Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.

All- trans-retinoic acid (RA), a vitamin A metabolite, is an important signaling molecule required for the proper development of the heart. The epicardium is the main source of RA in the embryonic heart, yet the cardiogenic functions of epicardial-produced RA are not fully understood. Here, we investigated the roles of RA signaling in the embryonic epicardium using in vivo and in vitro models of excess or deficiency of RA. Our results suggested that RA signaling facilitates the cytoskeletal rearrangement required for the epicardial-to-mesenchymal transition of epicardial cells. In vivo treatment with an inhibitor of RA synthesis delayed the migration of epicardial-derived precursor cells (EPDCs) into the myocardium; the opposite was seen in the case of dehydrogenase/reductase superfamily (DHRS)3-deficient embryos, a mouse model of RA excess. Analysis of the behavior of epicardial cells exposed to RA receptor agonists or inhibitors of RA synthesis in vitro revealed that appropriate levels of RA are important in orchestrating the platelet-derived growth factor-induced loss of epithelial character, cytoskeletal remodeling, and migration, necessary for the infiltration of the myocardium by EPDCs. To understand the molecular mechanisms by which RA regulates epicardial cytoskeletal rearrangement, we used a whole transcriptome profiling approach, which in combination with pull-down and inhibition assays, demonstrated that the Ras homolog gene family, member A (RhoA) pathway is required for the morphologic changes induced by RA in epicardial cells. Collectively, these data demonstrate that RA regulates the cytoskeletal rearrangement of epicardial cells via a signaling cascade that involves the RhoA pathway.-Wang, S., Yu, J., Jones, J. W., Pierzchalski, K., Kane, M. A., Trainor, P. A., Xavier-Neto, J., Moise, A. R. Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.

Alterations in retinoic acid signaling affect the development of the mouse coronary vasculature.

BACKGROUND: During the final stages of heart development the myocardium grows and becomes vascularized by means of paracrine factors and cell progenitors derived from the epicardium. There is evidence to suggest that retinoic acid (RA), a metabolite of vitamin A, plays an important role in epicardial-based developmental programming. However, the consequences of altered RA-signaling in coronary development have not been systematically investigated. RESULTS: We explored the developmental consequences of altered RA-signaling in late cardiogenic events that involve the epicardium. For this, we used a model of embryonic RA excess based on mouse embryos deficient in the retinaldehyde reductase DHRS3, and a complementary model of embryonic RA deficiency based on pharmacological inhibition of RA synthesis. We found that alterations in embryonic RA signaling led to a thin myocardium and aberrant coronary vessel formation and remodeling. Both excess, and deficient RA-signaling are associated with reductions in ventricular coverage and density of coronary vessels, altered vessel morphology, and impaired recruitment of epicardial-derived mural cells. Using a combined transcriptome and proteome profiling approach, we found that RA treatment of epicardial cells influenced key signaling pathways relevant for cardiac development. CONCLUSIONS: Epicardial RA-signaling plays critical roles in the development of the coronary vasculature needed to support myocardial growth. Developmental Dynamics 247:976-991, 2018. © 2018 Wiley Periodicals, Inc.

Signaling through retinoic acid receptors in cardiac development: Doing the right things at the right times.

Retinoic acid (RA) is a terpenoid that is synthesized from vitamin A/retinol (ROL) and binds to the nuclear receptors retinoic acid receptor (RAR)/retinoid X receptor (RXR) to control multiple developmental processes in vertebrates. The available clinical and experimental data provide uncontested evidence for the pleiotropic roles of RA signaling in development of multiple embryonic structures and organs such eyes, central nervous system, gonads, lungs and heart. The development of any of these above-mentioned embryonic organ systems can be effectively utilized to showcase the many strategies utilized by RA signaling. However, it is very likely that the strategies employed to transfer RA signals during cardiac development comprise the majority of the relevant and sophisticated ways through which retinoid signals can be conveyed in a complex biological system. Here, we provide the reader with arguments indicating that RA signaling is exquisitely regulated according to specific phases of cardiac development and that RA signaling itself is one of the major regulators of the timing of cardiac morphogenesis and differentiation. We will focus on the role of signaling by RA receptors (RARs) in early phases of heart development. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Repulsive Guidance Molecules a, b and c Are Skeletal Muscle Proteins, and Repulsive Guidance Molecule a Promotes Cellular Hypertrophy and Is Necessary for Myotube Fusion.

Repulsive guidance molecules (RGMs) compose a family of glycosylphosphatidylinositol (GPI)-anchored axon guidance molecules and perform several functions during neural development. New evidence has suggested possible new roles for these axon guidance molecules during skeletal muscle development, which has not been investigated thus far. In the present study, we show that RGMa, RGMb and RGMc are all induced during skeletal muscle differentiation in vitro. Immunolocalization performed on adult skeletal muscle cells revealed that RGMa, RGMb and RGMc are sarcolemmal proteins. Additionally, RGMa was found to be a sarcoplasmic protein with a surprisingly striated pattern. RGMa colocalization with known sarcoplasmic proteins suggested that this axon guidance molecule is a skeletal muscle sarcoplasmic protein. Western blot analysis revealed two RGMa fragments of 60 and 33 kDa, respectively, in adult skeletal muscle samples. RGMa phenotypes in skeletal muscle cells (C2C12 and primary myoblasts) were also investigated. RGMa overexpression produced hypertrophic cells, whereas RGMa knockdown resulted in the opposite phenotype. RGMa knockdown also blocked myotube formation in both skeletal muscle cell types. Our results are the first to show an axon guidance molecule as a skeletal muscle sarcoplasmic protein and to include RGMa in a system that regulates skeletal muscle cell size and differentiation.

Dact gene expression profiles suggest a role for this gene family in integrating Wnt and TGF-ß signaling pathways during chicken limb development.

BACKGROUND: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF-ß signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. RESULTS: During early limb development (HH24-HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31-HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co-localize with regions enriched in the nuclear ß-catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR-1b expression, a TGF-ß receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. CONCLUSIONS: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF-ß signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases.

KVFinder: steered identification of protein cavities as a PyMOL plugin.

BACKGROUND: The characterization of protein binding sites is a major challenge in computational biology. Proteins interact with a wide variety of molecules and understanding of such complex interactions is essential to gain deeper knowledge of protein function. Shape complementarity is known to be important in determining protein-ligand interactions. Furthermore, these protein structural features have been shown to be useful in assisting medicinal chemists during lead discovery and optimization. RESULTS: We developed KVFinder, a highly versatile and easy-to-use tool for cavity prospection and spatial characterization. KVFinder is a geometry-based method that has an innovative customization of the search space. This feature provides the possibility of cavity segmentation, which alongside with the large set of customizable parameters, allows detailed cavity analyses. Although the main focus of KVFinder is the steered prospection of cavities, we tested it against a benchmark dataset of 198 known drug targets in order to validate our software and compare it with some of the largely accepted methods. Using the one click mode, we performed better than most of the other methods, staying behind only of hybrid prospection methods. When using just one of KVFinder's customizable features, we were able to outperform all other compared methods. KVFinder is also user friendly, as it is available as a PyMOL plugin, or command-line version. CONCLUSION: KVFinder presents novel usability features, granting full customizable and highly detailed cavity prospection on proteins, alongside with a friendly graphical interface. KVFinder is freely available on http://lnbio.cnpem.br/bioinformatics/main/software/.

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans.

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

Unraveling the evolutionary origin of the complex Nuclear Receptor Element (cNRE), a cis-regulatory module required for preferential expression in the atrial chamber.

Cardiac function requires appropriate proteins in each chamber. Atria requires slow myosin to act as reservoirs, while ventricles demand fast myosin for swift pumping. Myosins are thus under chamber-biased cis-regulation, with myosin gene expression imbalances leading to congenital heart dysfunction. To identify regulatory inputs leading to cardiac chamber-biased expression, we computationally and molecularly dissected the quail Slow Myosin Heavy Chain III (SMyHC III) promoter that drives preferential expression to the atria. We show that SMyHC III gene states are orchestrated by a complex Nuclear Receptor Element (cNRE) of 32 base pairs. Using transgenesis in zebrafish and mice, we demonstrate that preferential atrial expression is achieved by a combinatorial regulatory input composed of atrial activation motifs and ventricular repression motifs. Using comparative genomics, we show that the cNRE might have emerged from an endogenous viral element through infection of an ancestral host germline, revealing an evolutionary pathway to cardiac chamber-specific expression.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy.

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.