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SUMMARYT-Cell Factors (TCFs) are the main transcriptional effectors of Wnt/β-catenin signaling. TCF responsiveness is a hallmark of self-renewal in mouse embryonic, and adult, neural stem cells (NSC). However,in vivocontribution(s) of TCF activities in long-lived NSCs are poorly understood. Granule neuron progenitors (GNP) in the upper rhombic lip (URL) are long-lived NSCs which expressAtoh1and generate cerebellar granule neurons. Using functional and transcriptomic approaches in amphibian, we demonstrate that TCFs are active in the URL, and are strictly necessary for the emergence and maintenance of the GNP germinative zone. We identify BarH-like 1 (Barhl1), a direct target of Atoh1, as a gate keeper for GNP exit from the URL, through silencing of TCF transcriptional activity. Our transcriptomic andin silicoanalysis identifies Barhl1/TCF URL target genes, and confirms our functional data. Our study providesin vivoevidence that inhibition of TCF repressive activity is necessary for maintenance of the URL, a long-lived neural germinative niche.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

International audience; CRISPR/Cas9-based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)-induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock-ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9-targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock-in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.

Background The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. Results We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. Conclusions To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (http://crispor.org) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

International audience; The majority of angiosperms are syncarpous- their gynoecium is composed of two or more fused carpels. In Arabidopsis thaliana, this fusion is regulated through the balance of expression between CUP SHAPED COTYLEDON (CUC) genes, which are orthologs of the Petunia hybrida transcription factor NO APICAL MERISTEM (NAM), and their post transcriptional regulator miR164. Accordingly, the expression of a miR164-insensitive form of A. thallana CUC2 causes a radical breakdown of carpel fusion. Here, we investigate the role of the NAM/miR164 genetic module in carpel closure in monocarpous plants. We show that the disruption of this module in monocarpous flowers of A. thaliana aux1-22 mutants causes a failure of carpel closure, similar to the failure of carpel fusion observed in the wild-type genetic background. This observation suggested that closely related mechanisms may bring about carpel closure and carpel fusion, at least in A. thaliana. We therefore tested whether these mechanisms were conserved in a eurosid species that is monocarpous in its wild type form. We observed that expression of MtNAM, the NAM ortholog in the monocarpous eurosid Medicago truncatula, decreases during carpel margin fusion, suggesting a role for the NAM/miR164 module in this process. We transformed M. truncatula with a miR164-resistant form of MtNAM and observed, among other phenotypes, incomplete carpel closure in the resulting transformants. These data confirm the underlying mechanistic similarity between carpel closure and carpel fusion which we observed in A. thaliana, Our observations suggest that the role of the NAM/miR164 module in the fusion of carpel margins has been conserved at least since the most recent common ancestor of the eurosid clade, and open the possibility that a similar mechanism may have been responsible for carpel closure at much earlier stages of angiosperm evolution. We combine our results with studies of early diverging angiosperms to speculate on the role of the NAM/miR164 module in the origin and further evolution of the angiosperm carpel.

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11::GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

Transposable elements have been widely used as mutagens in many organisms. Among them, the maize transposable element En/Spm has been shown to transpose efficiently in several plant species including the model plant Arabidopsis, where it has been used for large-scale mutagenesis. To determine whether we could use this transposon as a mutagen in the model legume plant Medicago truncatula, we tested the activity of the autonomous element, as well as two defective elements, in this plant, and in Arabidopsis as a positive control. In agreement with previous reports, we observed efficient excision of the autonomous En/Spm element in A. thaliana. This element was also active in M. truncatula, but the transposition activity was low and was apparently restricted to the tissue culture step necessary for the production of transgenic plants. The activity of one of the defective transposable elements, dSpm, was very low in A. thaliana and even lower in M. truncatula. The use of different sources of transposases suggested that this defect in transposition was associated with the dSpm element itself. Transposition of the other defective element, I6078, was also detected in M. truncatula, but, as observed with the autonomous element, transposition events were very rare and occurred during tissue culture. These results suggest that the En/Spm element is rapidly inactivated in the regenerated plants and their progeny, and therefore is not suitable for routine insertion mutagenesis in M. truncatula.

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.

Legumes have been for a long time recalcitrant to efficient Agrobacterium transformation. The choice and use of model legume plants (Medicago truncatula and Lotus japonicus) for molecular studies has triggered extensive studies devoted to the development of efficient Agrobacterium-mediated transformation protocols for these two plants. In M. truncatula, transformation protocols rely on the use of highly regenerable lines obtained by recurrent in vitro culture selection. These protocols are based on Agrobacterium-mediated transformation of M. truncatula followed by somatic embryogenesis-mediated plant regeneration. We describe here the protocol developed for M. truncatula R108-1 (c3).

International audience; Rhizobia and legumes establish symbiotic interactions leading to the production of root nodules, in which bacteria fix atmospheric nitrogen for the plant's benefit. This symbiosis is efficient because of the high rhizobia population within nodules. Here, we investigated how legumes accommodate such bacterial colonization. We used a reverse genetic approach to identify a Medicago truncatula gene, SymCRK, which encodes a cysteine-rich receptor-like kinase that is required for rhizobia maintenance within the plant cells, and performed detailed phenotypic analyses of the corresponding mutant. The Medicago truncatula symCRK mutant developed nonfunctional and necrotic nodules. A nonarginine asparate (nonRD) motif, typical of receptors involved in innate immunity, is present in the SymCRK kinase domain. Similar to the dnf2 mutant, bacteroid differentiation defect, defense-like reactions and early senescence were observed in the symCRK nodules. However, the dnf2 and symCRK nodules differ by their degree of colonization, which is higher in symCRK. Furthermore, in contrast to dnf2, symCRK is not a conditional mutant. These results suggest that in M. truncatula at least two genes are involved in the symbiotic control of immunity. Furthermore, phenotype differences between the two mutants suggest that two distinct molecular mechanisms control suppression of plant immunity during nodulation.

Wine strains of Saccharomyces cerevisiae have no to weak natural pectinase activity, despite their genetic ability to secrete an endo-polygalacturonase. The addition of external pectinase of fungal origin has therefore become a common step of winemaking in order to enhance the extraction of compounds located in the grape berry skins during maceration and to ease wine clarification after maturation. Recently, the strong pectinase activity of a wine strain of Saccharomyces paradoxus has been reported. In this study, the endo-polygalacturonase-encoding gene of S. paradoxus was sequenced and its activity was characterised, compared with that of S. cerevisiae and tested under winemaking conditions through overexpression of both genes individually in S. cerevisiae. A few differences in the amino acids sequences between the two proteins were revealed and the activity of the Pgu1 enzyme of S. paradoxus was shown to be weaker under winemaking conditions. Clear indicators of extracellular activity were observed in the wines made with both recombinant strains (i.e. enzyme activity in cell-free wine, higher methanol concentration and higher free-run wine), but the actual composition of the wines fermented with the mutants was only sparingly altered. Although unexpectedly found in lower concentrations in the latter wines, phenolic compounds were shown to be the most discriminatory components. Overexpressing the PGU1 gene of S. paradoxus or that of S. cerevisiae did not make much difference, showing that the higher activity of S. paradoxus strains under laboratory conditions could be due to a different regulation mechanism rather than to a different sequence of PGU1.

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.