XBtg2 is required for notochord differentiation during early Xenopus development

The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochord tissue, which did not undergo vacuolation. The XBtg2-depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5-D-4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.     

BMP signaling is required for normal thymus development

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.     

BMP signaling is required for heart formation in vertebrates

In these studies, we have taken advantage of a transient transgenic strategy in Xenopus embryos to demonstrate that BMP signaling is required in vivo for heart formation in vertebrates. Ectopic expression of dominant negative Type I (tALK3) or Type II (tBMPRII) BMP receptors in developing Xenopus embryos results in reduction or absence of heart formation. Additionally, blocking BMP signaling in this manner downregulates expression of XNkx2-5, a homeobox gene required for cardiac specification, prior to differentiation. Notably, however, initial expression of XNkx2-5 is not affected. Mutant phenotypes can be rescued by co-injection of mutant with wild-type receptors or co-injection of mutant receptors with XSmad1, a downstream mediator of BMP signaling. Whole-mount in situ analyses indicate that ALK3 and XSmad1 are coexpressed in cardiogenic regions. Together, our results demonstrate that BMP signaling is required for maintenance of XNkx2-5 expression and heart formation and suggest that ALK3, BMPRII, and XSmad1 may mediate this signaling.     

BMP type II receptor is required for gastrulation and early development of mouse embryos

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, play a variety of roles during mouse development. BMP type II receptor (BMPR-II) is a type II serine/threonine kinase receptor, which transduces signals for BMPs through heteromeric complexes with type I receptors, including activin receptor-like kinase 2 (ALK2), ALK3/BMPR-IA, and ALK6/BMPR-IB. To elucidate the function of BMPR-II in mammalian development, we generated BMPR-II mutant mice by gene targeting. Homozygous mutant embryos were arrested at the egg cylinder stage and could not be recovered at 9.5 days postcoitum. Histological analysis revealed that homozygous mutant embryos failed to form organized structure and lacked mesoderm. The BMPR-II mutant embryos are morphologically very similar to the ALK3/BMPR-IA mutant embryos, suggesting that BMPR-II is important for transducing BMP signals during early mouse development. Moreover, the epiblast of the BMPR-II mutant embryo exhibited an undifferentiated character, although the expression of tissue-specific genes for the visceral endoderm was essentially normal. Our results suggest that the function of BMPR-II is essential for epiblast differentiation and mesoderm induction during early mouse development.     

Cadherin-6 is required for zebrafish nephrogenesis during early development

We performed functional analyses of cadherin-6 (cdh6) in zebrafish nephrogenesis using antisense morpholino oligonucleotide (MO) inhibition combined with in situ hybridization. We have cloned a zebrafish homolog (accession number AB193290) of human K-cadherin (CDH6), which showed 6063% identity and 7678% similarity to the human, mouse, chicken and Xenopus homologs. Whole-mount in situ hybridization showed that cdh6 is expressed in the pronephric ducts and nephron primordia in addition to the central and peripheral nervous systems. Expression of cdh6 in the pronephric ducts was first detected at 14 hours post-fertilization (hpf) and increased to 24 hpf. Embryos injected with MOs directed against cdh6 (cdh6MOs) showed developmental defects, including a small head, body axis curvature, short yolk extension and a short bent tail by 30 hpf and edema appeared in the thorax by 42 hpf. Such defects and edema became more marked by 52 hpf and most of the affected embryos died by 5 days post-fertilization. Embryos injected with cdh6MOs were subjected to in situ hybridization with probes for the pronephric markers, wt1 and pax2.1, to examine disturbed development of the anterior region of the pronephric ducts and the nephron primordia. Histological studies showed malformation of the pronephros as abnormally fused glomerulus primordia, fused or abnormally bent pronephric tubule anlagen and coarctated pronephric ducts. These results suggest that cdh6 plays pivotal roles in the development of the pronephros in zebrafish embryos.     

BMP signaling is required for development of the ciliary body

The ciliary body in the eye secretes aqueous humor and glycoproteins of the vitreous body and maintains the intraocular pressure. The ciliary muscle controls the shape of the lens through the ciliary zonules to focus the image onto the retina. During embryonic development, the ciliary epithelium is derived from the optic vesicle, but the molecular signals that control morphogenesis of the ciliary body are unknown. We report that lens-specific expression of a transgenic protein, Noggin, can block BMP signaling in the mouse eye and result in failure in formation of the ciliary processes. Co-expression of transgenic BMP7 restores normal development of the ciliary epithelium. Ectopic expression of Noggin also promotes differentiation of retinal ganglion cells. These results indicate that BMP signaling is required for development of the ciliary body and may also play a role in regulation of neuronal differentiation in the developing eye.     

Elfin is expressed during early heart development

Elfin (previously named CLIM1) is a protein that possesses an N-terminal PDZ domain and a C-terminal LIM domain. It belongs to the family of Enigma proteins. Enigma proteins are a family of cytoplasmic proteins that contain an N-terminal PDZ domain and a series of C-terminal LIM domains. By virtue of these two protein interacting domains, Enigma proteins are capable of protein-protein interactions. It has been proposed that Enigma proteins may act as adapters between kinases and the cytoskeleton. We have previously shown that Elfin is most abundantly expressed in the heart and it colocalizes with alpha-actinin 2 at the Z-disks of the myocardium. In this report, Elfin was shown to localize at the actin stress fibers of myoblasts, as revealed by green fluorescent protein (GFP) tagging. In situ hybridization and immunostaining showed that Elfin expression begins at an early stage in mouse development and is present throughout the developing heart. Taken together, our experimental results suggest that Elfin may play an important role in myofibrillogenesis and heart development.     

Lrrc10 is required for early heart development and function in zebrafish

Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease.     

NXT2 is required for embryonic heart development in zebrafish

Background  

NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes.     

The polycomb-group gene Ezh2 is required for early mouse development

Polycomb-group (Pc-G) genes are required for the stable repression of the homeotic selector genes and other developmentally regulated genes, presumably through the modulation of chromatin domains. Among the Drosophila Pc-G genes, Enhancer of zeste [E(z)] merits special consideration since it represents one of the Pc-G genes most conserved through evolution. In addition, the E(Z) protein family contains the SET domain, which has recently been linked with histone methyltransferase (HMTase) activity. Although E(Z)-related proteins have not (yet) been directly associated with HMTase activity, mammalian Ezh2 is a member of a histone deacetylase complex. To investigate its in vivo function, we generated mice deficient for Ezh2. The Ezh2 null mutation results in lethality at early stages of mouse development. Ezh2 mutant mice either cease developing after implantation or initiate but fail to complete gastrulation. Moreover, Ezh2-deficient blastocysts display an impaired potential for outgrowth, preventing the establishment of Ezh2-null embryonic stem cells. Interestingly, Ezh2 is up-regulated upon fertilization and remains highly expressed at the preimplantation stages of mouse development. Together, these data suggest an essential role for Ezh2 during early mouse development and genetically link Ezh2 with eed and YY1, the only other early-acting Pc-G genes.     

FGF-16 is required for embryonic heart development

Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth.     

RBFOX2 is required for establishing RNA regulatory networks essential for heart development

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.     

G2E3 is a dual function ubiquitin ligase required for early embryonic development

G2E3 is a putative ubiquitin ligase (E3) identified in a microarray screen for mitotic regulatory proteins. It shuttles between the cytoplasm and nucleus, concentrating in nucleoli and relocalizing to the nucleoplasm in response to DNA damage. In this study, we demonstrate that G2E3 is an unusual ubiquitin ligase that is essential in early embryonic development to prevent apoptotic death. This protein has a catalytically inactive HECT domain and two distinct RING-like ubiquitin ligase domains that catalyze lysine 48-linked polyubiquitination. To address in vivo function, we generated a knock-out mouse model of G2E3 deficiency that incorporates a beta-galactosidase reporter gene under control of the endogenous promoter. Animals heterozygous for G2E3 inactivation are phenotypically normal with no overt change in development, growth, longevity, or fertility, whereas G2E3 null embryos die prior to implantation. Although normal numbers of G2E3(-/-) blastocysts are present at embryonic day 3.5, these blastocysts involute in culture as a result of massive apoptosis. Using beta-galactosidase staining as a marker for protein expression, we demonstrate that G2E3 is predominantly expressed within the central nervous system and the early stages of limb bud formation of the developing embryo. In adult animals, the most intense staining is found in Purkinje cell bodies and cells lining the ductus deferens. In summary, G2E3 is a dual function ubiquitin ligase essential for prevention of apoptosis in early embryogenesis.     

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.     

BMP signaling is required for septation of the outflow tract of the mammalian heart

Bone morphogenetic proteins (BMPs) constitute a family of approximately 20 growth factors involved in a tremendous variety of embryonic inductive processes. BMPs elicit dose-dependent effects on patterning during gastrulation and gradients of BMP activity are thought to be established through regulation of the relative concentrations of BMP receptors, ligands and antagonists. We tested whether later developmental events also are sensitive to reduced levels of BMP signaling. We engineered a knockout mouse that expresses a BMP type II receptor that lacks half of the ligand-binding domain. This altered receptor is expressed at levels comparable with the wild-type allele, but has reduced signaling capability. Unlike Bmpr2-null mice, mice homozygous for this hypomorphic receptor undergo normal gastrulation, providing genetic evidence of the dose-dependent effects of BMPs during mammalian development. Mutants, however, die at midgestation with cardiovascular and skeletal defects, demonstrating that the development of these tissues requires wild-type levels of BMP signaling. The most striking defects occur in the outflow tract of the heart, with absence of septation of the conotruncus below the valve level and interrupted aortic arch, a phenotype known in humans as persistent truncus arteriosus (type A4). In addition, semilunar valves do not form in mutants, while the atrioventricular valves appear unaffected. Abnormal septation of the heart and valve anomalies are the most frequent forms of congenital cardiac defects in humans; however, most mouse models display broad defects throughout cardiac tissues. The more restricted spectrum of cardiac anomalies in Bmpr2(deltaE2) mutants makes this strain a key murine model to understand the embryonic defects of persistent truncus arteriosus and impaired semilunar valve formation in humans.