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The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders., OFD1 (oro‐facial digital type I syndrome protein) resides in a centrosomal complex that links GPCR signalling to ubiquitylation and degradation of OFD1, controlling cilium morphology and dynamics and vertebrate development.

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MicroRNAs (miRNAs), a class of non-coding RNAs, are essential key players in the control of biological processes in both physiological and pathological conditions. miRNAs play important roles in fine tuning the expression of many genes, which often have roles in common molecular networks. miRNA dysregulation thus renders cells vulnerable to aberrant fluctuations in genes, resulting in degenerative diseases. The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells that resides between the light-sensitive photoreceptors (PR) and the choriocapillaris. The demanding physiological functions of RPE cells require precise gene regulation for the maintenance of retinal homeostasis under stress conditions and the preservation of vision. Thus far, our understanding of how miRNAs function in the homeostasis and maintenance of the RPE has been poorly addressed, and advancing our knowledge is central to harnessing their potential as therapeutic agents to counteract visual impairment. This review focuses on the emerging roles of miRNAs in the function and health of the RPE and on the future exploration of miRNA-based therapeutic approaches to counteract blinding diseases.

The retinal pigment epithelium (RPE) is a highly specialized monolayer of polarized, pigmented epithelial cells that resides between the vessels of the choriocapillaris and the neural retina. The RPE is essential for the maintenance and survival of overlying light-sensitive photoreceptors, as it participates in the formation of the outer blood-retinal barrier, phagocytosis, degradation of photoreceptor outer segment (POS) tips, maintenance of the retinoid cycle, and protection against light and oxidative stress. Autophagy is an evolutionarily conserved 'self-eating' process, designed to maintain cellular homeostasis. The daily autophagy demands in the RPE require precise gene regulation for the digestion and recycling of intracellular and POS components in lysosomes in response to light and stress conditions. In this review, we discuss selective autophagy and focus on the recent advances in our understanding of the mechanism of cell clearance in the RPE for visual function. Understanding how this catabolic process is regulated by both transcriptional and post-transcriptional mechanisms in the RPE will promote the recognition of pathological pathways in genetic disease and shed light on potential therapeutic strategies to treat visual impairments in patients with retinal disorders associated with lysosomal dysfunction.

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER‐phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER‐phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER‐phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3—master regulators of lysosomal biogenesis and autophagy—control ER‐phagy by inducing the expression of the ER‐phagy receptor FAM134B. The TFEB/TFE3‐FAM134B axis promotes ER‐phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK‐dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K‐PKB/Akt‐mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER‐phagy to respond to both metabolic and developmental cues., Nuclear translocation of TFEB/TFE3 transcription factors and expression of FAM134B downstream of FGF18 signalling induce ER‐phagy in chondrocytes to promote bone development.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light‐responsive miR‐211 controls lysosomal biogenesis at the beginning of light–dark transitions in the RPE by targeting Ezrin, a cytoskeleton‐associated protein essential for the regulation of calcium homeostasis. miR‐211‐mediated down‐regulation of Ezrin leads to Ca(2+) influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light‐mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR‐211(−/−) mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR‐211(−/−) phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development.

Inherited retinal diseases (IRDs) represent a frequent cause of genetic blindness. Their high genetic heterogeneity hinders the application of gene-specific therapies to the vast majority of patients. We recently demonstrated that the microRNA miR-204 is essential for retinal function, although the underlying molecular mechanisms remain poorly understood. Here, we investigated the therapeutic potential of miR-204 in IRDs. We subretinally delivered an adeno-associated viral (AAV) vector carrying the miR-204 precursor to two genetically different IRD mouse models. The administration of AAV-miR-204 preserved retinal function in a mouse model for a dominant form of retinitis pigmentosa (RHO-P347S). This was associated with a reduction of apoptotic photoreceptor cells and with a better preservation of photoreceptor marker expression. Transcriptome analysis showed that miR-204 shifts expression profiles of transgenic retinas toward those of healthy retinas by the downregulation of microglia activation and photoreceptor cell death. Delivery of miR-204 exerted neuroprotective effects also in a mouse model of Leber congenital amaurosis, due to mutations of the Aipl1 gene. Our study highlights the mutation-independent therapeutic potential of AAV-miR204 in slowing down retinal degeneration in IRDs and unveils the previously unreported role of this miRNA in attenuating microglia activation and photoreceptor cell death.

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.

Ultradian oscillators are cyclically expressed genes with a period of less than 24h, found in the major signalling pathways. The Notch effector hairy and enhancer of split Hes genes are ultradian oscillators. The physiological signals that synchronise and entrain Hes oscillators remain poorly understood. We investigated whether cellular metabolism modulates Hes1 cyclic expression. We demonstrated that, in mouse myoblasts (C2C12), Hes1 oscillation depends on reactive oxygen species (ROS), which are generated by the mitochondria electron transport chain and by NADPH oxidases NOXs. In vitro, the regulation of Hes1 by ROS occurs via the calcium-mediated signalling. The modulation of Hes1 by ROS was relevant in vivo, since perturbing ROS homeostasis was sufficient to alter Medaka (Oryzias latipes) somitogenesis, a process that is dependent on Hes1 ultradian oscillation during embryo development. Moreover, in a Medaka model for human microphthalmia with linear skin lesions syndrome, in which mitochondrial ROS homeostasis was impaired, we documented important somitogenesis defects and the deregulation of Hes homologues genes involved in somitogenesis. Notably, both molecular and developmental defects were rescued by antioxidant treatments. Our studies provide the first evidence of a coupling between cellular redox metabolism and an ultradian biological oscillator with important pathophysiological implication for somitogenesis.

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in the regulation of gene expression. The roles of individual miRNAs in controlling vertebrate eye development remain, however, largely unexplored. Here, we show that a single miRNA, miR-204, regulates multiple aspects of eye development in the medaka fish ( Oryzias latipes ). Morpholino-mediated ablation of miR-204 expression resulted in an eye phenotype characterized by microphthalmia, abnormal lens formation, and altered dorsoventral (D-V) patterning of the retina, which is associated with optic fissure coloboma. Using a variety of in vivo and in vitro approaches, we identified the transcription factor Meis2 as one of the main targets of miR-204 function. We show that, together with altered regulation of the Pax6 pathway, the abnormally elevated levels of Meis2 resulting from miR-204 inactivation are largely responsible for the observed phenotype. These data provide an example of how a specific miRNA can regulate multiple events in eye formation; at the same time, they uncover an as yet unreported function of Meis2 in the specification of D-V patterning of the retina.

A well integrated and hierarchically organized gene regulatory network is responsible for the progressive specification of the forebrain. The transcription factor Six3 is one of the central components of this network. As such, Six3 regulates several components of the network, but its upstream regulators are still poorly characterized. Here we have systematically identified such regulators, taking advantage of the detailed functional characterization of the regulatory region of the medaka fish Six3.2 ortholog and of a time/cost-effective trans-regulatory screening, which complemented and overcame the limitations of in silico prediction approaches. The candidates resulting from this search were validated with dose-response luciferase assays and expression pattern criteria. Reconfirmed candidates with a matching expression pattern were also tested with chromatin immunoprecipitation and functional studies. Our results confirm the previously proposed direct regulation of Pax6 and further demonstrate that Msx2 and Pbx1 are bona fide direct regulators of early Six3.2 distribution in distinct domains of the medaka fish forebrain. They also point to other transcription factors, including Tcf3, as additional regulators of different spatial-temporal domains of Six3.2 expression. The activity of these regulators is discussed in the context of the gene regulatory network proposed for the specification of the forebrain., Spanish Ministerio de Economía y Competitividad (MINECO) Grants BFU2010-16031 and BFU2013-43213-P, cofounded by FEDER Funds; Comunidad Autónoma de Madrid (CAM) Grant CELL-DD S2010/BMD-2315; Fundaluce; Fundación ONCE; the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) del Instituto Carlos III (ISCIII); and an Institutional Grant from the Fundación Ramón Areces.

Genomes are transcribed well beyond the conventionally annotated protein-encoding genes and produce many thousands of regulatory non-coding RNAs (ncRNAs). In the last few years, ncRNAs, especially microRNAs and long non-coding RNA, have received increasing attention because of their implication in the function of chromatin-modifying complexes and in the regulation of transcriptional and post-transcriptional events. The morphological events and the genetic networks responsible for the development of sensory organs have been well delineated and therefore sensory organs have provided a useful scenario to address the role of ncRNAs. In this review, we summarize the current information on the importance of microRNAs and long non-coding RNAs during the development of the eye, inner ear, and olfactory system in vertebrates. We will also discuss those cases in which alteration of ncRNA expression has been linked to pathological conditions affecting these organs. © 2013 Springer Basel., Spanish MINECO (BFU2010-16031); Comunidad Autonoma de Madrid (CAM, S2010/BMD-2315); Fundaluce; Fundación ONCE; CIBERER; MAE/MOST Israel–Italy Joint Innovation Program; Italian Telethon Foundation (TGM11SB2)

MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation.

Retinal axon specification and growth are critically sensitive to the dosage of numerous signaling molecules and transcription factors. Subtle variations in the expression levels of key molecules may result in a variety of axonal growth anomalies. miR-181a and miR-181b are two eye-enriched microRNAs whose inactivation in medaka fish leads to alterations of the proper establishment of connectivity and function in the visual system. miR-181a/b are fundamental regulators of MAPK signaling and their role in retinal axon growth and specification is just beginning to be elucidated. Here we demonstrate that miR-181a/b are key nodes in the interplay between TGF-β and MAPK/ERK within the functional pathways that control retinal axon specification and growth. Using a variety of in vivo and in vitro approaches in medaka fish, we demonstrate that TGF-β signaling controls the miR-181/ERK regulatory network, which in turn strengthens the TGF-β-mediated regulation of RhoA degradation. Significantly, these data uncover the role of TGF-β signaling in vivo, for the first time, in defining the correct wiring and assembly of functional retina neural circuits and further highlight miR-181a/b as key factors in axon specification and growth.

Vax2 is an eye-specific homeobox gene, the inactivation of which in mouse leads to alterations in the establishment of a proper dorsoventral eye axis during embryonic development. To dissect the molecular pathways in which Vax2 is involved, we performed a transcriptome analysis of Vax2–/– mice throughout the main stages of eye development. We found that some of the enzymes involved in retinoic acid (RA) metabolism in the eye show significant variations of their expression levels in mutant mice. In particular, we detected an expansion of the expression domains of the RA-catabolizing enzymes Cyp26a1 and Cyp26c1, and a downregulation of the RA-synthesizing enzyme Raldh3. These changes determine a significant expansion of the RA-free zone towards the ventral part of the eye. At postnatal stages of eye development, Vax2 inactivation led to alterations of the regional expression of the cone photoreceptor genes Opn1sw (S-Opsin) and Opn1mw (M-Opsin), which were significantly rescued after RA administration. We confirmed the above described alterations of gene expression in the Oryzias latipes (medaka fish) model system using both Vax2 gain- and loss-of-function assays. Finally, a detailed morphological and functional analysis of the adult retina in mutant mice revealed that Vax2 is necessary for intraretinal pathfinding of retinal ganglion cells in mammals. These data demonstrate for the first time that Vax2 is both necessary and sufficient for the control of intraretinal RA metabolism, which in turn contributes to the appropriate expression of cone opsins in the vertebrate eye.

Timely generation of distinct neural cell types in appropriate numbers is fundamental for the generation of a functional retina. In vertebrates, the transcription factor Six6 is initially expressed in multipotent retina progenitors and then becomes restricted to differentiated retinal ganglion and amacrine cells. How Six6 expression in the retina is controlled and what are its precise functions are still unclear. To address this issue, we used bioinformatic searches and transgenic approaches in medaka fish (Oryzias latipes) to characterise highly conserved regulatory enhancers responsible for Six6 expression. One of the enhancers drove gene expression in the differentiating and adult retina. A search for transcription factor binding sites, together with luciferase, ChIP assays and gain-of-function studies, indicated that NeuroD, a bHLH transcription factor, directly binds an ‘E-box’ sequence present in this enhancer and specifically regulates Six6 expression in the retina. NeuroD-induced Six6 overexpression in medaka embryos promoted unorganized retinal progenitor proliferation and, most notably, impaired photoreceptor differentiation, with no apparent changes in other retinal cell types. Conversely, Six6 gain- and loss-of-function changed NeuroD expression levels and altered the expression of the photoreceptor differentiation marker Rhodopsin. In addition, knockdown of Six6 interfered with amacrine cell generation. Together, these results indicate that Six6 and NeuroD control the expression of each other and their functions coordinate amacrine cell generation and photoreceptor terminal differentiation.

Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of in vivo and in vitro approaches, we demonstrate that these actions are mediated by the direct targeting of the Ankrd13A gene, which in turn controls focal cell adhesion formation and distribution. Significantly, in vivo restoration of abnormally elevated levels of Ankrd13A resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time in vivo, the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a "master regulator" of the molecular networks that regulate lens morphogenesis in vertebrates.

A. Indrieri, V. van Rahden, V. Tiranti, I. Conte, J. Quartararo, M. Morleo, D. Iaconis, R. Tammaro, G. Chesi , M. Cermola, R. Tate, I. Maystadt, S. Demuth , A. Zvulunov, I. D’Amato, P. Goffrini, I. Ferrero, P. Bovolenta, K. Kutsche , M. Zeviani, B. Franco Telethon Institute of Genetics and Medicine (TIGEM), Napoli Institut fur Humangenetik, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Germany UO Neurogenetica Molecolare, Fondazione IRCCS Istituto Neurologico "C. Besta", Milano Dip di Genetica, Biologia dei Microrganismi, Antropologia, Evoluzione, Universita degli Studi di Parma, Parma Microscopia Integrata, Instituto di Genetica e Biofisica "Adriano Buzzati Traverso", CNR, Napoli Centre de Genetique Humaine, Institut de Pathologie et de Genetique, Gosselies (Charleroi), Belgium Gemeinschaftspraxis fur Humangenetik, Erfurt, Germany; Schneider Children's Medical Center of Israel, Ben-Gurion University of the Negev, Beer-Sheva, Israel Centro de Biologia Molecular “Severo Ochoa”, CSIC-UAM and CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Telethon Institute of Genetics and Medicine (TIGEM), Napoli, Dip di Pediatria, Universita degli Studi di Napoli "Federico II", Napoli.