Dkk3 is required for TGF-beta signaling during Xenopus mesoderm induction

Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.     

The extracellular adenosine deaminase growth factor, ADGF/CECR1, plays a role in Xenopus embryogenesis via the adenosine/P1 receptor

Adenosine deaminase-related growth factors (ADGF), also known as CECR1 in vertebrates, are a novel family of growth factors with sequence similarity to classical cellular adenosine deaminase. Although genes for ADGF/CECR1 have been identified in both invertebrates as well as vertebrates, their in vivo functions in vertebrates remain unknown. We isolated cDNA clones for two cerc 1s from Xenopus laevis. Both recombinant Xenopus CECR1s exhibited adenosine deaminase and growth factor activity, and the adenosine deaminase activity was found to be indispensable for growth factor activity. The Xenopus cerc 1s are expressed in the somites, pronephros, eyes, cement gland, neural tube, and neural floor plate of the embryos. Knock-down of these two genes using morpholino oligonucleotides caused a reduction in the body size and abnormalities of the body axis in the Xenopus embryos, accompanied by selective changes in the expression of developmental marker genes. Injection of adenosine, agonists for adenosine/P1 receptors, or adenosine deaminase inhibitor into late gastrula archenteron embryos resulted in developmental defects similar to those caused by morpholino oligonucleotide injection. These results show, for the first time, the involvement of CECR1s via the adenosine/P1 receptors in vertebrate embryogenesis via regulation of extracellular adenosine concentrations.     

Identification and characterization of Xenopus kctd15, an ectodermal gene repressed by the FGF pathway

The FGF pathway regulates a variety of developmental processes in animals through activation and/or repression of numerous target genes. Here we have identified a Xenopus homolog of potassium channel tetramerization domain containing 15 (KCTD15) as an FGF-repressed gene. Kctd15 expression is first detected at the gastrula stage and gradually increases until the tadpole stage. Whole-mount in situ hybridization reveals that the spatial expression of kctd15 is tightly regulated during early embryogenesis. While kctd15 is uniformly expressed throughout the presumptive ectoderm at the early gastrula stage, its expression becomes restricted to the non-neural ectoderm and is excluded from the neural plate at the early neurula stage. At the mid-neurula stage, kctd15 shows a more restricted distribution pattern in regions that are located at the anterior, lateral or medial edge of the neural fold, including the preplacodal ectoderm, the craniofacial neural crest and the prospective roof plate. At the tailbud stage, kctd15 expression is mainly detected in neural crest- or placode-derived tissues that are located around the eye, including the mandibular arch, trigeminal ganglia and the olfactory placode. FGF represses kctd15 expression in ectodermal explants, and the inhibition of FGF receptor with a chemical compound dramatically expands the region expressing kctd15 in whole embryos. Dorsal depletion of kctd15 in Xenopus embryos leads to bent axes with reduced head structures, defective eyes and abnormal somites, while ventral depletion causes defects in ventral and caudal morphologies. These results suggest that kctd15 is an FGF-repressed ectodermal gene required for both dorsal and ventral development.     

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

Directionality of heart looping: effects of Pitx2c misexpression on flectin asymmetry and midline structures

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating head formation in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of beta-catenin. Thus, the IGF pathway is required for head formation during embryogenesis.     

Ccm1 is required for arterial morphogenesis: implications for the etiology of human cavernous malformations

Hemorrhagic stroke is a significant cause of morbidity and mortality in children, and is frequently associated with intracranial vascular malformations. One prevalent form of these vascular malformations, cerebral cavernous malformation, is characterized by thin-walled vascular cavities that hemorrhage and has been linked to loss-of-function mutations in CCM1. The neural and epithelial expression of CCM1 in adulthood suggests that cavernous malformations may be the result of primary neural defects. In this study, we generated mice lacking Ccm1 and demonstrate that Ccm1 is ubiquitously expressed early in embryogenesis and is essential for vascular development. Homozygous mutant embryos die in mid-gestation and the first detectable defects are exclusively vascular in nature. The precursor vessels of the brain become dilated starting at E8.5, reminiscent of the intracranial vascular defects observed in the human disease. In addition, there is marked enlargement and increased endothelial proliferation of the caudal dorsal aorta, as well as variable narrowing of the branchial arch arteries and proximal dorsal aorta. These vascular defects are not secondary to primary neural defects, as neural morphology and marker expression are normal even subsequent to the onset of vascular pathology. The defects in the vascular structure of embryos lacking Ccm1 are associated with early downregulation of artery-specific markers, including the Efnb2- and Notch-related genes. Finally, consistent with the murine data, we found that there is an analogous reduction in Notch gene expression in arterioles from humans with mutations in CCM1. Our studies suggest that cavernous malformations result from primary vascular rather than neural defects.     

Misexpression of the RNA-binding protein ELRB in Xenopus presumptive neurectoderm induces proliferation arrest and programmed cell death.

Proteins of the ELAV/Hu family share the presence of three RNA binding domains. In Xenopus, three nervous system-specific elav/Hu related genes, elrB, elrC and elrD, have been identified so far. The temporally regulated expression patterns of elrB, elrC and elrD suggest their involvement at different steps of neural differentiation. In the present study we misexpressed elrB by RNA injection in early Xenopus embryos and analyzed morphologically and molecularly its effects on neural development. We showed that heterochronous expression of elrB in presumptive neurectoderm down-regulates the expression of neural markers, such as N-tubulin, as well as that of other Xenopus elav-like genes, elrC and elrD, whereas ectopic expression of elrB in presumptive mesoderm has no effect on MyoD. Misexpression of elrB also induces severe defects in neural tube development, associated with massive cell loss resulting from early cell cycle arrest and programmed cell death. Our results are discussed in the context of early neural differentiation.     

Cyclin E2 is required for embryogenesis in Xenopus laevis

In mammalian cells, E-type cyclins (E1 and E2) are generally believed to be required for entry into S phase. However, in mice, cyclin E is largely dispensable for normal embryogenesis. Moreover, Drosophila cyclin E plays a critical role in cell fate determination in neural lineages independently of proliferation. Thus, the functions of cyclin E, particularly during early development, remain elusive. Here, we investigated the requirement for E-type cyclins during Xenopus embryogenesis. Although cyclin E1 has been reported as a maternal cyclin, inhibition of its translation in the embryo caused no serious defects. We isolated a Xenopus homologue of human cyclin E2, which was zygotically expressed. Sufficient inhibition of its expression led to death at late gastrula, while partial inhibition allowed survival. These observations indicate distinct roles for Xenopus cyclins E1 and E2, and an absolute requirement of cyclin E2 for Xenopus embryogenesis.     

Degradation of hyaluronan by a Hyal2-type hyaluronidase affects pattern formation of vitelline vessels during embryogenesis of Xenopus laevis.

A Hyal2-type hyaluronidase of Xenopus laevis (Xhyal2) was characterized by molecular cloning, biochemical analysis and ectopic overexpression in embryos. When expressed in Xenopus oocytes, Xhyal2 exists as a soluble protein in the extracellular space and in intercellular compartments as well as being attached to the cell surface through a glycosyl-phosphatidyl-inositol anchor. This enzyme specifically degrades hyaluronan not only at acidic pH values but more slowly also under physiological conditions. Xhyal2 is differentially expressed during embryogenesis. Particularly striking is the high level of expression in the developing brain, the head mesenchyme and the pronephros. Elevated levels of mRNA were also found in endothelial cells which will later form vascular structures. Ectopic overexpression of Xhyal2 in frog embryos causes loss of hyaluronan in the cellular environment. This causes severe defects in the assembly of the highly structured plexus of the vitelline vessels from prevascular endothelial cells. Our data support the notion that the level of Xhyal2 expression determines the organization of the extracellular environment so that cells can merge and/or migrate within an originally impenetrable matrix.     

Distinct roles for Distal-less genes Dlx3 and Dlx5 in regulating ectodermal development in Xenopus

In vertebrates, there are six or more copies of genes related to the Drosophila pattern formation homeodomain gene Distal-less. Among this family, Dlx3 and Dlx5 share extensive sequence homology and have similar, but distinctive, expression patterns, suggesting that these two factors may have substantially redundant developmental functions. Here we show that at the earliest phases of embryogenesis in Xenopus, there are significant differences between Dlx3 and Dlx5 expression and that this correlates with different functions in the restriction of neural crest and neural plate boundaries, respectively.     

Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse.

Integrins alpha6beta1 and alpha6beta4 are cell surface receptors for laminins. Integrin alpha6-null mice die at birth with severe skin blistering and defects in the cerebral cortex and in the retina. Integrin alpha3beta1 can associate with laminins and other ligands. Integrin alpha3-null mice also die at birth, with kidney and lung defects at late stages of development, and moderate skin blistering. To investigate possible overlapping functions between alpha3 and alpha6 integrins, we analyzed the phenotype of compound alpha3-/-/alpha6-/- mutant embryos. Double homozygous mutant embryos were growth-retarded and displayed several developmental defects not observed in the single mutant animals. First, limb abnormalities characterized by an absence of digit separation and the fusion of preskeletal elements were observed. Further analyses indicated a defect in the apical ectodermal ridge, an essential limb organizing center. In the double mutant, the ridge appeared flattened, and ridge cells did not show a columnar morphology. A strong reduction in ridge cell proliferation and alterations of the basal lamina underlying the ectoderm were observed. These results suggest that alpha3 and alpha6 integrins are required for the organization or compaction of presumptive apical ectodermal ridge cells into a distinct differentiated structure. Additional defects were present: an absence of neural tube closure, bilateral lung hypoplasia, and several abnormalities in the urogenital tract. Finally, an aggravation of brain and eye lamination defects was observed. The presence of novel phenotypes in double mutant embryos demonstrates the synergism between alpha3 and alpha6 integrins and their essential roles in multiple processes during embryogenesis.     

Gadd45a and Gadd45g regulate neural development and exit from pluripotency in Xenopus

Gadd45 genes encode a small family of multifunctional stress response proteins, mediating cell proliferation, apoptosis, DNA repair and DNA demethylation. Their role during embryonic development is incompletely understood. Here we identified Xenopus Gadd45b, compared Gadd45a, Gadd45b and Gadd45g expression during Xenopus embryogenesis, and characterized their gain and loss of function phenotypes. Gadd45a and Gadd45g act redundantly and double Morpholino knock down leads to pleiotropic phenotypes, including shortened axes, head defects and misgastrulation. In contrast, Gadd45b, which is expressed at very low levels, shows little effect upon knock down or overexpression. Gadd45ag double Morphants show reduced neural cell proliferation and downregulation of pan-neural and neural crest markers. In contrast, Gadd45ag Morphants display increased expression of multipotency marker genes including Xenopus oct4 homologs as well as gastrula markers, while mesodermal markers are downregulated. The results indicate that Gadd45ag are required for early embryonic cells to exit pluripotency and enter differentiation.