Nodal/Activin Pathway is a Conserved Neural Induction Signal in Chordates.

Neural induction is the process through which pluripotent cells are committed to a neural fate. This first step of Central Nervous System formation is triggered by the "Spemann organizer" in amphibians and by homologous embryonic regions in other vertebrates. Studies in classical vertebrate models have produced contrasting views about the molecular nature of neural inducers and no unifying scheme could be drawn. Moreover, how this process evolved in the chordate lineage remains an unresolved issue. In this work, by using graft and micromanipulation experiments, we definitively establish that the dorsal blastopore lip of the cephalochordate amphioxus is homologous to the vertebrate organizer and is able to trigger the formation of neural tissues in a host embryo. In addition, we demonstrate that Nodal/Activin is the main signal eliciting neural induction in amphioxus, and that it also functions as a bona fide neural inducer in the classical vertebrate model Xenopus. Altogether, our results allow us to propose that Nodal/Activin was a major player of neural induction in the ancestor of chordates. This study further reveals the diversity of neural inducers deployed during chordate evolution and advocates against a universally conserved molecular explanation for this process.

Hypogonadism Associated with Cyp19a1 (Aromatase) Posttranscriptional Upregulation in Celf1 Knockout Mice.

CELF1 is a multifunctional RNA-binding protein that controls several aspects of RNA fate. The targeted disruption of the Celf1 gene in mice causes male infertility due to impaired spermiogenesis, the postmeiotic differentiation of male gametes. Here, we investigated the molecular reasons that underlie this testicular phenotype. By measuring sex hormone levels, we detected low concentrations of testosterone in Celf1-null mice. We investigated the effect of Celf1 disruption on the expression levels of steroidogenic enzyme genes, and we observed that Cyp19a1 was upregulated. Cyp19a1 encodes aromatase, which transforms testosterone into estradiol. Administration of testosterone or the aromatase inhibitor letrozole partly rescued the spermiogenesis defects, indicating that a lack of testosterone associated with excessive aromatase contributes to the testicular phenotype. In vivo and in vitro interaction assays demonstrated that CELF1 binds to Cyp19a1 mRNA, and reporter assays supported the conclusion that CELF1 directly represses Cyp19a1 translation. We conclude that CELF1 downregulates Cyp19a1 (Aromatase) posttranscriptionally to achieve high concentrations of testosterone compatible with spermiogenesis completion. We discuss the implications of these findings with respect to reproductive defects in men, including patients suffering from isolated hypogonadotropic hypogonadism and myotonic dystrophy type I.

BMP signalling controls the construction of vertebrate mucociliary epithelia.

Despite the importance of mucociliary epithelia in animal physiology, the mechanisms controlling their establishment are poorly understood. Using the developing Xenopus epidermis and regenerating human upper airways, we reveal the importance of BMP signalling for the construction of vertebrate mucociliary epithelia. In Xenopus, attenuation of BMP activity is necessary for the specification of multiciliated cells (MCCs), ionocytes and small secretory cells (SSCs). Conversely, BMP activity is required for the proper differentiation of goblet cells. Our data suggest that the BMP and Notch pathways interact to control fate choices in the developing epidermis. Unexpectedly, BMP activity is also necessary for the insertion of MCCs, ionocytes and SSCs into the surface epithelium. In human, BMP inhibition also strongly stimulates the formation of MCCs in normal and pathological (cystic fibrosis) airway samples, whereas BMP overactivation has the opposite effect. This work identifies the BMP pathway as a key regulator of vertebrate mucociliary epithelium differentiation and morphogenesis.

A gene regulation network controlled by Celf1 protein-rbpj mRNA interaction in Xenopus somite segmentation.

Somite segmentation is impaired in Xenopus celf1 morphant embryos. The Celf1 RNA-binding protein targets bound mRNAs for rapid degradation, and antisense approaches demonstrated that segmentation defects in celf1 morphants were due to a derepression of rbpj mRNA. Rbpj protein is a key player of Notch signalling. Because segmentation involves complex cross-talk between several signalling pathways, we analysed how rbpj derepression impacted these pathways. We found that rbpj derepression stimulated the Notch pathway. Notch positively controlled the expression of cyp26a, which encodes a retinoic acid (RA)-degrading enzyme. Thus, rbpj derepression led to cyp26a overexpression and RA attenuation. It also repressed fgf8, consistent with an inhibition of FGF signalling. Pharmacological inhibition of the FGF pathway repressed cyp26a, but rbpj derepression was sufficient to restore cyp26a expression. Hence, while it was known that the FGF pathway antagonized RA signalling through expression of cyp26a, our results suggest that Rbpj mediates this antagonism. Furthermore, they show that the post-transcriptional repression exerted by Celf1 on rbpj mRNA is required to keep cyp26a expression under the control of FGF signalling. We conclude that rbpj repression by Celf1 is important to couple the FGF and RA pathways in Xenopus segmentation.

Inactivation of the Celf1 gene that encodes an RNA-binding protein delays the first wave of spermatogenesis in mice.

BACKGROUND: The first wave of spermatogenesis in mammals is characterized by a sequential and synchronous appearance of germ cells in the prepubertal testis. Post-transcriptional controls of gene expression play important roles in this process but the molecular actors that underlie them are poorly known. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the requirement for the RNA-binding protein CELF1 during the first wave of spermatogenesis in mice. Mice inactivated for Celf1 gene were not viable on pure genetic backgrounds. On a mixed background, we observed by histology and gene profiling by RT-qPCR that the testes of inactivated prepubertal mice were characterized by several features. (i) Spermiogenesis (differentiation of post-meiotic cells) was blocked in a subset of prepubertal inactivated mice. (ii) The appearance of the different stages of germ cell development was delayed by several days. (iii) The expression of markers of Leydig cells functions was similarly delayed. CONCLUSIONS/SIGNIFICANCE: Celf1 disruption is responsible for a blockage of spermiogenesis both in adults and in prepubertal males. Hence, the spermiogenesis defects found in Celf1-inactivated adults appear from the first wave of spermiogenesis. The disruption of Celf1 gene is also responsible for a fully penetrant delayed first wave of spermatogenesis, and a delay of steroidogenesis may be the cause for the delay of germ cells differentiation.

Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/Notch pathway.

Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.

Protein tyrosine kinase 7 has a conserved role in Wnt/ß-catenin canonical signalling.

The receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with ß-catenin in a yeast two-hybrid assay and mammalian cells. PTK7-deficient cells exhibit weakened ß-catenin/T-cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require ß-catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway.

Post-transcriptional controls - adding a new layer of regulation to clock gene expression.

Living organisms undergo biochemical, physiological and behavioral cycles with periods ranging from seconds to years. Cycles with intermediate periods are governed by endogenous clocks that depend on oscillating gene expression. Here we illustrate the modalities and specific functions of post-transcriptional control of gene expression (exerted on pre-mRNAs and mRNAs) in biological clocks through two examples: the circadian clock and the vertebrate somite segmentation clock, an embryonic clock with a period far below a day. We conclude that both constitutive and cyclic post-transcriptional controls underpin clock function.

A strategy to analyze the phenotypic consequences of inhibiting the association of an RNA-binding protein with a specific RNA.

Targeted inactivations of RNA-binding proteins (RNA-BPs) can lead to huge phenotypical defects. These defects are due to the deregulation of certain mRNAs. However, we generally do not know, among the hundreds of mRNAs that are normally controlled by one RNA-BP, which are responsible for the observed phenotypes. Here, we designed an antisense oligonucleotide ("target protector") that masks the binding site of the RNA-BP CUG-binding protein 1 (CUGBP1) on the mRNA Suppressor of Hairless [Su(H)] that encodes a key player of Notch signaling. We showed that injecting this oligonucleotide into Xenopus embryos specifically inhibited the binding of CUGBP1 to the mRNA. This caused the derepression of Su(H) mRNA, the overexpression of Su(H) protein, and a phenotypic defect, loss of somitic segmentation, similar to that caused by a knockdown of CUGBP1. To demonstrate a causal relationship between Su(H) derepression and the segmentation defects, a rescue experiment was designed. Embryonic development was restored when the translation of Su(H) mRNA was re-repressed and the level of Su(H) protein was reduced to a normal level. This "target protector and rescue assay" demonstrates that the phenotypic defects associated with CUGBP1 inactivation in Xenopus are essentially due to the deregulation of Su(H) mRNA. Similar approaches may be largely used to uncover the links between the phenotype caused by the inactivation of an RNA-BP and the identity of the RNAs associated with that protein.

Analysis of splicing patterns by pyrosequencing.

Several different mRNAs can be produced from a given pre-mRNA by regulated alternative splicing, or as the result of deregulations that may lead to pathological states. Analysing splicing patterns is therefore of importance to describe and understand developmental programs, cellular responses to internal or external cues, or human diseases. We describe here a method, Pyrosequencing Analysis of Splicing Patterns (PASP), that combines RT-PCR and pyrosequencing of PCR products. We demonstrated that: (i) Ratios of two pure RNAs mixed in various proportions were accurately measured by PASP; (ii) PASP can be adapted to virtually any splicing event, including mutually exclusive exons, complex patterns of exon skipping or inclusion, and alternative 3' terminal exons; (iii) In extracts from different organs, the proportions of RNA isoforms measured by PASP reflected those measured by other methods. The PASP method is therefore reliable for analysing splicing patterns. All steps are done in 96-wells microplates, without gel electrophoresis, opening the way to high-throughput comparisons of RNA from several sources.

Identification of new subgroup of HSP70 in Bythograeidae (hydrothermal crabs) and Xanthidae.

Crabs of the Bythograeidae family (Crustacea: Brachyura: Bythogreoidea) are the only endemic crab family living in hydrothermal fields. The hydrothermal environment is characterized by unique ecological parameters, such as the high temperature gradient around the hydrothermal chimney (2-350 degrees C), a fluid environment containing high levels of metals and numerous gases. The 70-kDa Heat Shock Protein (HSP70) group is the most-studied HSP, because it is ubiquitous, and a strong positive correlation has been found between the amounts of HSP70 produced in response to stress, and the ability of the organism to withstand stressful conditions. The 70-kDa heat shock protein genes from Bythograeids (species analyzed: Bythograea thermydron, Cyanagraea praedator and Segonzacia mesatlantica) were characterized. Our results revealed that Bythograeidae possess genes which are similar with those present in Xanthids (coastal crabs). The deduced protein sequences displayed motifs distinct from those in the other crustacean HSC70/HSP70s available in the databases. Phylogenetic analysis showed that these members of HSP70 family identified in Bythograeidae and Xanthidae constitute a new subgroup within this family.