Cardiac Development and Animal Models of Congenital Heart Defects.

The major events of cardiac development, including early heart formation, chamber morphogenesis and septation, and conduction system and coronary artery development, are briefly reviewed together with a short introduction to the animal species commonly used to study heart development and model congenital heart defects (CHDs).

Hyperglycemia impairs left-right axis formation and thereby disturbs heart morphogenesis in mouse embryos.

Congenital heart defects with heterotaxia are associated with pregestational diabetes mellitus. To provide insight into the mechanisms underlying such diabetes-related heart defects, we examined the effects of high-glucose concentrations on formation of the left-right axis in mouse embryos. Expression of Pitx2, which plays a key role in left-right asymmetric morphogenesis and cardiac development, was lost in the left lateral plate mesoderm of embryos of diabetic dams. Embryos exposed to high-glucose concentrations in culture also failed to express Nodal and Pitx2 in the left lateral plate mesoderm. The distribution of phosphorylated Smad2 revealed that Nodal activity in the node was attenuated, accounting for the failure of left-right axis formation. Consistent with this notion, Notch signal-dependent expression of Nodal-related genes in the node was also down-regulated in association with a reduced level of Notch signaling, suggesting that high-glucose concentrations impede Notch signaling and thereby hinder establishment of the left-right axis required for heart morphogenesis.

Relation of Nodal expression to the specification of the dorsal-ventral axis and tissue patterning in the starfish Patiria pectinifera.

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid-blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left-right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF-beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

BACKGROUND: Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. RESULTS: Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. CONCLUSIONS: Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Left-right asymmetry: lessons from Cancún.

The satellite symposium on 'Making and breaking the left-right axis: implications of laterality in development and disease' was held in June 2013 in conjunction with the 17th International Society for Developmental Biology meeting in Cancún, Mexico. As we summarize here, leaders in the field gathered at the symposium to discuss recent advances in understanding how left-right asymmetry is generated and utilized across the animal kingdom.

Is VACTERL a laterality defect?

To date the etiology of the association called VACTERL remains a mystery. Interestingly, clues as to the origin of this collection of defects may reside in an old hypothesis concerning the midline as a developmental field as postulated by Dr. John Opitz. This theory suggested that the midline was not a separate entity, but could be influenced by other developmental signals. With new information concerning the origin of the left-right axis (laterality) and the importance of communications between this axis and the cranio-caudal (anterior-posterior) axis for normal development, it has become clear that coordination of the molecular signals responsible for specification of these domains is essential for normal development. In fact, if the signals regulating laterality are disrupted, then midline and other defects can occur as has been observed in cases of heterotaxy, presumably because of a disruption in this coordinated signaling effort. Thus, the origins of the defects commonly observed in the VACTERL association may be due to altered signaling responsible for establishing the left-right axis.

Wnt signaling regulates left-right axis formation in the node of mouse embryos.

The node triggers formation of the left-right axis in mouse embryos by establishing local asymmetry of Nodal and Cerl2 expression. We found that Wnt3 is expressed in perinodal crown cells preferentially on the left side. The enhancer responsible for Wnt3 expression was identified and found to be regulated by Foxa2 and Rbpj under the control of Notch signaling. Rbpj binding sites suppress enhancer activity in pit cells of the node, thereby ensuring crown cell-specific expression. In addition, we found that the expression of Gdf1 and Cerl2 is also regulated by Notch signaling, suggesting that such signaling may induce the expression of genes related to left-right asymmetry as a set. Furthermore, Cerl2 expression became symmetric in response to inhibition of Wnt-ß-catenin signaling. Our results suggest that Wnt signaling regulates the asymmetry of Cerl2 expression, which likely generates a left-right difference in Nodal activity at the node for further amplification in lateral plate mesoderm.

ccdc141 is required for left-right axis development by regulating cilia formation in the Kupffer's vesicle of zebrafish.

Laterality is a crucial physiological process intricately linked to the cilium-centrosome complex during embryo development. Defects in the process can result in severe organ mispositioning. Coiled-coil domain containing 141 (CCDC141) has been previously known as a centrosome-related gene, but its role in left-right (LR) asymmetry has not been characterized. In this study, we utilize the zebrafish model and human exome analysis to elucidate the function of ccdc141 in laterality defects. The knockdown of ccdc141 in zebrafish disrupts early LR signaling pathways, cilia function, and Kupffer's vesicle formation. Unlike ccdc141-knockdown embryos exhibiting aberrant LR patterns, ccdc141-null mutants show no apparent abnormality, suggesting a genetic compensation response effect. In parallel, we observe a marked reduction in α-tubulin acetylation levels in the ccdc141 crispants. The treatment with histone deacetylase (HDAC) inhibitors, particularly the HDAC6 inhibitor, rescues the ccdc141 crispant phenotypes. Furthermore, exome analysis of 70 patients with laterality defects reveals an increased burden of CCDC141 mutations, with in-vivo studies verifying the pathogenicity of the patient mutation CCDC141-R123G. Our findings highlight the critical role of ccdc141 in ciliogenesis and demonstrate that CCDC141 mutations lead to abnormal LR patterns, identifying it as a candidate gene for laterality defects.

Canonical and Non-Canonical Wnt Signaling Generates Molecular and Cellular Asymmetries to Establish Embryonic Axes.

The formation of embryonic axes is a critical step during animal development, which contributes to establishing the basic body plan in each particular organism. Wnt signaling pathways play pivotal roles in this fundamental process. Canonical Wnt signaling that is dependent on ß-catenin regulates the patterning of dorsoventral, anteroposterior, and left-right axes. Non-canonical Wnt signaling that is independent of ß-catenin modulates cytoskeletal organization to coordinate cell polarity changes and asymmetric cell movements. It is now well documented that components of these Wnt pathways biochemically and functionally interact to mediate cell-cell communications and instruct cellular polarization in breaking the embryonic symmetry. The dysfunction of Wnt signaling disrupts embryonic axis specification and proper tissue morphogenesis, and mutations of Wnt pathway genes are associated with birth defects in humans. This review discusses the regulatory roles of Wnt pathway components in embryonic axis formation by focusing on vertebrate models. It highlights current progress in decoding conserved mechanisms underlying the establishment of asymmetry along the three primary body axes. By providing an in-depth analysis of canonical and non-canonical pathways in regulating cell fates and cellular behaviors, this work offers insights into the intricate processes that contribute to setting up the basic body plan in vertebrate embryos.

Clinical Developmental Cardiology for Understanding Etiology of Congenital Heart Disease.

Congenital heart diseases (CHD) result from abnormal development of the cardiovascular system and usually involve defects in specific steps or structural components of the developing heart and vessels. The determination of left-right patterning of our body proceeds by the steps involving the leftward "nodal flow" by motile cilia in the node and molecules that are expressed only on the left side of the embryo, eventually activating the molecular pathway for the left-side specific morphogenesis. Disruption of any of these steps may result in left-right patterning defects or heterotaxy syndrome. As for the outflow tract development, neural crest cells migrate into the cardiac outflow tract and contribute to form the septum of the outflow tract that divides the embryonic single truncus arteriosus into the aortic and the pulmonary trunk. Reciprocal signaling between neural crest cells and another population of myocardial precursor cells originated from the second heart field are essential for the steps of outflow tract development. To better understand the etiology of CHD, it is important to consider what kind of CHD is caused by abnormalities in each step during the complex development of the cardiovascular system.

The maternal coordinate system: Molecular-genetics of embryonic axis formation and patterning in the zebrafish.

Axis specification of the zebrafish embryo begins during oogenesis and relies on proper formation of well-defined cytoplasmic domains within the oocyte. Upon fertilization, maternally-regulated cytoplasmic flow and repositioning of dorsal determinants establish the coordinate system that will build the structure and developmental body plan of the embryo. Failure of specific genes that regulate the embryonic coordinate system leads to catastrophic loss of body structures. Here, we review the genetic principles of axis formation and discuss how maternal factors orchestrate axis patterning during zebrafish early embryogenesis. We focus on the molecular identity and functional contribution of genes controlling critical aspects of oogenesis, egg activation, blastula, and gastrula stages. We examine how polarized cytoplasmic domains form in the oocyte, which set off downstream events such as animal-vegetal polarity and germ line development. After gametes interact and form the zygote, cytoplasmic segregation drives the animal-directed reorganization of maternal determinants through calcium- and cell cycle-dependent signals. We also summarize how maternal genes control dorsoventral, anterior-posterior, mesendodermal, and left-right cell fate specification and how signaling pathways pattern these axes and tissues during early development to instruct the three-dimensional body plan. Advances in reverse genetics and phenotyping approaches in the zebrafish model are revealing positional patterning signatures at the single-cell level, thus enhancing our understanding of genotype-phenotype interactions in axis formation. Our emphasis is on the genetic interrogation of novel and specific maternal regulatory mechanisms of axis specification in the zebrafish.

The value of right-hand rule in determining left-right axis in fetal MRI / 中华放射学杂志

Objective:To investigate the application value of right-hand rule in determining left-right axis and fetal situs in prenatal MRI.Methods:The prenatal MRI data of 254 fetuses were included retrospectively in West China Second University Hospital, Sichuan University from December 2017 to February 2021, which were followed by clear postpartum diagnosis. Fetal left-right axis and situs (orientation of gastric vacuole, liver and heart) were determined by the same radiologist blindly using general experience and right-hand rule respectively, meanwhile, results and time of each case were recorded. The right-hand rule was described in detail below: use your right hand to make a fist, and with thumb outstretched; the fetal head was represented by your right fist, the forearm represented the fetal body, the dorsal side of your fist represented the fetal occiput and spine column, the palm side represented the fetal belly, and your four fingers represented the fetal face, then the left side of fetus was exactly the direction your thumb pointing to. The postpartum imaging was used as the gold standard for situs anomalies. The time to determine the fetal left-right axis was compared using paired t test for statistical analysis. Results:Four fetuses were found situs anomalies in postpartum imaging. There were 1 missed case and 1 misdiagnosed case when using general experience to determine fetal situs, neither missed case nor misdiagnosis were present when using right-hand rule. The average time of using general experience or right-hand rule to determine the fetal left-right axis and situs were (35±6)s and (24±9)s respectively, and the difference was statistically significant ( t=20.65, P<0.001). Conclusions:The right-hand rule is an accurate and efficient method for determining left-right axis and situs in prenatal MRI, which can effectively reduce missed diagnosis and misdiagnosis of situs anomalies, especially in situs inversus totalis.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

Embryonic chirality and the evolution of spiralian left-right asymmetries.

The group Spiralia includes species with one of the most significant cases of left-right asymmetries in animals: the coiling of the shell of gastropod molluscs (snails). In this animal group, an early event of embryonic chirality controlled by cytoskeleton dynamics and the subsequent differential activation of the genes nodal and Pitx determine the left-right axis of snails, and thus the direction of coiling of the shell. Despite progressive advances in our understanding of left-right axis specification in molluscs, little is known about left-right development in other spiralian taxa. Here, we identify and characterize the expression of nodal and Pitx orthologues in three different spiralian animals-the brachiopod Novocrania anomala, the annelid Owenia fusiformis and the nemertean Lineus ruber-and demonstrate embryonic chirality in the biradial-cleaving spiralian embryo of the bryozoan Membranipora membranacea We show asymmetric expression of nodal and Pitx in the brachiopod and annelid, respectively, and symmetric expression of Pitx in the nemertean. Our findings indicate that early embryonic chirality is widespread and independent of the cleavage programme in the Spiralia. Additionally, our study illuminates the evolution of nodal and Pitx signalling by demonstrating embryonic asymmetric expression in lineages without obvious adult left-right asymmetries.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Left-right patterning in the C. elegans embryo: Unique mechanisms and common principles.

The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.