Retinoic acid regulates olfactory progenitor cell fate and differentiation.

BACKGROUND: In order to fulfill their chemosensory function, olfactory neurons are in direct contact with the external environment and are therefore exposed to environmental aggressive factors. Olfaction is maintained through life because, unlike for other sensory neuroepithelia, olfactory neurons have a unique capacity to regenerate after trauma. The mechanisms that control the ontogenesis and regenerative ability of these neurons are not fully understood. Here, we used various experimental approaches in two model systems (chick and mouse) to assess the contribution of retinoic acid signaling in the induction of the olfactory epithelium, the generation and maintenance of progenitor populations, and the ontogenesis and differentiation of olfactory neurons. RESULTS: We show that retinoic acid signaling, although dispensable for initial induction of the olfactory placode, plays a key role in neurogenesis within this neuroepithelium. Retinoic acid depletion in the olfactory epithelium, both in chick and mouse models, results in a failure of progenitor cell maintenance and, consequently, differentiation of olfactory neurons is not sustained. Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid. CONCLUSIONS: Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells. Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium.

Retinoic acid deficiency impairs the vestibular function.

The retinaldehyde dehydrogenase 3 (Raldh3) gene encodes a major retinoic acid synthesizing enzyme and is highly expressed in the inner ear during embryogenesis. We found that mice deficient in Raldh3 bear severe impairment in vestibular functions. These mutant mice exhibited spontaneous circling/tilted behaviors and performed poorly in several vestibular-motor function tests. In addition, video-oculography revealed a complete loss of the maculo-ocular reflex and a significant reduction in the horizontal angular vestibulo-ocular reflex, indicating that detection of both linear acceleration and angular rotation were compromised in the mutants. Consistent with these behavioral and functional deficiencies, morphological anomalies, characterized by a smaller vestibular organ with thinner semicircular canals and a significant reduction in the number of otoconia in the saccule and the utricle, were consistently observed in the Raldh3 mutants. The loss of otoconia in the mutants may be attributed, at least in part, to significantly reduced expression of Otop1, which encodes a protein known to be involved in calcium regulation in the otolithic organs. Our data thus reveal a previously unrecognized role of Raldh3 in structural and functional development of the vestibular end organs.

Integrin-linked kinase deletion in the developing lens leads to capsule rupture, impaired fiber migration and non-apoptotic epithelial cell death.

PURPOSE: The lens is a powerful model system to study integrin-mediated cell-matrix interaction in an in vivo context, as it is surrounded by a true basement membrane, the lens capsule. To characterize better the function of integrin-linked kinase (ILK), we examined the phenotypic consequences of its deletion in the developing mouse lens. METHODS: ILK was deleted from the embryonic lens either at the time of placode invagination using the Le-Cre line or after initial lens formation using the Nestin-Cre line. RESULTS: Early deletion of ILK leads to defects in extracellular matrix deposition that result in lens capsule rupture at the lens vesicle stage (E13.5). If ILK was deleted at a later time-point after initial establishment of the lens capsule, rupture was prevented. Instead, ILK deletion resulted in secondary fiber migration defects and, most notably, in cell death of the anterior epithelium (E18.5-P0). Remarkably, dying cells did not stain positively for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or activated-caspase 3, suggesting that they were dying from a non-apoptotic mechanism. Moreover, cross to a Bax(fl/fl)/Bak⁻/⁻ mouse line that is resistant to most forms of apoptosis failed to promote cell survival in the ILK-deleted lens epithelium. Electron microscopy revealed the presence of numerous membranous vacuoles containing degrading cellular material. CONCLUSIONS. Our study reveals a role for ILK in extracellular matrix organization, fiber migration, and cell survival. Furthermore, to our knowledge we show for the first time that ILK disruption results in non-apoptotic cell death in vivo.

Retinoic acid receptor (RAR)-alpha is not critically required for mediating retinoic acid effects in the developing mouse retina.

PURPOSE: To determine the functional contribution of retinoic acid receptor (RAR)-alpha in the developing murine neural retina, through a phenotypic analysis of the corresponding null mutants. METHODS: RARalpha mutant (Rara(-/-)) mice were compared with wild-type littermates at several stages of pre- and postnatal development. An RA-response element (RARE)-containing reporter transgene was used to assess the contribution of RARalpha to retinoid signaling in the retina. In situ hybridization was performed on serial eye sections to investigate the expression of main developmental regulators. Immunofluorescence was used to detect differentiated cell types in the adult retina. Mutants were also subjected to clinical observation and visual function evaluation with the optomotor test and electroretinography. RESULTS: Both isoform transcripts of RARalpha were expressed throughout the neural retina at various stages of pre- and postnatal development. In the Rara(-/-) mice the RARE-reporter transgene consistently failed to activate in the developing neural retina. However, they did not exhibit any alteration of the expression patterns of molecular determinants and had a normal organization of retinal cell types at postnatal stages. Their performance in visual tests was indistinguishable from that of control littermates. CONCLUSIONS: Although RARalpha mediates RARE reporter transgene activity in the neural retina, its function is not necessary for the retina to develop and function normally. These data suggest that retinoic acid regulates neural retinal development through other, possibly RAR-independent, pathways.

Dynamic expression of the retinoic acid-synthesizing enzyme retinol dehydrogenase 10 (rdh10) in the developing mouse brain and sensory organs.

Organs develop through many tissue interactions during embryogenesis, involving numerous signaling cascades and gene products. One of these signaling molecules is retinoic acid (RA), an active vitamin A derivative, which in mammalian embryos is synthesized from maternal retinol by two oxidative reactions involving alcohol/retinol dehydrogenases (ADH/RDHs) and retinaldehyde dehydrogenases (RALDHs), respectively. The activity of RALDHs is known to be crucial for RA synthesis; however, recently a retinol dehydrogenase (RDH10) has been shown to represent a new limiting factor in this synthesis. We investigated the spatiotemporal distribution of Rdh10 gene transcripts by in situ hybridization and quantitative polymerase chain reaction (PCR) during development of the brain and sensory organs. Although Rdh10 relative mRNA levels decline throughout brain development, we show a strong and lasting expression in the meninges and choroid plexuses. Rdh10 expression is also specifically seen in the striatum, a known site of retinoid signaling. In the eye, regional expression is observed both in the prospective pigmented epithelium and neural retina. In the inner ear Rdh10 expression is specific to the endolymphatic system and later the stria vascularis, both organs being involved in endolymph homeostasis. Furthermore, in the peripheral olfactory system and the vibrissae follicles, expression is present from early stages in regions where sensory receptors appear and mesenchymal/epithelial interactions take place. The distribution of Rdh10 transcripts during brain and sensory organ development is consistent with a role of this enzyme in generating region-specific pools of retinaldehyde that will be used by the various RALDHs to refine the patterns of RA synthesis.

Expression of the murine retinol dehydrogenase 10 (Rdh10) gene correlates with many sites of retinoid signalling during embryogenesis and organ differentiation.

Retinoic acid acts as a signalling molecule regulating many developmental events in vertebrates. As this molecule directly influences gene expression by activating nuclear receptors, its patterns of synthesis have to be tightly regulated, and it is well established that at least three retinaldehyde dehydrogenases (RALDHs) are involved in such tissue-specific synthesis. Whereas embryos from oviparous species can obtain retinaldehyde by metabolizing carotenoids stored in the yolk, placental embryos rely on retinol transferred from the maternal circulation. Here, we show that the gene encoding one of the murine retinol dehydrogenases, Rdh10, is expressed according to complex profiles both during early embryogenesis and organ differentiation. Many of its expression sites correlate with regions of active retinoid signalling and Raldh gene expression, especially with Raldh2 in the early presomitic and somitic mesoderm, retrocardiac and posterior branchial arch region, or later in the pleural mesothelium and kidney cortical region. Rdh10 also shows cell-type and/or regional specificity during development of the palate, teeth, and olfactory system. During limb bud development, it may participate in retinoic acid production in proximal/posterior cells, and eventually in interdigital mesenchyme. These data implicate the retinol to retinaldehyde conversion as the first step in the tissue-specific regulation of retinoic acid synthesis, at least in mammalian embryos.