miRNAs in Heart Development and Disease.

Cardiovascular diseases (CVD) are a group of disorders that affect the heart and blood vessels. They include conditions such as myocardial infarction, coronary artery disease, heart failure, arrhythmia, and congenital heart defects. CVDs are the leading cause of death worldwide. Therefore, new medical interventions that aim to prevent, treat, or manage CVDs are of prime importance. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the posttranscriptional level and play important roles in various biological processes, including cardiac development, function, and disease. Moreover, miRNAs can also act as biomarkers and therapeutic targets. In order to identify and characterize miRNAs and their target genes, scientists take advantage of computational tools such as bioinformatic algorithms, which can also assist in analyzing miRNA expression profiles, functions, and interactions in different cardiac conditions. Indeed, the combination of miRNA research and bioinformatic algorithms has opened new avenues for understanding and treating CVDs. In this review, we summarize the current knowledge on the roles of miRNAs in cardiac development and CVDs, discuss the challenges and opportunities, and provide some examples of recent bioinformatics for miRNA research in cardiovascular biology and medicine.

Myocardial RNA Sequencing Reveals New Potential Therapeutic Targets in Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) represents a global health challenge, with limited therapies proven to enhance patient outcomes. This makes the elucidation of disease mechanisms and the identification of novel potential therapeutic targets a priority. Here, we performed RNA sequencing on ventricular myocardial biopsies from patients with HFpEF, prospecting to discover distinctive transcriptomic signatures. A total of 306 differentially expressed mRNAs (DEG) and 152 differentially expressed microRNAs (DEM) were identified and enriched in several biological processes involved in HF. Moreover, by integrating mRNA and microRNA expression data, we identified five potentially novel miRNA-mRNA relationships in HFpEF: the upregulated hsa-miR-25-3p, hsa-miR-26a-5p, and has-miR4429, targeting HAPLN1; and NPPB mRNA, targeted by hsa-miR-26a-5p and miR-140-3p. Exploring the predicted miRNA-mRNA interactions experimentally, we demonstrated that overexpression of the distinct miRNAs leads to the downregulation of their target genes. Interestingly, we also observed that microRNA signatures display a higher discriminative power to distinguish HFpEF sub-groups over mRNA signatures. Our results offer new mechanistic clues, which can potentially translate into new HFpEF therapies.

CCBE1 Is Essential for Epicardial Function during Myocardium Development.

The epicardium is a single cell layer of mesothelial cells that plays a critical role during heart development contributing to different cardiac cell types of the developing heart through epithelial-to-mesenchymal transition (EMT). Moreover, the epicardium is a source of secreted growth factors that promote myocardial growth. CCBE1 is a secreted extracellular matrix protein expressed by epicardial cells that is required for the formation of the primitive coronary plexus. However, the role of CCBE1 during epicardial development was still unknown. Here, using a Ccbe1 knockout (KO) mouse model, we observed that loss of CCBE1 leads to congenital heart defects including thinner and hyper-trabeculated ventricular myocardium. In addition, Ccbe1 mutant hearts displayed reduced proliferation of cardiomyocyte and epicardial cells. Epicardial outgrowth culture assay to assess epicardial-derived cells (EPDC) migration showed reduced invasion of the collagen gel by EPDCs in Ccbe1 KO epicardial explants. Ccbe1 KO hearts also displayed fewer nonmyocyte/nonendothelial cells intramyocardially with a reduced proliferation rate. Additionally, RNA-seq data and experimental validation by qRT-PCR showed a marked deregulation of EMT-related genes in developing Ccbe1 mutant hearts. Together, these findings indicate that the myocardium defects in Ccbe1 KO mice arise from disruption of epicardial development and function.

DAND5 Inactivation Enhances Cardiac Differentiation in Mouse Embryonic Stem Cells.

Deciphering the clues of a regenerative mechanism for the mammalian adult heart would save millions of lives in the near future. Heart failure due to cardiomyocyte loss is still one of the significant health burdens worldwide. Here, we show the potential of a single molecule, DAND5, in mouse pluripotent stem cell-derived cardiomyocytes specification and proliferation. Dand5 loss-of-function generated the double of cardiac beating foci compared to the wild-type cells. The early formation of cardiac progenitor cells and the increased proliferative capacity of Dand5 KO mESC-derived cardiomyocytes contribute to the observed higher number of derived cardiac cells. Transcriptional profiling sequencing and quantitative RT-PCR assays showed an upregulation of early cardiac gene networks governing cardiomyocyte differentiation, cell cycling, and cardiac regenerative pathways but reduced levels of genes involved in cardiomyocyte maturation. These findings prompt DAND5 as a key driver for the generation and expansion of pluripotent stem cell-derived cardiomyocytes systems with further clinical application purposes.

Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells.

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.

Novel triblock co-polymer nanofibre system as an alternative support for embryonic stem cells growth and pluripotency.

Conventionally, embryonic stem cells (ESCs) are cultured on gelatin or over a mitotically inactivated monolayer of mouse embryonic fibroblasts (MEFsi). Considering the lack of versatile, non-animal-derived and inexpensive materials for that purpose, we aimed to find a biomaterial able to support ESC growth in a pluripotent state that avoids the need for laborious and time-consuming MEFsi culture in parallel with mouse ESC (mESC) culture. Undifferentiated mESCs were cultured in a new nanofibre material designed for ESC culture, which is based on the self-assembly of a triblock co-polymer, poly(ethyleneglycol-ß-trimethylsilyl methacrylate-ß-methacrylic acid), conjugated with the peptide glycine-arginine-glycine-aspartate-serine, to evaluate its potential application in ESC research. The morphology, proliferation, viability, pluripotency and differentiation potential of mESCs were assessed. Compared to conventional stem cell culture methodologies, the nanofibres promoted a higher increase in mESCs number, enhanced pluripotency and were able to support differentiation after long-term culture. This newly developed synthetic system allows the elimination of animal-derived matrices and provides an economic method of ESC culture, made of a complex network of nanofibres in a scale similar to native extracellular matrices, where the functional properties of the cells can be observed and manipulated. Copyright © 2013 John Wiley & Sons, Ltd.

Xenopus Pkdcc1 and Pkdcc2 Are Two New Tyrosine Kinases Involved in the Regulation of JNK Dependent Wnt/PCP Signaling Pathway.

Protein Kinase Domain Containing, Cytoplasmic (PKDCC) is a protein kinase which has been implicated in longitudinal bone growth through regulation of chondrocytes formation. Nevertheless, the mechanism by which this occurs remains unknown. Here, we identified two new members of the PKDCC family, Pkdcc1 and Pkdcc2 from Xenopus laevis. Interestingly, our knockdown experiments revealed that these two proteins are both involved on blastopore and neural tube closure during gastrula and neurula stages, respectively. In vertebrates, tissue polarity and cell movement observed during gastrulation and neural tube closure are controlled by Wnt/Planar Cell Polarity (PCP) molecular pathway. Our results showed that Pkdcc1 and Pkdcc2 promote the recruitment of Dvl to the plasma membrane. But surprisingly, they revealed different roles in the induction of a luciferase reporter under the control of Atf2 promoter. While Pkdcc1 induces Atf2 expression, Pkdcc2 does not, and furthermore inhibits its normal induction by Wnt11 and Wnt5a. Altogether our data show, for the first time, that members of the PKDCC family are involved in the regulation of JNK dependent Wnt/PCP signaling pathway.

Expression and function of Ccbe1 in the chick early cardiogenic regions are required for correct heart development.

During the course of a differential screen to identify transcripts specific for chick heart/hemangioblast precursor cells, we have identified Ccbe1 (Collagen and calcium-binding EGF-like domain 1). While the importance of Ccbe1 for the development of the lymphatic system is now well demonstrated, its role in cardiac formation remained unknown. Here we show by whole-mount in situ hybridization analysis that cCcbe1 mRNA is initially detected in early cardiac progenitors of the two bilateral cardiogenic fields (HH4), and at later stages on the second heart field (HH9-18). Furthermore, cCcbe1 is expressed in multipotent and highly proliferative cardiac progenitors. We characterized the role of cCcbe1 during early cardiogenesis by performing functional studies. Upon morpholino-induced cCcbe1 knockdown, the chick embryos displayed heart malformations, which include aberrant fusion of the heart fields, leading to incomplete terminal differentiation of the cardiomyocytes. cCcbe1 overexpression also resulted in severe heart defects, including cardia bifida. Altogether, our data demonstrate that although cardiac progenitors cells are specified in cCcbe1 morphants, the migration and proliferation of cardiac precursors cells are impaired, suggesting that cCcbe1 is a key gene during early heart development.

Targeted inactivation of Cerberus like-2 leads to left ventricular cardiac hyperplasia and systolic dysfunction in the mouse.

Previous analysis of the Cerberus like 2 knockout (Cerl2-/-) mouse revealed a significant mortality during the first day after birth, mostly due to cardiac defects apparently associated with randomization of the left-right axis. We have however, identified Cerl2-associated cardiac defects, particularly a large increase in the left ventricular myocardial wall in neonates that cannot be explained by laterality abnormalities. Therefore, in order to access the endogenous role of Cerl2 in cardiogenesis, we analyzed the embryonic and neonatal hearts of Cerl2 null mutants that did not display a laterality phenotype. Neonatal mutants obtained from the compound mouse line Cer2-/-::Mlc1v-nLacZ24+, in which the pulmonary ventricle is genetically marked, revealed a massive enlargement of the ventricular myocardium in animals without laterality defects. Echocardiography analysis in Cerl2-/- neonates showed a left ventricular systolic dysfunction that is incompatible with a long lifespan. We uncovered that the increased ventricular muscle observed in Cerl2-/- mice is caused by a high cardiomyocyte mitotic index in the compact myocardium which is mainly associated with increased Ccnd1 expression levels in the left ventricle at embryonic day (E) 13. Interestingly, at this stage we found augmented left ventricular expression of Cerl2 levels when compared with the right ventricle, which may elucidate the regionalized contribution of Cerl2 to the left ventricular muscle formation. Importantly, we observed an increase of phosphorylated Smad2 (pSmad2) levels in embryonic (E13) and neonatal hearts indicating a prolonged TGFßs/Nodal-signaling activation. Concomitantly, we detected an increase of Baf60c levels, but only in Cerl2-/- embryonic hearts. These results indicate that independently of its well-known role in left-right axis establishment Cerl2 plays an important role during heart development in the mouse, mediating Baf60c levels by exerting an important control of the TGFßs/Nodal-signaling pathway.

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture.

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro differentiation assay revealed that mouse ESCs cultured in LBG preserve their tri-lineage differentiation capacity. In conclusion, our data indicate that LBG coating promotes mouse ESC growth in an undifferentiated state demonstrating to be a viable, non-animal derived alternative to gelatin to support pluripotent mouse ESCs in culture.

Transthoracic echocardiography reference values in juvenile and adult 129/Sv mice.

BACKGROUND: In the recent years, the use of Doppler-echocardiography has become a standard non-invasive technique in the analysis of cardiac malformations in genetically modified mice. Therefore, normal values have to be established for the most commonly used inbred strains in whose genetic background those mutations are generated. Here we provide reference values for transthoracic echocardiography measurements in juvenile (3 weeks) and adult (8 weeks) 129/Sv mice. METHODS: Echocardiographic measurements were performed using B-mode, M-mode and Doppler-mode in 15 juvenile (3 weeks) and 15 adult (8 weeks) mice, during isoflurane anesthesia. M-mode measurements variability of left ventricle (LV) was determined. RESULTS: Several echocardiographic measurements significantly differ between juvenile and adult mice. Most of these measurements are related with cardiac dimensions. All B-mode measurements were different between juveniles and adults (higher in the adults), except for fractional area change (FAC). Ejection fraction (EF) and fractional shortening (FS), calculated from M-mode parameters, do not differ between juvenile and adult mice. Stroke volume (SV) and cardiac output (CO) were significantly different between juvenile and adult mice. SV was 31.93 ± 8.67 µl in juveniles vs 70.61 ± 24.66 µl in adults, ρ < 0.001. CO was 12.06 ± 4.05 ml/min in juveniles vs 29.71 ± 10.13 ml/min in adults, ρ < 0.001. No difference was found in mitral valve (MV) and tricuspid valve (TV) related parameters between juvenile and adult mice. It was demonstrated that variability of M-mode measurements of LV is minimal. CONCLUSIONS: This study suggests that differences in cardiac dimensions, as wells as in pulmonary and aorta outflow parameters, were found between juvenile and adult mice. However, mitral and tricuspid inflow parameters seem to be similar between 3 weeks and 8 weeks mice. The reference values established in this study would contribute as a basis to future studies in post-natal cardiovascular development and diagnosing cardiovascular disorders in genetically modified mouse mutant lines.

The dynamic right-to-left translocation of Cerl2 is involved in the regulation and termination of Nodal activity in the mouse node.

The determination of left-right body asymmetry in mouse embryos depends on the interplay of molecules in a highly sensitive structure, the node. Here, we show that the localization of Cerl2 protein does not correlate to its mRNA expression pattern, from 3-somite stage onwards. Instead, Cerl2 protein displays a nodal flow-dependent dynamic behavior that controls the activity of Nodal in the node, and the transmission of the laterality information to the left lateral plate mesoderm (LPM). Our results indicate that Cerl2 initially localizes and prevents the activation of Nodal genetic circuitry on the right side of the embryo, and later its right-to-left translocation shutdowns Nodal activity in the node. The consequent prolonged Nodal activity in the node by the absence of Cerl2 affects local Nodal expression and prolongs its expression in the LPM. Simultaneous genetic removal of both Nodal node inhibitors, Cerl2 and Lefty1, sustains even longer and bilateral this LPM expression.

Fluid flow and interlinked feedback loops establish left-right asymmetric decay of Cerl2 mRNA.

Breaking of left-right symmetry in mouse embryos requires fluid flow at the node, but the precise action of the flow has remained unknown. Here we show that the left-right asymmetry of Cerl2 expression around the node, a target of the flow, is determined post-transcriptionally by decay of Cerl2 mRNA in a manner dependent on its 3' untranslated region. Cerl2 mRNA is absent specifically from the apical region of crown cells on the left side of the node. Preferential decay of Cerl2 mRNA on the left is initiated by the leftward flow and further enhanced by the operation of Wnt-Cerl2 interlinked feedback loops, in which Wnt3 upregulates Wnt3 expression and promotes Cerl2 mRNA decay, whereas Cerl2 promotes Wnt degradation. Mathematical modelling and experimental data suggest that these feedback loops behave as a bistable switch that can amplify in a noise-resistant manner a small bias conferred by fluid flow.

Cilia at the node of mouse embryos sense fluid flow for left-right determination via Pkd2.

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

Identification and functional analysis of novel genes expressed in the Anterior Visceral Endoderm.

During early vertebrate development, the correct establishment of the body axes is critical. The anterior pole of the mouse embryo is established when Distal Visceral Endoderm (DVE) cells migrate to form the Anterior Visceral Endoderm (AVE). Symmetrical expression of Lefty1, Cer1 and Dkk1 determines the direction of DVE migration and the future anterior side. In addition to the establishment of the Anterior-Posterior axis, the AVE has also been implicated in anterior neural specification. To better understand the role of the AVE in these processes, we have performed a differential screening using Affymetrix GeneChip technology with AVE cells isolated from cer1P-EGFP transgenic mouse embryos. We found 175 genes which were upregulated in the AVE and 36 genes in the Proximal-posterior sample. Using DAVID software, we characterized the AVE cell population regarding cellular component, molecular function and biological processes. Among the genes that were found to be upregulated in the AVE, several novel genes were identified. Four of these transcripts displaying high-fold change in the AVE were further characterized by in situ hybridization in early stages of development in order to validate the screening. From those four selected genes, one, denominated Adtk1, was chosen to be functionally characterized by targeted inactivation in ES cells. Adtk1 encodes for a serine/threonine kinase. Adtk1 null mutants are smaller and present short limbs due to decreased mineralization, suggesting a potential role in chondrogenesis during limb development. Taken together, these data point to the importance of reporting novel genes present in the AVE.

Characterization of Cer-1 cis-regulatory region during early Xenopus development.

Cerberus-related molecules are well-known Wnt, Nodal, and BMP inhibitors that have been implicated in different processes including anterior­posterior patterning and left­right asymmetry. In both mouse and frog, two Cerberus-related genes have been isolated, mCer-1 and mCer-2, and Xcer and Xcoco, respectively. Until now, little is known about the mechanisms involved in their transcriptional regulation. Here, we report a heterologous analysis of the mouse Cerberus-1 gene upstream regulatory regions, responsible for its expression in the visceral endodermal cells. Our analysis showed that the consensus sequences for a TATA, CAAT, or GC boxes were absent but a TGTGG sequence was present at position -172 to -168 bp, relative to the ATG. Using a series of deletion constructs and transient expression in Xenopus embryos, we found that a fragment of 1.4 kb of Cer-1 promoter sequence could reproduce the endogenous expression pattern of Xenopus cerberus. A 0.7-kb mcer-1 upstream region was able to drive reporter expression to the involuting mesendodermal cells, while further deletions abolished reporter gene expression. Our results suggest that although no sequence similarity was found between mouse and Xenopus cerberus cis-regulatory regions, the signaling cascades regulating cerberus expression, during gastrulation, is conserved.

Left-right asymmetry in the level of active Nodal protein produced in the node is translated into left-right asymmetry in the lateral plate of mouse embryos.

Left-right (L-R) asymmetry in the mouse embryo is generated in the node and is dependent on cilia-driven fluid flow, but how the initial asymmetry is transmitted from the node to the lateral plate has remained unknown. We have now identified a transcriptional enhancer (ANE) in the human LEFTY1 gene that exhibits marked L>R asymmetric activity in perinodal cells of the mouse embryo. Dissection of ANE revealed that it is activated in the perinodal cells on the left side by Nodal signaling, suggesting that Nodal activity in the node is asymmetric at a time when Nodal expression is symmetric. Phosphorylated Smad2/3 (pSmad2) indeed manifested an L-R asymmetric distribution at the node, being detected in perinodal cells preferentially on the left side. This asymmetry in pSmad2 distribution was found to be generated not by unidirectional transport of Nodal but rather as a result of LR distribution of active Nodal in the node is translated into the asymmetry in LPM.

Ccbe1 expression marks the cardiac and lymphatic progenitor lineages during early stages of mouse development.

The mammalian heart is a complex organ composed of diverse components and various cell types. Heart organogenesis requires the contribution of distinct pools of heart progenitors positioned in separate embryonic regions and subject to particular developmental signals. Moreover, these embryonic heart lineages have different transcriptional profiles expressing specific genes which activate pathways involved in heart lineage specification. Understanding the molecular control of heart organogenesis has major implications for treating congenital and adult heart diseases since specific heart lineages have been associated with particular human cardiovascular malformations. Collagen and calcium-binding EGF-like domain 1 (Ccbe1) was identified in our laboratory using an Affymetrix GeneChip system approach to identify the transcriptome of chick heart/hemangioblast precursor cells. Here, we present a detailed and systematic analysis of the expression of Ccbe1 during early mouse development using whole-mount in situ hybridization (WISH), immunohistochemistry and histological techniques. Ccbe1 mRNA was initially detected in the early cardiac progenitors of the two bilateral cardiogenic fields (E7.0) and in the cardiogenic mesoderm (E7.5 to E8.0). Ccbe1 mRNA was then persistently detected in the pericardium and transiently expressed in the myocardial tissue of the primitive heart tube (E8.25), being later expressed in the proepicardium. By E9.5, the Ccbe1 and Prox1 proteins were found to be expressed in common regions, including the septum transversum and in the proximity of the anterior cardinal vein. Here, it is shown that Ccbe1 is expressed in the FHF, SHF and proepicardium during heart organogenesis (E7.0 to E8.75). Later in development, Ccbe1 expression is localized in the septum transversum and in the vicinity of the anterior cardinal vein, embryonic structures related to hepatic and lymphatic development, respectively.

Reversal of left-right asymmetry induced by aberrant Nodal signaling in the node of mouse embryos.

The node at the anterior tip of the primitive streak serves as an initial generator of the left-right (L-R) axis in mammalian embryos. We now show that a small disturbance in molecular signaling at the node is responsible for the L-R reversal of visceral organs in the inv mutant mouse. In the node of wild-type embryos, the expression of Nodal and Cerl2 (Dand5), which encodes an inhibitor of Nodal, is asymmetric, with the level of Nodal expression being higher on the left side and that of Cerl2 expression higher on the right. In inv/inv embryos, however, a localized reduction in the level of Cerl2 expression results in upregulation of the Nodal signal and a consequent induction of Lefty expression in the node. The ectopic expression of Lefty1 delays the onset of Nodal expression in the lateral plate mesoderm. L-R asymmetry of Cerl2 expression in the node also becomes reversed in a manner dependent on the Nodal signal. Nodal expression in the lateral plate mesoderm then appears on the right side, probably reflecting the balance between Nodal and Cerl2 in the node. The inhibition of Cerl2 expression by the Nodal signal suggests a mechanism for amplification of the cue for L-R asymmetry provided by nodal flow and for stabilization of asymmetric gene expression around the node. In inv/inv embryos, this system may function in reverse as a result of ectopic production of Lefty, which inhibits the Nodal signal on the left side in a manner dependent on leftward nodal flow.