Tcf7l1 (formerly Tcf3) proteins are conserved transcription factors whose function as transcriptional repressors is relieved through interactions with β-catenin. Although the functions of Tcf7l1 proteins have been studied in many developmental contexts, whether this conserved mediator of Wnt signaling is required for appropriate cardiomyocyte (CM) development has not been investigated. We find that Tcf7l1 proteins are necessary during two developmental periods to limit CM number in zebrafish embryos: prior to gastrulation and after the initial wave of CM differentiation. In contrast to partially redundant roles in anterior neural patterning, we find that Tcf7l1a and Tcf7l1b have non-redundant functions with respect to restricting CM specification during anterior mesodermal patterning, suggesting that between the two zebrafish Tcf7l1 paralogs there is a limit to the transcriptional repression provided during early CM specification. Using cell transplantation experiments, we determine that the Tcf7l1 paralogs are required cell autonomously to restrict CM specification and promote endothelial cell (EC) specification, which is overtly similar to the ability of Wnt signaling to direct a transformation between these progenitors in embryonic stem cells. Therefore, these results argue that during anterior-posterior patterning of the mesoderm Tcf7l1 proteins are cell autonomously required to limit Wnt signaling, which balances CM and EC progenitor specification within the anterior lateral plate mesoderm. This study expands our understanding of the in vivo developmental requirements of Tcf7l1 proteins and the mechanisms directing CM development in vertebrates.

Immature fruits from Punica granatum L. thinning are a neglected side product of pomegranate production with cumbersome disposal costs for farmers. To explore value potential of immature fruits from pomegranate 'Wonderful' cultivars, the compositional landscapes and antitumorigenic activities of pomegranate extracts from two different stages of maturation were assessed. Cancer cell proliferation and cytotoxicity was quantified in human lung H1299 and colon HCT116 adenocarcinomas by crystal violet staining, MTS assay and caspase-3 activity. High performance liquid chromatography-diode array detector (HPLC/DAD) and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC/ESI-MS) analyses indicate that immature fruits are rich sources of gallotannins and ellagitannins, with the highest amounts contained in immature fruit peels. Biological investigations reveal a robust anticancer activity by those immature P. granatum fruit extracts, which reflected induction of tumor cytotoxicity and cell death mechanisms. Together, present observations suggest P. granatum byproducts from the thinning process may provide unexplored values for virtuous circular economy.

In clementine, failure of fertilization can result in parthenocarpic fruit development, which has several advantages, such as seedless fruit, longer shelf-life, and greater consumer appeal. Recently, S-RNases have been identified in Citrus grandis, thus revealing that the self-incompatibility (SI) reaction relies on the S-RNase gametophytic mechanism. The fundamental role of environmental factors, mostly temperature, in determining the numbers of pollen tubes reaching the ovary is also well established in Citrus. In the present work, temperature-dependent pollen-pistil interactions in C. clementina were analyzed, focusing on several morphological aspects, as well as on polyamine (PA) content and the activity and distribution of transglutaminase (TGase), both reported to be involved in the SI response in pear and in pummelo. Results clearly indicate that temperature contributed to a different activation of the SI response, which occurs at optimal temperature of 25°C but was by-passed at 15°C. TGase activity was stimulated during the SI response, and it localized differently in the compatible and incompatible interaction: in compatible pollinated styles, TGase localized inside the style canal, while it was detected all around it in incompatible crosses. TGase localization and activity were congruent with the levels of soluble and insoluble conjugated PAs and with morphological evidences, which highlighted cell wall modification occurring as a result of SI.

How two-chambered hearts in basal vertebrates have evolved from single-chamber hearts found in ancestral chordates remains unclear. Here, we show that the teleost sinus venosus (SV) is a chamber-like vessel comprised of an outer layer of smooth muscle cells. We find that in adult zebrafish nr2f1a mutants, which lack atria, the SV comes to physically resemble the thicker bulbus arteriosus (BA) at the arterial pole of the heart through an adaptive, hypertensive response involving smooth muscle proliferation due to aberrant hemodynamic flow. Single cell transcriptomics show that smooth muscle and endothelial cell populations within the adapting SV also take on arterial signatures. Bulk transcriptomics of the blood sinuses flanking the tunicate heart reinforce a model of greater equivalency in ancestral chordate BA and SV precursors. Our data simultaneously reveal that secondary complications from congenital heart defects can develop in adult zebrafish similar to those in humans and that the foundation of equivalency between flanking auxiliary vessels may remain latent within basal vertebrate hearts.

The actin-depolymerizing factor (ADF) gene family regulates changes in actin. However, the entire ADF family in the sweet orange Citrus sinensis has not been systematically identified, and their expressions in different organs and biotic stress have not been determined. In this study, through phylogenetic analysis of the sweet orange ADF gene family, seven CsADFs were found to be highly conserved and sparsely distributed across the four chromosomes. Analysis of the cis-regulatory elements in the promoter region showed that the CsADF gene had the potential to impact the development of sweet oranges under biotic or abiotic stress. Quantitative fluorescence analysis was then performed. Seven CsADFs were differentially expressed against the invasion of Xcc and CLas pathogens. It is worth noting that the expression of CsADF4 was significantly up-regulated at 4 days post-infection. Subcellular localization results showed that CsADF4 was localized in both the nucleus and the cytoplasm. Overexpression of CsADF4 enhanced the sensitivity of sweet orange leaves to Xcc. These results suggest that CsADFs may regulate the interaction of C. sinensis and bacterial pathogens, providing a way to further explore the function and mechanisms of ADF in the sweet orange.

Anthocyanins are the most important compounds for nutritional quality and economic values of blood orange. However, there are few reports on the pre-harvest treatment accelerating the accumulation of anthocyanins in postharvest blood orange fruit. Here, we performed a comparative transcriptome and metabolomics analysis to elucidate the underlying mechanism involved in seasonal drought (SD) treatment during the fruit expansion stage on anthocyanin accumulation in postharvest 'Tarocco' blood orange fruit.

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.

The nuclear Peroxisome Proliferator Activated Receptors (PPARs) are ligand-activated transcription factors playing a fundamental role in energy homeostasis and metabolism. Consequently, functional impairment or dysregulation of these receptors lead to a variety of metabolic diseases. While some phytocannabinoids (pCBs) are known to activate PPARγ, no data have been reported so far on their possible activity at PPARα.

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.

Canonical Wnt/β-catenin (Wnt) signaling plays multiple conserved roles during fate specification of cardiac progenitors in developing vertebrate embryos. Although lineage analysis in ascidians and mice has indicated there is a close relationship between the cardiac second heart field (SHF) and pharyngeal muscle (PM) progenitors, the signals underlying directional fate decisions of the cells within the cardio-pharyngeal muscle field in vertebrates are not yet understood. Here, we examined the temporal requirements of Wnt signaling in cardiac and PM development. In contrast to a previous report in chicken embryos that suggested Wnt inhibits PM development during somitogenesis, we find that in zebrafish embryos Wnt signaling is sufficient to repress PM development during anterior-posterior patterning. Importantly, the temporal sensitivity of dorso-anterior PMs to increased Wnt signaling largely overlaps with when Wnt signaling promotes specification of the adjacent cardiac progenitors. Furthermore, we find that excess early Wnt signaling can cell autonomously promote expansion of the first heart field (FHF) progenitors at the expense of PM and SHF within the anterior lateral plate mesoderm (ALPM). Our study provides insight into an antagonistic developmental mechanism that balances the sizes of the adjacent cardiac and PM progenitor fields in early vertebrate embryos.

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.

Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.

Retinoic acid (RA) signaling is required to restrict heart size through limiting the posterior boundary of the vertebrate cardiac progenitor field within the anterior lateral plate mesoderm (ALPM). However, we still do not fully understand how different cardiac progenitor populations that contribute to the developing heart, including earlier-differentiating first heart field (FHF), later-differentiating second heart field (SHF), and neural crest-derived progenitors, are each affected in RA-deficient embryos. Here, we quantified the number of cardiac progenitors and differentiating cardiomyocytes (CMs) in RA-deficient zebrafish embryos. While Nkx2.5+ cells were increased overall in the nascent hearts of RA-deficient embryos, unexpectedly, we found that the major effect within this population was a significant expansion in the number of differentiating FHF CMs. In contrast to the expansion of the FHF, there was a progressive decrease in SHF progenitors at the arterial pole as the heart tube elongated. Temporal differentiation assays and immunostaining in RA-deficient embryos showed that the outflow tracts (OFTs) of the hearts were significantly smaller, containing fewer differentiated SHF-derived ventricular CMs and a complete absence of SHF-derived smooth muscle at later stages. At the venous pole of the heart, pacemaker cells of the sinoatrial node also failed to differentiate in RA-deficient embryos. Interestingly, genetic lineage tracing showed that the number of neural-crest derived CMs was not altered within the enlarged hearts of RA-deficient zebrafish embryos. Altogether, our data show that the enlarged hearts in RA-deficient zebrafish embryos are comprised of an expansion in earlier differentiating FHF-derived CMs coupled with a progressive depletion of the SHF, suggesting RA signaling determines the relative ratios of earlier- and later-differentiation cardiac progenitors within an expanded cardiac progenitor pool.

The nuclear receptors (NRs) RARα, RXRα, PPARα, and PPARγ represent promising pharmacological targets for the treatment of neurodegenerative diseases. In the search for molecules able to simultaneously target all the above-mentioned NRs, we screened an in-house developed molecular database using a ligand-based approach, identifying (-)-Muqubilin (Muq), a cyclic peroxide norterpene from a marine sponge, as a potential hit. The ability of this compound to stably and effectively bind these NRs was assessed by molecular docking and molecular dynamics simulations. Muq recapitulated all the main interactions of a canonical full agonist for RXRα and both PPARα and PPARγ, whereas the binding mode toward RARα showed peculiar features potentially impairing its activity as full agonist. Luciferase assays confirmed that Muq acts as a full agonist for RXRα, PPARα, and PPARγ with an activity in the low- to sub-micromolar range. On the other hand, in the case of RAR, a very weak agonist activity was observed in the micromolar range. Quite surprisingly, we found that Muq is a positive allosteric modulator for RARα, as both luciferase assays and in vivo analysis using a zebrafish transgenic retinoic acid (RA) reporter line showed that co-administration of Muq with RA produced a potent synergistic enhancement of RARα activation and RA signaling.

Appropriate levels of retinoic acid (RA) signaling are critical for normal heart development in vertebrates. A fascinating property of RA signaling is the thoroughness by which positive and negative feedback are employed to promote proper embryonic RA levels. In the present short review, we first cover the advancement of hypotheses regarding the impact of RA signaling on cardiac specification. We then discuss our current understanding of RA signaling feedback mechanisms and the implications of recent studies, which have indicated improperly maintained RA signaling feedback can be a contributing factor to developmental malformations.

Cardiomelic or heart-hand syndromes include congenital defects affecting both the forelimb and heart, suggesting a hypothesis where similar signals may coordinate their development. In support of this hypothesis, we have recently defined a mechanism by which retinoic acid (RA) signaling acts on the forelimb progenitors to indirectly restrict cardiac cell number. However, we still do not have a complete understanding of the mechanisms downstream of RA signaling that allow for the coordinated development of these structures. Here, we test the hypothesis that appropriate Fgf signaling in the cardiac progenitor field downstream of RA signaling is required for the coordinated development of the heart and forelimb. Consistent with this hypothesis, we find that increasing Fgf signaling can autonomously increase cardiac cell number and non-autonomously inhibit forelimb formation over the same time period that embryos are sensitive to loss of RA signaling. Furthermore, we find that Fgf8a, which is expressed in the cardiac progenitors, is expanded into the posterior in RA signaling-deficient zebrafish embryos. Reducing Fgf8a function in RA signaling-deficient embryos is able to rescue both heart and forelimb development. Together, these results are the first to directly support the hypothesis that RA signaling is required shortly after gastrulation in the forelimb field to temper Fgf8a signaling in the cardiac field, thus coordinating the development of the heart and forelimb.

The pear (genus Pyrus) is one of the most ancient and widely cultivated tree fruit crops in temperate climates. The Mount Etna area claims a large number of pear varieties differentiated due to a long history of cultivation and environmental variability, making this area particularly suitable for genetic studies. Ninety-five pear individuals were genotyped using the simple sequence repeat (SSR) methodology interrogating both the nuclear (nDNA) and chloroplast DNA (cpDNA) to combine an investigation of maternal inheritance of chloroplast SSRs (cpSSRs) with the high informativity of nuclear SSRs (nSSRs). The germplasm was selected ad hoc to include wild genotypes, local varieties, and national and international cultivated varieties. The objectives of this study were as follows: (i) estimate the level of differentiation within local varieties; (ii) elucidate the phylogenetic relationships between the cultivated genotypes and wild accessions; and (iii) estimate the potential genetic flow and the relationship among the germplasms in our analysis. Eight nSSRs detected a total of 136 alleles with an average minor allelic frequency and observed heterozygosity of 0.29 and 0.65, respectively, whereas cpSSRs allowed identification of eight haplotypes (S4 Table). These results shed light on the genetic relatedness between Italian varieties and wild genotypes. Among the wild species, compared with P. amygdaliformis, few P. pyraster genotypes exhibited higher genetic similarity to local pear varieties. Our analysis revealed the presence of genetic stratification with a 'wild' subpopulation characterizing the genetic makeup of wild species and the international cultivated varieties exhibiting the predominance of the 'cultivated' subpopulation.

Determination of appropriate chamber size is critical for normal vertebrate heart development. Although Nr2f transcription factors promote atrial maintenance and differentiation, how they determine atrial size remains unclear. Here, we demonstrate that zebrafish Nr2f1a is expressed in differentiating atrial cardiomyocytes. Zebrafish nr2f1a mutants have smaller atria due to a specific reduction in atrial cardiomyocyte (AC) number, suggesting it has similar requirements to Nr2f2 in mammals. Furthermore, the smaller atria in nr2f1a mutants are derived from distinct mechanisms that perturb AC differentiation at the chamber poles. At the venous pole, Nr2f1a enhances the rate of AC differentiation. Nr2f1a also establishes the atrial-atrioventricular canal (AVC) border through promoting the differentiation of mature ACs. Without Nr2f1a, AVC markers are expanded into the atrium, resulting in enlarged endocardial cushions (ECs). Inhibition of Bmp signaling can restore EC development, but not AC number, suggesting that Nr2f1a concomitantly coordinates atrial and AVC size through both Bmp-dependent and independent mechanisms. These findings provide insight into conserved functions of Nr2f proteins and the etiology of atrioventricular septal defects (AVSDs) associated with NR2F2 mutations in humans.

Coordinated transcriptional and epigenetic mechanisms that direct development of the later differentiating second heart field (SHF) progenitors remain largely unknown. Here, we show that a novel zebrafish histone deacetylase 1 (hdac1) mutant allele cardiac really gone (crg) has a deficit of ventricular cardiomyocytes (VCs) and smooth muscle within the outflow tract (OFT) due to both cell and non-cell autonomous loss in SHF progenitor proliferation. Cyp26-deficient embryos, which have increased retinoic acid (RA) levels, have similar defects in SHF-derived OFT development. We found that nkx2.5+ progenitors from Hdac1 and Cyp26-deficient embryos have ectopic expression of ripply3, a transcriptional co-repressor of T-box transcription factors that is normally restricted to the posterior pharyngeal endoderm. Furthermore, the ripply3 expression domain is expanded anteriorly into the posterior nkx2.5+ progenitor domain in crg mutants. Importantly, excess ripply3 is sufficient to repress VC development, while genetic depletion of Ripply3 and Tbx1 in crg mutants can partially restore VC number. We find that the epigenetic signature at RA response elements (RAREs) that can associate with Hdac1 and RA receptors (RARs) becomes indicative of transcriptional activation in crg mutants. Our study highlights that transcriptional repression via the epigenetic regulator Hdac1 facilitates OFT development through directly preventing expression of the RA-responsive gene ripply3 within SHF progenitors.

Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate toward the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid-induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system.