Angiosperm flowers reached their highest morphological diversity early in their evolutionary history.

Flowers are the complex and highly diverse reproductive structures of angiosperms. Because of their role in sexual reproduction, the evolution of flowers is tightly linked to angiosperm speciation and diversification. Accordingly, the quantification of floral morphological diversity (disparity) among angiosperm subgroups and through time may give important insights into the evolutionary history of angiosperms as a whole. Based on a comprehensive dataset focusing on 30 characters describing floral structure across angiosperms, we used 1201 extant and 121 fossil flowers to measure floral disparity and explore patterns of floral evolution through time and across lineages. We found that angiosperms reached their highest floral disparity in the Early Cretaceous. However, decreasing disparity toward the present likely has not precluded the innovation of other complex traits at other morphological levels, which likely played a key role in the outstanding angiosperm species richness. Angiosperms occupy specific regions of the theoretical morphospace, indicating that only a portion of the possible floral trait combinations is observed in nature. The ANA grade, the magnoliids, and the early-eudicot grade occupy large areas of the morphospace (higher disparity), whereas nested groups occupy narrower regions (lower disparity).

Integrating Fossil Flowers into the Angiosperm Phylogeny Using Molecular and Morphological Evidence.

Fossils are essential to infer past evolutionary processes. The assignment of fossils to extant clades has traditionally relied on morphological similarity and on apomorphies shared with extant taxa. The use of explicit phylogenetic analyses to establish fossil affinities has so far remained limited. In this study, we built a comprehensive framework to investigate the phylogenetic placement of 24 exceptionally preserved fossil flowers. For this, we assembled a new species-level data set of 30 floral traits for 1201 extant species that were sampled to capture the stem and crown nodes of all angiosperm families. We explored multiple analytical approaches to integrate the fossils into the phylogeny, including different phylogenetic estimation methods, topological-constrained analyses, and combining molecular and morphological data of extant and fossil species. Our results were widely consistent across approaches and showed minor differences in the support of fossils at different phylogenetic positions. The placement of some fossils agrees with previously suggested relationships, but for others, a new placement is inferred. We also identified fossils that are well supported within particular extant families, whereas others showed high phylogenetic uncertainty. Finally, we present recommendations for future analyses combining molecular and morphological evidence, regarding the selection of fossils and appropriate methodologies, and provide some perspectives on how to integrate fossils into the investigation of divergence times and the temporal evolution of morphological traits. [Angiosperms; fossil flowers; phylogenetic uncertainty; RoguePlots.].

Utrophin modulator drugs as potential therapies for Duchenne and Becker muscular dystrophies.

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin-deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co-localise in human foetal muscle, in the dystrophin-competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.

Phylogenetic analysis of fossil flowers using an angiosperm-wide data set: proof-of-concept and challenges ahead.

PREMISE: Significant paleobotanical discoveries in recent decades have considerably improved our understanding of the early evolution of angiosperms and their flowers. However, our ability to test the systematic placement of fossil flowers on the basis of phylogenetic analyses has remained limited, mainly due to the lack of an adequate, angiosperm-wide morphological data set for extant taxa. Earlier attempts to place fossil flowers phylogenetically were, therefore, forced to make prior qualitative assessments of the potential systematic position of fossils and to restrict phylogenetic analyses to selected angiosperm subgroups. METHODS: We conduct angiosperm-wide molecular backbone analyses of 10 fossil flower taxa selected from the Cretaceous record. Our analyses make use of a floral trait data set built within the framework of the eFLOWER initiative. We provide an updated version of this data set containing data for 28 floral and two pollen traits for 792 extant species representing 372 angiosperm families. RESULTS: We find that some fossils are placed congruently with earlier hypotheses while others are found in positions that had not been suggested previously. A few take up equivocal positions, including the stem branches of large clades. CONCLUSIONS: Our study provides an objective approach to test for the phylogenetic position of fossil flowers across angiosperms. Such analyses may provide a complementary tool for paleobotanical studies, allowing for a more comprehensive understanding of fossil phylogenetic relationships in angiosperms. Ongoing work focused on extending the sampling of extant taxa and the number of floral traits will further improve the applicability and accuracy of our approach.

Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model.

BACKGROUND: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. METHODS: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status. RESULTS: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. CONCLUSIONS: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology.

Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy.

Several exon skipping antisense oligonucleotides (eteplirsen, golodirsen, viltolarsen, and casimersen) have been approved for the treatment of Duchenne muscular dystrophy, but many more are in development targeting an array of different DMD exons. Preclinical screening of the new oligonucleotide sequences is routinely performed using patient-derived cell cultures, and evaluation of their efficacy may be performed at RNA and/or protein level. While several methods to assess exon skipping and dystrophin expression in cell culture have been developed, the choice of methodology often depends on the availability of specific research equipment.In this chapter, we describe and indicate the relevant bibliography of all the methods that may be used in this evaluation and describe in detail the protocols routinely followed at our institution, one to evaluate the efficacy of skipping at RNA level (nested PCR) and the other the restoration of protein expression (myoblot ), which provide good results using equipment largely available to most research laboratories.

Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening.

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient's immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and ß-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

An Overview of Alternative Splicing Defects Implicated in Myotonic Dystrophy Type I.

Myotonic dystrophy type I (DM1) is the most common form of adult muscular dystrophy, caused by expansion of a CTG triplet repeat in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase (DMPK) gene. The pathological CTG repeats result in protein trapping by expanded transcripts, a decreased DMPK translation and the disruption of the chromatin structure, affecting neighboring genes expression. The muscleblind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) are two families of tissue-specific regulators of developmentally programmed alternative splicing that act as antagonist regulators of several pre-mRNA targets, including troponin 2 (TNNT2), insulin receptor (INSR), chloride channel 1 (CLCN1) and MBNL2. Sequestration of MBNL proteins and up-regulation of CELF1 are key to DM1 pathology, inducing a spliceopathy that leads to a developmental remodelling of the transcriptome due to an adult-to-foetal splicing switch, which results in the loss of cell function and viability. Moreover, recent studies indicate that additional pathogenic mechanisms may also contribute to disease pathology, including a misregulation of cellular mRNA translation, localization and stability. This review focuses on the cause and effects of MBNL and CELF1 deregulation in DM1, describing the molecular mechanisms underlying alternative splicing misregulation for a deeper understanding of DM1 complexity. To contribute to this analysis, we have prepared a comprehensive list of transcript alterations involved in DM1 pathogenesis, as well as other deregulated mRNA processing pathways implications.