Endocardial Cushion Formation Research Articles

Impairment of endothelial-mesenchymal transformation during atrioventricular cushion formation in Tmem100 null embryos.

Endothelial-mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin-dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up-regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively-active calcineurin A, suggesting dysregulation of myocardial calcineurin-VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium-derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial-specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium-related signaling events.

Abstract 332: Loss of beta-catenin Leads to Induction of a Chondrogenic Phenotype in Adult Mouse Heart Aortic Valves

Objectives: Altered Wnt/beta-catenin signaling has been implicated in human heart valve disease; however, a causal relation has not been established. This study is aimed at determining the role of Wnt/beta-catenin signaling in adult valve homeostasis and disease. In endochondral bone development, Wnt/beta-catenin signaling inhibits early chondrogenesis and promotes osteogenesis. Therefore, we hypothesize that loss of Wnt/beta-catenin signaling promotes chondrogenic differentiation of valvular interstitial cells (VICs) resembling myxomatous valve disease. Methods & Results: Periostin (Postn)Cre was used for conditional loss of beta-catenin (Ctnnb1) specifically in VICs in mice. PostnCre is active in valve mesenchymal progenitors by embryonic day (E)13.5, therefore bypassing requirements for beta-catenin in endothelial-to-mesenchymal transition and endocardial cushion formation. Histology of adult heart valves was analyzed by Masson’s Trichrome and Movat’s Pentachrome staining, and the expression of extracellular matrix (ECM) genes was examined at both mRNA and protein level. Heart valve development appears normal at both fetal and neonatal stages with loss of beta-catenin. However proteoglycan-rich nodules form in adult aortic valves (AoVs) lacking beta-catenin at 3 months with 100% penetrance. These nodular cells are enlarged and express nuclear Sox9 and chondrogenic ECM genes, including Aggrecan, Collagen type 2 (Col1) and Col10, reminiscent of hypertrophic cartilage. Fibrillar collagens 1 and 3, as well as Versican, are excluded from the nodules, accompanied by abnormal ECM deposition throughout the AoV leaflets. Likewise, treatment of cultured porcine aortic VICs with the Wnt pathway inhibitor XAV also induces Aggrecan and Col2 gene expression in vitro. This work demonstrates that beta-catenin is required for the maintenance of normal ECM deposition and composition in adult mice, and, therefore, is indispensible for adult heart valve homeostasis. Conclusions: We have demonstrated that beta-catenin is a key factor in maintaining normal valve ECM organization in adult AoV. In the absence of beta-catenin, VICs are susceptible to chondrogenic differentiation related to myxomatous valve disease.

RNA-seq analysis to identify novel roles of scleraxis during embryonic mouse heart valve remodeling.

Heart valve disease affects up to 30% of the population and has been shown to have origins during embryonic development. Valvulogenesis begins with formation of endocardial cushions in the atrioventricular canal and outflow tract regions. Subsequently, endocardial cushions remodel, elongate and progressively form mature valve structures composed of a highly organized connective tissue that provides the necessary biomechanical function throughout life. While endocardial cushion formation has been well studied, the processes required for valve remodeling are less well understood. The transcription factor Scleraxis (Scx) is detected in mouse valves from E15.5 during initial stages of remodeling, and expression remains high until birth when formation of the highly organized mature structure is complete. Heart valves from Scx-/- mice are abnormally thick and develop fibrotic phenotypes similar to human disease by juvenile stages. These phenotypes begin around E15.5 and are associated with defects in connective tissue organization and valve interstitial cell differentiation. In order to understand the etiology of this phenotype, we analyzed the transcriptome of remodeling valves isolated from E15.5 Scx-/- embryos using RNA-seq. From this, we have identified a profile of protein and non-protein mRNAs that are dependent on Scx function and using bioinformatics we can predict the molecular functions and biological processes affected by these genes. These include processes and functions associated with gene regulation (methyltransferase activity, DNA binding, Notch signaling), vitamin A metabolism (retinoic acid biosynthesis) and cellular development (cell morphology, cell assembly and organization). In addition, several mRNAs are affected by alternative splicing events in the absence of Scx, suggesting additional roles in post-transcriptional modification. In summary, our findings have identified transcriptome profiles from abnormal heart valves isolated from E15.5 Scx-/- embryos that could be used in the future to understand mechanisms of heart valve disease in the human population.

Complex cardiac defects after ethanol exposure during discrete cardiogenic events in zebrafish: Prevention with folic acid

Fetal alcohol spectrum disorder (FASD) describes a range of birth defects including various congenital heart defects (CHDs). Mechanisms of FASD-associated CHDs are not understood. Whether alcohol interferes with a single critical event or with multiple events in heart formation is not known. Our zebrafish embryo experiments showed that ethanol interrupts different cardiac regulatory networks and perturbs multiple steps of cardiogenesis (specification, myocardial migration, looping, chamber morphogenesis, and endocardial cushion formation). Ethanol exposure during gastrulation until cardiac specification or during myocardial midline migration did not produce severe or persistent heart development defects. However, exposure comprising gastrulation until myocardial precursor midline fusion or during heart patterning stages produced aberrant heart looping and defective endocardial cushions. Continuous exposure during entire cardiogenesis produced complex cardiac defects leading to severely defective myocardium, endocardium, and endocardial cushions. Supplementation of retinoic acid with ethanol partially rescued early heart developmental defects, but the endocardial cushions did not form correctly. In contrast, supplementation of folic acid rescued normal heart development, including the endocardial cushions. Our results indicate that ethanol exposure interrupted divergent cardiac morphogenetic events causing heart defects. Folic acid supplementation was effective in preventing a wide spectrum of ethanol-induced heart developmental defects.

Abstract 172: Notch Signaling Induces Endocardial Hematopoiesis And Endocardial Cushion Formation Downstream Of Nkx2-5

A subset of cardiac progenitors display multipotency to give rise to major cardiovascular lineages during cardiogenesis. In addition, we have recently identified Nkx2-5+ endocardial cells in the outflow and atria that contribute to definitive hematopoietic cells in vivo, which represents phylogenetically conserved pathway of cardio-vasculo-hematopoietic differentiation. Here, we have examined the molecular mechanism underlying the endocardial hematopoiesis. Immunostaining and colony assay suggest that Nkx2-5 mutant mice showed significant decrease in CD31+/41+ hemogenic endocardial cells, while lineage tracing on Nkx2-5-null background revealed that Nkx2-5-derived endocardial cells are present. These data indicate that Nkx2-5 is required not for the formation of the Nkx2-5-derived endocardium but for the hemogenic activity of the endocardium. Notch signaling is also active in cushion endocardium and its mutant embryos show a similar defect with Nkx2-5 mutants as previously reported. Inspired by these similarities between the function of Nkx2-5 and Notch signaling during cardiogenesis, we next investigated the impact of Notch activation on the endocardial hematopoiesis. In vivo forced activation of Notch in the developing heart using Nkx2-5-Cre mice led to massive increase in the endocardial hematopoiesis. As Nkx2-5 binding sites are found in the regulatory region of Notch1, RBPjk, and Jag1 genes, we tested whether Notch regulates endocardial hematopoiesis downstream of Nkx2-5. Surprisingly, Notch activation restored the defective endocardial hematopoiesis as well as endocardial cushion formation in Nkx2-5 knockout mice, suggesting that Notch signaling mediates the Nkx2-5-dependent endocardial hematopoiesis and cushion development. Drosophila dorsal vessel and lymph gland are closely related and regulated by tinman and notch. Therefore, Nkx2-5-Notch regulatory pathway represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway.

Tbx20 acts upstream of Wnt signaling to regulate endocardial cushion formation and valve remodeling during mouse cardiogenesis

Cardiac valves are essential to direct forward blood flow through the cardiac chambers efficiently. Congenital valvular defects are prevalent among newborns and can cause an immediate threat to survival as well as long-term morbidity. Valve leaflet formation is a rigorously programmed process consisting of endocardial epithelial-mesenchymal transformation (EMT), mesenchymal cell proliferation, valve elongation and remodeling. Currently, little is known about the coordination of the diverse signals that regulate endocardial cushion development and valve elongation. Here, we report that the T-box transcription factor Tbx20 is expressed in the developing endocardial cushions and valves throughout heart development. Ablation of Tbx20 in endocardial cells causes severe valve elongation defects and impaired cardiac function in mice. Our study reveals that endocardial Tbx20 is crucial for valve endocardial cell proliferation and extracellular matrix development, but is not required for initiation of EMT. Elimination of Tbx20 also causes aberrant Wnt/β-catenin signaling in the endocardial cushions. In addition, Tbx20 regulates Lef1, a key transcriptional mediator for Wnt/β-catenin signaling, in this developmental process. Our study suggests a model in which Tbx20 regulates the Wnt pathway to direct endocardial cushion maturation and valve elongation, and provides new insights into the etiology of valve defects in humans.

Endothelial deletion of murine Jag1 leads to valve calcification and congenital heart defects associated with Alagille syndrome

The Notch signaling pathway is an important contributor to the development and homeostasis of the cardiovascular system. Not surprisingly, mutations in Notch receptors and ligands have been linked to a variety of hereditary diseases that impact both the heart and the vasculature. In particular, mutations in the gene encoding the human Notch ligand jagged 1 result in a multisystem autosomal dominant disorder called Alagille syndrome, which includes tetralogy of Fallot among its more severe cardiac pathologies. Jagged 1 is expressed throughout the developing embryo, particularly in endothelial cells. Here, we demonstrate that endothelial-specific deletion of Jag1 leads to cardiovascular defects in both embryonic and adult mice that are reminiscent of those in Alagille syndrome. Mutant mice display right ventricular hypertrophy, overriding aorta, ventricular septal defects, coronary vessel abnormalities and valve defects. Examination of mid-gestational embryos revealed that the loss of Jag1, similar to the loss of Notch1, disrupts endothelial-to-mesenchymal transition during endocardial cushion formation. Furthermore, adult mutant mice exhibit cardiac valve calcifications associated with abnormal matrix remodeling and induction of bone morphogenesis. This work shows that the endothelium is responsible for the wide spectrum of cardiac phenotypes displayed in Alagille Syndrome and it demonstrates a crucial role for Jag1 in valve morphogenesis.

The W-Loop of Alpha-Cardiac Actin Is Critical for Heart Function and Endocardial Cushion Morphogenesis in Zebrafish

Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physiology and results in the development of distinct congenital disorders. A zebrafish mutant (s434 mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation. We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at the W-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434 mutants show loss of polymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addition, actc1a mutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregulation or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of the W-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTC mutations in human congenital disorders.

Wnt3a/β-catenin increases proliferation in heart valve interstitial cells

Valve interstitial cells (VICs), the most prevalent cells in the heart valve, mediate normal valve function and repair in valve injury and disease. The Wnt3a/β-catenin pathway, important for proliferation and endothelial-to-mesenchymal transition in endocardial cushion formation in valve development, is up-regulated in adult valves with calcific aortic stenosis. Therefore, we tested the hypothesis that Wnt3a/β-catenin signaling regulates proliferation in adult VICs. Porcine VICs were treated with 150 ng/ml of exogenous Wnt3a. To measure proliferation, cells were counted on day 4 posttreatment and stained for bromodeoxyuridine (BrdU) at 24 h posttreatment. β-Catenin small interfering RNA (siRNA) was used to knock down β-catenin expression. Apoptosis was measured with terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To assess changes in β-catenin, cells were stained for β-catenin at days 1, 3, 6, and 9 posttreatment. Western blot for β-catenin was performed on whole cell, cytoplasmic, and nuclear extracts at day 4 posttreatment. To measure β-catenin-mediated transcription, TOPFLASH/FOPFLASH reporter assay was performed at 24 h posttreatment. Wnt3a produced a significant increase in cell number at day 4 posttreatment and in the percentage of BrdU-positive nuclei at 24 h posttreatment. The increase in proliferation was abolished by β-catenin siRNA. Apoptosis was minimal in all conditions. Wnt3a produced progressively greater β-catenin staining as treatment length increased from 1 to 9 days. Wnt3a produced a significant increase in β-catenin protein in both whole cell and nuclear lysates after 4 days of treatment. Wnt3a significantly increased TOPFLASH/FOPFLASH reporter activity after 24 h of treatment. Wnt3a/β-catenin signaling pathway is an important regulator of proliferation in adult VICs.

Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGFβ3.

BackgroundValvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor β (TGFβ) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFβ-dependent endocardial cell invasion.ResultsIn this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFβ3 after it is no longer required to induce basal levels of invasion. TGFβ3, but not TGFβ2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFβ3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFβ3 production in the endocardium. More TGFβ3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFβ3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFβ3 secretion, and early exposure to excess TGFβ3 induces precocious invasion. MBNL1 expression precedes TGFβ3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFβ3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFβ is necessary and sufficient to mediate this effect.ConclusionsTaken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFβ3 production.

Conditional Ablation of Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion Formation, Cardiomyocyte Proliferation and Survival

Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.

Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation

A key step in heart development is the coordinated development of the atrioventricular canal (AVC), the constriction between the atria and ventricles that electrically and physically separates the chambers, and the development of the atrioventricular valves that ensure unidirectional blood flow. Using knock-out and inducible overexpression mouse models, we provide evidence that the developmentally important T-box factors Tbx2 and Tbx3, in a functionally redundant manner, maintain the AVC myocardium phenotype during the process of chamber differentiation. Expression profiling and ChIP-sequencing analysis of Tbx3 revealed that it directly interacts with and represses chamber myocardial genes, and induces the atrioventricular pacemaker-like phenotype by activating relevant genes. Moreover, mutant mice lacking 3 or 4 functional alleles of Tbx2 and Tbx3 failed to form atrioventricular cushions, precursors of the valves and septa. Tbx2 and Tbx3 trigger development of the cushions through a regulatory feed-forward loop with Bmp2, thus providing a mechanism for the co-localization and coordination of these important processes in heart development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-011-0884-2) contains supplementary material, which is available to authorized users.

Tbx20 Transcription Factor Is a Downstream Mediator for Bone Morphogenetic Protein-10 in Regulating Cardiac Ventricular Wall Development and Function

Bone morphogenetic protein 10 (BMP10) belongs to the TGFβ-superfamily. Previously, we had demonstrated that BMP10 is a key regulator for ventricular chamber formation, growth, and maturation. Ablation of BMP10 leads to hypoplastic ventricular wall formation, and elevated levels of BMP10 are associated with abnormal ventricular trabeculation/compaction and wall maturation. However, the molecular mechanism(s) by which BMP10 regulates ventricle wall growth and maturation is still largely unknown. In this study, we sought to identify the specific transcriptional network that is potentially mediated by BMP10. We analyzed and compared the gene expression profiles between α-myosin heavy chain (αMHC)-BMP10 transgenic hearts and nontransgenic littermate controls using Affymetrix mouse exon arrays. T-box 20 (Tbx20), a cardiac transcription factor, was significantly up-regulated in αMHC-BMP10 transgenic hearts, which was validated by quantitative RT-PCR and in situ hybridization. Ablation of BMP10 reduced Tbx20 expression specifically in the BMP10-expressing region of the developing ventricle. In vitro promoter analysis demonstrated that BMP10 was able to induce Tbx20 promoter activity through a conserved Smad binding site in the Tbx20 promoter proximal region. Furthermore, overexpression of Tbx20 in myocardium led to dilated cardiomyopathy that exhibited ventricular hypertrabeculation and an abnormal muscular septum, which phenocopied genetically modified mice with elevated BMP10 levels. Taken together, our findings demonstrate that the BMP10-Tbx20 signaling cascade is important for ventricular wall development and maturation.

GATA5 interacts with GATA4 and GATA6 in outflow tract development

Members of the GATA family of transcription factors are critical regulators of heart development and mutations in 2 of them, GATA4 and GATA6 are associated with outflow tract and septal defects in human. The heart expresses 3 GATA factors, GATA4, 5 and 6 in a partially overlapping pattern. Here, we report that compound Gata4/ Gata5 and Gata5/ Gata6 mutants die embryonically or perinatally due to severe congenital heart defects. Almost all Gata4 +/− Gata5 +/− mutant embryos have double outlet right ventricles (DORV), large ventricular septal defects (VSD) as well as hypertrophied mitral and tricuspid valves. Only 25% of double compound Gata4/ Gata5 heterozygotes survive to adulthood and these mice have aortic stenosis. Compound loss of a Gata5 and a Gata6 allele also leads to DORVs associated with subaortic VSDs. Expression of several transcription factors important for endocardial and myocardial cell differentiation, such as Tbx20, Mef2c, Hey1 and Hand2, was reduced in compound heterozygote embryos. These findings suggest the existence of important genetic interactions between Gata5 and the 2 other cardiac GATA factors in endocardial cushion formation and outflow tract morphogenesis. The data identify GATA5 as a potential genetic modifier of congenital heart disease and provide insight for elucidating the genetic basis of an important class of human birth defects.

MicroRNA-23 Restricts Cardiac Valve Formation by Inhibiting Has2 and Extracellular Hyaluronic Acid Production

Since their discovery almost 20 years ago, microRNAs have been shown to perform essential roles during tissue development and disease. Although roles for microRNAs in the myocardium during embryo development and cardiac disease have been demonstrated, very little is know about their role in the endocardium or during cardiac valve formation. To study the role of microRNAs in cardiac valve formation. We show that zebrafish dicer mutant embryos, lacking mature miRNAs, form excessive endocardial cushions. By screening miRNAs expressed in the heart, we found that miR-23 is both necessary and sufficient for restricting the number of endocardial cells that differentiate into endocardial cushion cells. In addition, in mouse endothelial cells, miR-23 inhibited a transforming growth factor-β-induced endothelial-to-mesenchymal transition. By in silico screening of expression data with predicted miR-23 target sites combined with in vivo testing, we identified hyaluronic acid synthase 2 (Has2), Icat, and Tmem2 as novel direct targets of miR-23. Finally, we demonstrate that the upregulation of Has2, an extracellular remodeling enzyme required for endocardial cushion and valve formation, is responsible for the excessive endocardial cushion cell differentiation in dicer mutants. MiR-23 in the embryonic heart is required to restrict endocardial cushion formation by inhibiting Has2 expression and extracellular hyaluronic acid production.

Loss of Gata5 in mice leads to bicuspid aortic valve

Bicuspid aortic valve (BAV), the leading congenital heart disease, occurs in 1%-2% of the population. Genetic studies suggest that BAV is an autosomal-dominant disease with reduced penetrance. However, only 1 gene, NOTCH1, has been linked to cases of BAV. Here, we show that targeted deletion of Gata5 in mice leads to hypoplastic hearts and partially penetrant BAV formation. Endocardial cell-specific inactivation of Gata5 led to BAV, similar to that observed in Gata5-/- mice. In all cases, the observed BAVs resulted from fusion of the right-coronary and noncoronary leaflets, the subtype associated with the more severe valve dysfunction in humans. Neither endocardial cell proliferation nor cushion formation was altered in the absence of Gata5. Rather, defective endocardial cell differentiation, resulting from the deregulation of several components of the Notch pathway and other important endocardial cell regulators, may be the underlying mechanism of disease. The results unravel a critical cell-autonomous role for endocardial Gata5 in aortic valve formation and identify GATA5 as a potential gene responsible for congenital heart disease in humans. Mice with mutated Gata5 alleles represent unique models to dissect the mechanisms underlying degenerative aortic valve disease and to develop much-needed preventive and therapeutic interventions.

Transcriptomic analysis of cell-free fetal RNA suggests a specific molecular phenotype in trisomy 18.

Trisomy 18 is a common human aneuploidy that is associated with significant perinatal mortality. Unlike the well-characterized "critical region" in trisomy 21 (21q22), there is no corresponding region on chromosome 18 associated with its pathogenesis. The high morbidity and mortality of affected individuals has limited extensive investigations. In order to better understand the molecular mechanisms underlying the congenital anomalies observed in this condition, we investigated the in utero gene expression profile of second trimester fetuses affected with trisomy 18. Total RNA was extracted from cell-free amniotic fluid supernatant from aneuploid fetuses and euploid controls matched for gestational age and hybridized to Affymetrix U133 Plus 2.0 arrays. Individual differentially expressed transcripts were obtained by two-tailed t tests. Over-represented functional pathways among these genes were identified with DAVID and Ingenuity(®) Pathways Analysis. Results show that three hundred and fifty-two probe sets representing 251 annotated genes were statistically significantly differentially expressed between trisomy 18 and controls. Only 7 genes (2.8% of the annotated total) were located on chromosome 18, including ROCK1, an up-regulated gene involved in valvuloseptal and endocardial cushion formation. Pathway analysis indicated disrupted function in ion transport, MHCII/T cell mediated immunity, DNA repair, G-protein mediated signaling, kinases, and glycosylation. Significant down-regulation of genes involved in adrenal development was identified, which may explain both the abnormal maternal serum estriols and the pre and postnatal growth restriction in trisomy 18. Comparison of this gene set to one previously generated for trisomy 21 fetuses revealed only six overlapping differentially regulated genes. This study contributes novel information regarding functional developmental gene expression differences in fetuses with trisomy 18.

Progress of pregestational diabetes mellitus and congenital heart defects

Pregestational diabetes mellitus may cause fetal heart defects,and it is considered to be an important non-genetic risk factors for congenital heart defects. However, the pathogenesis is unclear. Recent studies have shown that hyperglycemia, which acts as a primary teratogen in pregestational diabetes mellitus, may affect the endocardial cushion formation and neural crest cell development. Cardiac oxidative stress damage, increased myocardial cell apoptosis,increased synthesis of extracellular matrix, as well as the alteration of heart development-related genes expression are important pathogenic mechanism. In this paper, we review the progress in the effect and the pathogenesis of pregestational diabetes mellitus on heart development. Key words: Pregestational diabetes mellitus; Congenital heart defects; Endocardial cushion; Neural crest cell

Endothelial SUR‐8 acts in an ERK‐independent pathway during atrioventricular cushion development

SUR-8, a conserved leucine-rich repeats protein, was first identified as a positive regulator of Ras pathway in Caenorhabditis elegans. Biochemical studies indicated that SUR-8 interacts with Ras and Raf, leading to the elevated ERK activity. However, the physiological role of SUR-8 during mammalian development remains unclear. Here we found that germline deletion of SUR-8 in mice resulted in early embryonic lethality. Inactivated SUR-8 specifically in mouse endothelial cells (ECs) revealed that SUR-8 is essential for embryonic heart development. SUR-8 deficiency in ECs resulted in late embryonic lethality, and the mutant mice displayed multiple cardiac defects. The reduced endothelial-mesenchymal transformation (EMT) and the reduced mesenchyme proliferation phase were observed in the atrioventricular canal (AVC) within the mutant hearts, leading to the formation of hypoplastic endocardial cushions. However, ERK activation did not appear to be affected in mutant ECs, suggesting that SUR-8 may act in an ERK-independent pathway to regulate AVC development. Developmental Dynamics 239:2005–2013, 2010 © 2010 Wiley-Liss, Inc.

Acide hyaluronique, embryogenèse et morphogenèse

Hyaluronan is a major carbohydrate component of the extracellular matrix. Besides its structural role, it also appears to regulate cell transformation and migration during embryogenesis in vertebrates. Hyaluronan is synthesized by hyaluronan synthetases, transmembrane proteins expressed at several embryonic stages, as early as gastrulation. Inactivation or upregulation of hyaluronan synthetases elicits cardiac or skeletal development anomalies (atrioventricular septal defects caused by abnormal endocardial cushion formation, impaired chondrogenesis). Hyaluronidases degrade hyaluronan and interact with cell surface receptors involved in cell activation. Hyluronan binds not only extracellular matrix glycoproteins, but also cell surface receptors (CD44, RHAMM) also involved in cell signalling, differentiation and proliferation pathways. It facilitates migration and transformation and decreases contact inhibition. Hence, hyaluronan has a central regulating role during embryogenesis.