Genetic variations in G-quadruplex forming sequences affect the transcription of human disease-related genes.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4s). G4s folded in proximal promoter regions (PPR) are associated either with positive or negative transcriptional regulation. Given that single nucleotide variants (SNVs) affecting G4 folding (G4-Vars) may alter gene transcription, and that SNVs are associated with the human diseases' onset, we undertook a novel comprehensive study of the G4-Vars genome-wide (G4-variome) to find disease-associated G4-Vars located into PPRs. We developed a bioinformatics strategy to find disease-related SNVs located into PPRs simultaneously overlapping with putative G4-forming sequences (PQSs). We studied five G4-Vars disturbing in vitro the folding and stability of the G4s located into PPRs, which had been formerly associated with sporadic Alzheimer's disease (GRIN2B), a severe familiar coagulopathy (F7), atopic dermatitis (CSF2), myocardial infarction (SIRT1) and deafness (LHFPL5). Results obtained in cultured cells for these five G4-Vars suggest that the changes in the G4s affect the transcription, potentially contributing to the development of the mentioned diseases. Collectively, data reinforce the general idea that G4-Vars may impact on the different susceptibilities to human genetic diseases' onset, and could be novel targets for diagnosis and drug design in precision medicine.

G-Quadruplexes Regulate miRNA Biogenesis in Live Zebrafish Embryos.

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesis in vivo during zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to fold in vitro as G4. MiR-150 controls the expression of myb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos with in vitro transcribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels of myb mRNA and stronger phenotypes associated with myb knock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related to myb knock-down. Overall, results suggest that the G4 formed in pre-miR-150 functions in vivo as a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis.

CNBP controls transcription by unfolding DNA G-quadruplex structures.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4). Experimental evidences suggest that G4-DNA surrounding transcription start sites act as cis-regulatory elements by either stimulating or inhibiting gene transcription. Therefore, proteins able to target and regulate specific G4 formation/unfolding are crucial for G4-mediated transcriptional control. Here we present data revealing that CNBP acts in vitro as a G4-unfolding protein over a tetramolecular G4 formed by the TG4T oligonucleotide, as well as over the G4 folded in the promoters of several oncogenes. CNBP depletion in cellulo led to a reduction in the transcription of endogenous KRAS, suggesting a regulatory role of CNBP in relieving the transcriptional abrogation due to G4 formation. CNBP activity was also assayed over the evolutionary conserved G4 enhancing the transcription of NOGGIN (NOG) developmental gene. CNBP unfolded in vitro NOG G4 and experiments performed in cellulo and in vivo in developing zebrafish showed a repressive role of CNBP on the transcription of this gene by G4 unwinding. Our results shed light on the mechanisms underlying CNBP way of action, as well as reinforce the notion about the existence and function of G4s in whole living organisms.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology.

Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome.

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patient's fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology.

A noncoding expansion in EIF4A3 causes Richieri-Costa-Pereira syndrome, a craniofacial disorder associated with limb defects.

Richieri-Costa-Pereira syndrome is an autosomal-recessive acrofacial dysostosis characterized by mandibular median cleft associated with other craniofacial anomalies and severe limb defects. Learning and language disabilities are also prevalent. We mapped the mutated gene to a 122 kb region at 17q25.3 through identity-by-descent analysis in 17 genealogies. Sequencing strategies identified an expansion of a region with several repeats of 18- or 20-nucleotide motifs in the 5' untranslated region (5' UTR) of EIF4A3, which contained from 14 to 16 repeats in the affected individuals and from 3 to 12 repeats in 520 healthy individuals. A missense substitution of a highly conserved residue likely to affect the interaction of eIF4AIII with the UPF3B subunit of the exon junction complex in trans with an expanded allele was found in an unrelated individual with an atypical presentation, thus expanding mutational mechanisms and phenotypic diversity of RCPS. EIF4A3 transcript abundance was reduced in both white blood cells and mesenchymal cells of RCPS-affected individuals as compared to controls. Notably, targeting the orthologous eif4a3 in zebrafish led to underdevelopment of several craniofacial cartilage and bone structures, in agreement with the craniofacial alterations seen in RCPS. Our data thus suggest that RCPS is caused by mutations in EIF4A3 and show that EIF4A3, a gene involved in RNA metabolism, plays a role in mandible, laryngeal, and limb morphogenesis.

CNBP modulates the transcription of Wnt signaling pathway components.

BACKGROUND: Cellular nucleic acid binding protein (CNBP) is a small and highly conserved protein with nucleic acid chaperone activity that binds single-stranded nucleic acids. Data collected so far suggests that CNBP is required for proper craniofacial development. Despite the advances achieved in the last decade, the identity of the molecular targets of CNBP responsible for its role in rostral head development remains elusive. METHODS: In this work we used the CNBP single-stranded DNA-consensus binding sequence to find out putative CNBP target genes present in the human, mouse, chicken, Xenopus and zebrafish genomes. RESULTS: Most of the identified genes are associated with embryonic developmental processes, being three of them (cdk14, ptk7 and tcf7l2) members of the Wnt signaling pathway. This finding, along with previous one showing that CNBP down-regulates the transcription of Wnt5, aimed our work to address the role of CNBP on the WNT signaling players and pathway regulation. Experiments carried out in zebrafish developing embryos revealed that craniofacial morphology was more adversely affected as CNBP abundance decreased. Furthermore, we observed that CNBP up-regulated in a dose-dependent fashion the transcription of cdk14, ptk7 and tcf7l2, which in turn was reflected in c-myc, ccnd1 and axin2 expression. CONCLUSIONS: RESULTS reveal a role of CNBP in transcriptional control of components of the Wnt signaling pathway, which might explain its requirement for proper craniofacial development.

Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.

Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advances made concerning CNBP biological roles, a full knowledge about the structure-function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here, we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan, which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and ß-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding.

Set-up of an infrared fast behavioral assay using zebrafish (Danio rerio) larvae, and its application in compound biotoxicity screening.

Zebrafish (Danio rerio) is increasingly employed for evaluating toxicity and drug discovery assays. Commonly experimental approaches for biotoxicity assessment are based on visual inspection or video recording. However, these techniques are limited for large-scale assays, as they demand either a time-consuming detailed inspection of the animals or intensive computing resources in order to analyze a considerable amount of screenshots. Recently, we have developed a simple methodology for tracking the locomotor activity of small animals cultured in microtiter plates. In this work, we implemented this automatic methodology, based on infrared (IR) microbeam scattering, for measuring behavioral activity in zebrafish larvae. We determined the appropriate culture conditions, number of animals and stage of development to get robust results. Furthermore, we validated this methodology as a rapid test for evaluating toxicity. By measuring the effects of reference compounds on larvae activity, we were able to estimate the concentration that could cause a 50% decrease in activity events values (AEC50), showing a strong linear correlation (R² = 0.91) with the LC50 values obtained with the standard DarT test. The toxicity order of the measured compounds was CuSO4 > 2,4-dinitrophenol > 3,4-dichloroaniline > SDS > sodium benzoate > EDTA > K2CrO4 ; regarding solvents, EtOH ≈ DMSO. In this study, we demonstrate that global swimming behavior could be a simple readout for toxicity, easy to scale-up in automated experiments. This approach is potentially applicable for fast ecotoxicity assays and whole-organism high-throughput compound screening, reducing the time and money required to evaluate unknown samples and to identify leading pharmaceutical compounds.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

ΔNp63 is regulated by BMP4 signaling and is required for early epidermal development in Xenopus.

BACKGROUND: It has been established in several models that the p63 gene has an important role in the development of the epidermis and its derivatives. In Xenopus, only the ΔNp63 isoform of this gene has been cloned and its role during epidermal development remains unknown. RESULTS: In this work, we showed that ΔNp63 is expressed in the nonneural ectoderm since the gastrula stage and that it is regulated by the bone morphogenetic protein 4 (BMP4) signaling pathway. Our in vivo and in vitro experiments demonstrated that ΔNp63 is required in the earliest inductive steps of epidermal development. The overexpression of ΔNp63 caused an increase in epidermal markers with a suppression of neural induction while the blocking of ΔNp63 led to the opposite results. Finally, we found that ΔNp63 acts as an anti-apoptotic gene, regulating the transcription of some apoptotic and anti-apoptotic factors. CONCLUSION: The results suggest that ΔNp63 is an essential gene in early epidermal specification under the control of BMP4.

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems.

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Deciphering the cellular and molecular roles of cellular nucleic acid binding protein during cranial neural crest development.

Cellular nucleic acid binding protein (Cnbp) is a highly conserved single-stranded nucleic acid binding protein required for rostral head development. The use of a morpholino that inhibits Cnbp mRNA translation previously revealed a role of Cnbp in balancing neural crest cell apoptosis and proliferation in the developing zebrafish. Here, we report the use of another morpholino that specifically modifies the splicing of Cnbp pre-mRNA resulting in a reduction of full-length mRNA levels along with the generation of a novel transcript coding for an isoform that may act as dominant negative proteins. The use of this morpholino resulted in more severe phenotypes that enabled us to demonstrate that Cnbp loss-of-function adversely affects the formation and survival of craniofacial cartilaginous structures not only controlling the ratio of cell proliferation and apoptosis but also defining skeletogenic neural crest cell fate.

Cellular nucleic-acid-binding protein, a transcriptional enhancer of c-Myc, promotes the formation of parallel G-quadruplexes.

G-rich sequences that contain stretches of tandem guanines can form four-stranded, intramolecular stable DNA structures called G-quadruplexes (termed G4s). Regulation of the equilibrium between single-stranded and G4 DNA in promoter regions is essential for control of gene expression in the cell. G4s are highly stable structures; however, their folding kinetics are slow under physiological conditions. CNBP (cellular nucleic-acid-binding protein) is a nucleic acid chaperone that binds the G4-forming G-rich sequence located within the NHE (nuclease hypersensitivity element) III of the c-Myc proto-oncogene promoter. Several reports have demonstrated that CNBP enhances the transcription of c-Myc in vitro and in vivo; however, none of these reports have assessed the molecular mechanisms responsible for this control. In the present study, by means of Taq polymerase stop assays, electrophoretic mobility-shift assays and CD spectroscopy, we show that CNBP promotes the formation of parallel G4s to the detriment of anti-parallel G4s, and its nucleic acid chaperone activity is required for this effect. These findings are the first to implicate CNBP as a G4-folding modulator and, furthermore, assign CNBP a novel mode-of-action during c-Myc transcriptional regulation.

What's new about CNBP? Divergent functions and activities for a conserved nucleic acid binding protein.

BACKGROUND: Cellular nucleic acid binding protein (CNBP) is a conserved single-stranded nucleic acid binding protein present in most eukaryotes, but not in plants. Expansions in the CNBP gene cause myotonic dystrophy type 2. Initially reported as a transcriptional regulator, CNBP was then also identified acting as a translational regulator. SCOPE OF REVIEW: The focus of this review was to link the CNBP structural features and newly reported biochemical activities with the recently described biological functions, in the context of its pathological significance. MAJOR CONCLUSIONS: Several post-translational modifications affect CNBP subcellular localization and activity. CNBP participates in the transcriptional and translational regulation of a wide range of genes by remodeling single-stranded nucleic acid secondary structures and/or by modulating the activity of trans-acting factors. CNBP is required for proper neural crest and heart development, and plays a role in cell proliferation control. Besides, CNBP has been linked with neurodegenerative, inflammatory, and congenital diseases, as well as with tumor processes. GENERAL SIGNIFICANCE: This review provides an insight into the growing functions of CNBP in cell biology. A unique and robust mechanistic or biochemical connection among these roles has yet not been elucidated. However, the ability of CNBP to dynamically integrate signaling pathways and to act as nucleic acid chaperone may explain most of the roles and functions identified so far.

Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development.

The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3'UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.

CNBP: a multifunctional nucleic acid chaperone involved in cell death and proliferation control.

Cellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases. In these seemingly unrelated biological processes, CNBP appears to be involved in controlling cell death and proliferation rates. Low levels of CNBP may reduce rate of global protein synthesis, thereby reducing proliferation and increasing apoptosis. Conversely, CNBP might affect transcription of genes required for cell proliferation. Experimental evidences gathered so far make it difficult to ascertain or rule out any of these possibilities. Moreover, both possibilities may not be mutually exclusive. CNBP is a small and strikingly conserved single-stranded nucleic acid binding protein that is able to bind DNA as well as RNA. CNBP has a broad spectrum of targets, ranging from regulatory sites in gene promoters to translational regulatory elements in mRNA untranslated regions. Biochemical experiments have recently shed light on the possible mechanism of action for CNBP, which may act as a nucleic acid chaperone catalyzing the rearrangement of G-rich nucleic acid secondary structures likely relevant for transcriptional and/or translational gene regulation. This review focuses on the involvement of CNBP in vertebrate craniofacial development and human DM2 and sIBM diseases, as well as on the biochemical and structural features of CNBP and its cellular and molecular mechanism of action.

Zebrafish cnbp intron1 plays a fundamental role in controlling spatiotemporal gene expression during embryonic development.

Cellular nucleic acid binding protein (CNBP) is a strikingly conserved zinc-finger nucleic acid chaperone required for forebrain development. Its depletion causes forebrain truncation mainly as a consequence of a reduction in size of craniofacial structures and neural crest derivatives. The CNBP expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. CNBP expression is highly conserved between all vertebrates characterized. In this study we have combined comparative sequence analysis and in vivo testing of DNA fragments in zebrafish to identify evolutionarily constrained regulatory motifs that likely control expression of the cnbp gene in embryos. We found a novel exon sequence located 5' upstream of the Exon1-sequence reported in most databases, and two transcription start sites that generate two primary-transcripts that differ in their 5'UTRs and expression profile during zebrafish embryonic development. Furthermore, we found a region inside the intron1 sequence that controls the cnbp developmental-specific transcriptional activation. Conserved binding sites for neural crest transcription factors were identified in this region. Mutagenesis analysis of the regulatory region revealed that Pax6/FoxD3 binding sites are required for proper zygotic cnbp expression. This is the first study that identifies, in vivo, cis-regulatory sequences inside intron sequences and typical neural crest transcription factors involved in cnbp spatiotemporal specific transcriptional control during vertebrate embryonic development.

Proteasomal inhibition attenuates craniofacial malformations in a zebrafish model of Treacher Collins Syndrome.

Treacher Collins Syndrome (TCS) is a congenital disease characterized by defects in the craniofacial skeleton and absence of mental alterations. Recently we modelled TCS in zebrafish (Danio rerio) embryos through the microinjection of Morpholino® oligonucleotides blocking the translation of the ortholog of the main causative gene (TCOF1). We showed that Cnbp, a key cytoprotective protein involved in normal rostral head development, was detected in lower levels (without changes in its mRNA expression) in TCS-like embryos. As previous reports suggested that Cnbp is degraded through the proteasomal pathway, we tested whether proteasome inhibitors (MG132 and Bortezomib (Velcade®, Millennium laboratories)) were able to ameliorate cranial skeleton malformations in TCS. Here we show that treatment with both proteasome inhibitors produced a robust craniofacial cartilage phenotype recovery. This recovery seems to be consequence of a decreased degradation of Cnbp in TCS-like embryos. Critical TCS manifestations, such as neuroepithelial cell death and cell redox imbalance were attenuated. Thus, proteasome inhibitors may offer an opportunity for TCS molecular and phenotypic manifestation's prevention. Although further development of new safe inhibitors compatible with administration during pregnancy is required, our results encourage this therapeutic approach.

Dicer1 is required for pigment cell and craniofacial development in zebrafish.

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1sa9205, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1sa9205 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1sa9205 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1sa9205 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10.