Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10's essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation 'machinery' that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

When Bigger Is Better: 3D RNA Profiling of the Developing Head in the Catshark Scyliorhinus canicula.

We report the adaptation of RNA tomography, a technique allowing spatially resolved, genome-wide expression profiling, to a species occupying a key phylogenetic position in gnathostomes, the catshark Scyliorhinus canicula. We focused analysis on head explants at an embryonic stage, shortly following neural tube closure and of interest for a number of developmental processes, including early brain patterning, placode specification or the establishment of epithalamic asymmetry. As described in the zebrafish, we have sequenced RNAs extracted from serial sections along transverse, horizontal and sagittal planes, mapped the data onto a gene reference taking advantage of the high continuity genome recently released in the catshark, and projected read counts onto a digital model of the head obtained by confocal microscopy. This results in the generation of a genome-wide 3D atlas, containing expression data for most protein-coding genes in a digital model of the embryonic head. The digital profiles obtained for candidate forebrain regional markers along antero-posterior, dorso-ventral and left-right axes reproduce those obtained by in situ hybridization (ISH), with expected relative organizations. We also use spatial autocorrelation and correlation as measures to analyze these data and show that they provide adequate statistical tools to extract novel expression information from the model. These data and tools allow exhaustive searches of genes exhibiting any predefined expression characteristic, such a restriction to a territory of interest, thus providing a reference for comparative analyses across gnathostomes. This methodology appears best suited to species endowed with large embryo or organ sizes and opens novel perspectives to a wide range of evo-devo model organisms, traditionally counter-selected on size criterion.

Prmt5 promotes vascular morphogenesis independently of its methyltransferase activity.

During development, the vertebrate vasculature undergoes major growth and remodeling. While the transcriptional cascade underlying blood vessel formation starts to be better characterized, little is known concerning the role and mode of action of epigenetic enzymes during this process. Here, we explored the role of the Protein Arginine Methyl Transferase Prmt5 in blood vessel formation as well as hematopoiesis using zebrafish as a model system. Through the combination of different prmt5 loss-of-function approaches we highlighted a key role of Prmt5 in both processes. Notably, we showed that Prmt5 promotes vascular morphogenesis through the transcriptional control of ETS transcription factors and adhesion proteins in endothelial cells. Interestingly, using a catalytic dead mutant of Prmt5 and a specific drug inhibitor, we found that while Prmt5 methyltransferase activity was required for blood cell formation, it was dispensable for vessel formation. Analyses of chromatin architecture impact on reporter genes expression and chromatin immunoprecipitation experiments led us to propose that Prmt5 regulates transcription by acting as a scaffold protein that facilitates chromatin looping to promote vascular morphogenesis.

CBP and P300 regulate distinct gene networks required for human primary myoblast differentiation and muscle integrity.

The acetyltransferases CBP and P300 have been implicated in myogenesis in mouse immortalized cell lines but these studies focused only on the expression of a handful of myogenic factors. Hence, the respective role of these two related cofactors and their impact at global scale on gene expression rewiring during primary myoblast differentiation remain unknown. Here, we characterised the gene networks regulated by these two epigenetic enzymes during human primary myoblast differentiation (HPM). We found that CBP and p300 play a critical role in the activation of the myogenic program and mostly regulate distinct gene sets to control several aspects of HPM biology, even though they also exhibit some degree of redundancy. Moreover, CBP or P300 knockdown strongly impaired muscle cell adhesion and resulted in the activation of inflammation markers, two hallmarks of dystrophic disease. This was further validated in zebrafish where inhibition of CBP and P300 enzymatic activities led to cell adhesion defects and muscle fiber detachment. Our data highlight an unforeseen link between CBP/P300 activity and the emergence of dystrophic phenotypes. They thereby identify CBP and P300 as mediators of adult muscle integrity and suggest a new lead for intervention in muscular dystrophy.

Robust Identification of Developmentally Active Endothelial Enhancers in Zebrafish Using FANS-Assisted ATAC-Seq.

Identification of tissue-specific and developmentally active enhancers provides insights into mechanisms that control gene expression during embryogenesis. However, robust detection of these regulatory elements remains challenging, especially in vertebrate genomes. Here, we apply fluorescent-activated nuclei sorting (FANS) followed by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to identify developmentally active endothelial enhancers in the zebrafish genome. ATAC-seq of nuclei from Tg(fli1a:egfp)y1 transgenic embryos revealed expected patterns of nucleosomal positioning at transcriptional start sites throughout the genome and association with active histone modifications. Comparison of ATAC-seq from GFP-positive and -negative nuclei identified more than 5,000 open elements specific to endothelial cells. These elements flanked genes functionally important for vascular development and that displayed endothelial-specific gene expression. Importantly, a majority of tested elements drove endothelial gene expression in zebrafish embryos. Thus, FANS-assisted ATAC-seq using transgenic zebrafish embryos provides a robust approach for genome-wide identification of active tissue-specific enhancer elements.

Vegfa signals through ERK to promote angiogenesis, but not artery differentiation.

Vascular endothelial growth factor a (Vegfa) is essential for blood vessel formation and can induce activation of numerous signaling effectors in endothelial cells. However, it is unclear how and where these function in developmental contexts during vascular morphogenesis. To address this issue, we have visualized activation of presumptive Vegfa effectors at single-cell resolution in zebrafish blood vessels. From these studies, we find that phosphorylation of the serine/threonine kinase ERK (pERK) preferentially occurs in endothelial cells undergoing angiogenesis, but not in committed arterial endothelial cells. pERK in endothelial cells was ectopically induced by Vegfa and lost in Vegfa signaling mutants. Both chemical and endothelial autonomous inhibition of ERK prevented endothelial sprouting, but did not prevent initial artery differentiation. Timed chemical inhibition during angiogenesis caused a loss of genes implicated in coordinating tip/stalk cell behaviors, including flt4 and, at later stages, dll4 ERK inhibition also blocked excessive angiogenesis and ectopic flt4 expression in Notch-deficient blood vessels. Together, these studies implicate ERK as a specific effector of Vegfa signaling in the induction of angiogenic genes during sprouting.

Distinct Notch signaling outputs pattern the developing arterial system.

Differentiation of arteries and veins is essential for the development of a functional circulatory system. In vertebrate embryos, genetic manipulation of Notch signaling has demonstrated the importance of this pathway in driving artery endothelial cell differentiation. However, when and where Notch activation occurs to affect endothelial cell fate is less clear. Using transgenic zebrafish bearing a Notch-responsive reporter, we demonstrate that Notch is activated in endothelial progenitors during vasculogenesis prior to blood vessel morphogenesis and is maintained in arterial endothelial cells throughout larval stages. Furthermore, we find that endothelial progenitors in which Notch is activated are committed to a dorsal aorta fate. Interestingly, some arterial endothelial cells subsequently downregulate Notch signaling and then contribute to veins during vascular remodeling. Lineage analysis, together with perturbation of both Notch receptor and ligand function, further suggests several distinct developmental windows in which Notch signaling acts to promote artery commitment and maintenance. Together, these findings demonstrate that Notch acts in distinct contexts to initiate and maintain artery identity during embryogenesis.

BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch.

A variety of signaling pathways have been shown to regulate specification of neuronal subtype identity. However, the mechanisms by which future neurons simultaneously process information from multiple pathways to establish their identity remain poorly understood. The zebrafish pineal gland offers a simple system with which to address questions concerning the integration of signaling pathways during neural specification as it contains only two types of neurons - photoreceptors and projection neurons. We have previously shown that Notch signaling inhibits the projection neuron fate. Here, we show that BMP signaling is both necessary and sufficient to promote the photoreceptor fate. We also demonstrate that crosstalk between BMP and Notch signaling is required for the inhibition of a projection neuron fate in future photoreceptors. In this case, BMP signaling is required as a competence factor for the efficient activation of Notch targets. Our results indicate that both the induction of a photoreceptor fate and the interaction with Notch relies on a canonical BMP/Smad5 pathway. However, the activation of Notch-dependent transcription does not require a canonical Smad5-DNA interaction. Our results provide new insights into how multiple signaling influences are integrated during cell fate specification in the vertebrate CNS.

Notch resolves mixed neural identities in the zebrafish epiphysis.

Manipulation of Notch activity alters neuronal subtype identity in vertebrate neuronal lineages. Nonetheless, it remains controversial whether Notch activity diversifies cell fate by regulating the timing of neurogenesis or acts directly in neuronal subtype specification. Here, we address the role of Notch in the zebrafish epiphysis, a simple structure containing only two neural subtypes: projection neurons and photoreceptors. Reducing the activity of the Notch pathway results in an excess of projection neurons at the expense of photoreceptors, as well as an increase in cells retaining a mixed identity. However, although forced activation of the pathway inhibits the projection neuron fate, it does not promote photoreceptor identity. As birthdating experiments show that projection neurons and photoreceptors are born simultaneously, Notch acts directly during neuronal specification rather than by controlling the timing of neurogenesis. Finally, our data suggest that two distinct signals are required for photoreceptor fate specification: one for the induction of the photoreceptor fate and the other, involving Notch, for the inhibition of projection neuron traits. We propose a novel model in which Notch resolves mixed neural identities by repressing an undesired genetic program.