</em> in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of <em>Atoh1</em> result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of <em>Atoh1, Barhl1</em> and <em>Pou4f3</em>, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that <em>Atoh1</em> is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that <em>Atoh1</em> expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered <em>Atoh1</em> CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with <em>Atoh1</em> therapy.</p> </div