ABSTRACT
Xenopus is used as a model animal for investigating the inductive events and organogenesis that occur during early vertebrate development. Given that they are easy to obtain in high numbers and are relatively large in size, Xenopus embryos are excellent specimens for performing manipulations such as microinjection and microsurgery. The animal cap, which is the area around the animal pole of the blastula, is destined to form the ectoderm during normal development. However, these cells retain pluripotentiality and upon exposure to specific inducers, the animal cap can differentiate into neural, mesodermal, and endodermal tissues. In this sense, the cells of the animal cap are equivalent to mammalian embryonic stem cells. In this unit, the isolation and differentiation of animal cap cells, the so-called animal cap assay, is described. Useful methods for analyzing the mechanism of animal cap differentiation at the molecular level are also described.
Subject(s)
Blastula/cytology , Cell Differentiation , Cell Separation , Animals , Cytological Techniques , Embryo, Nonmammalian , Models, Animal , Pluripotent Stem Cells/cytology , XenopusABSTRACT
Bone morphogenetic proteins (BMPs) regulate multiple biological processes, including cellular proliferation, adhesion, differentiation, and early development. In Xenopus development, inhibition of the BMP pathway is essential for neural induction. Here, we report that dullard, a gene involved in neural development, functions as a negative regulator of BMP signaling. We show that Dullard promotes the ubiquitin-mediated proteosomal degradation of BMP receptors (BMPRs). Dullard preferentially complexes with the BMP type II receptor (BMPRII) and partially colocalizes with the caveolin-1-positive compartment, suggesting that Dullard promotes BMPR degradation via the lipid raft-caveolar pathway. Dullard also associates with BMP type I receptors and represses the BMP-dependent phosphorylation of the BMP type I receptor. The phosphatase activity of Dullard is essential for the degradation of BMP receptors and neural induction in Xenopus. Together, these observations suggest that Dullard is an essential inhibitor of BMP receptor activation during Xenopus neuralization.
Subject(s)
Bone Morphogenetic Protein Receptors, Type II/metabolism , Bone Morphogenetic Protein Receptors, Type I/metabolism , Nerve Tissue Proteins/physiology , Neurons/metabolism , Ubiquitin/metabolism , Xenopus Proteins/physiology , Animals , Bone Morphogenetic Protein Receptors, Type I/genetics , Bone Morphogenetic Protein Receptors, Type II/genetics , Bone Morphogenetic Proteins , Caveolin 1/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Immunoblotting , Immunoprecipitation , Membrane Microdomains , Mutation , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/genetics , Neurons/cytology , Oligodeoxyribonucleotides, Antisense/pharmacology , Phosphoprotein Phosphatases , Phosphorylation , Plasmids , Polymerase Chain Reaction , Signal Transduction , Transcription Factors/metabolism , Xenopus Proteins/antagonists & inhibitors , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Xenopus laevis/genetics , Xenopus laevis/metabolismABSTRACT
Vertebrates use two or three forms of kidney successively during development and the nephric duct is essential for this succession of kidney induction. While transcripts of many Wnt ligands and Wnt receptor Frizzled genes have been localized in developing kidney, the relationship between Wnt signaling and nephric duct development remains unknown. This study investigated the role of Xenopus frizzled-8 (Xfz8) in pronephros development. Translational inhibition of Xfz8 caused a significant reduction in the staining of a duct-specific antibody, but did not affect the expression of early pronephric maker genes in the duct region. Defects in pronephric tubule branching were also observed following inhibition of Xfz8. Histological analysis revealed that the Xfz8-inhibited cells failed to form a normal epithelium structure. These results suggest that Xfz8 is involved in the process of normal epithelium formation in the developing pronephric duct and tubules after specification.
Subject(s)
Kidney/embryology , Kidney/metabolism , Receptors, Cell Surface/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/metabolism , Animals , Biomarkers/analysis , Female , Immunohistochemistry , In Situ Hybridization , Kidney/abnormalities , Kidney/cytology , Morphogenesis , Protein Biosynthesis , Proto-Oncogene Proteins/metabolism , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Signal Transduction , Wnt Proteins , Xenopus Proteins/biosynthesis , Xenopus Proteins/genetics , Xenopus laevis/embryologyABSTRACT
In a screen for genes expressed in neural tissues and pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of NLI-IF (Nuclear LIM Interactor-Interacting Factor), a protein whose function is not yet characterized. Dullard mRNA was maternally derived and localized to the animal hemisphere. At neurula stages, the expression was in neural regions and subsequently localized to neural tissues, branchial arches, and pronephroi. Using antisense morpholino oligonucleotide-mediated inhibition, we showed that dullard was required for neural development. The translational knock-down of dullard resulted in failure of neural tube development and the embryos consequently showed a reduction of head development. Expression of neural marker genes in dullard-inhibited embryos was also suppressed. These results suggest that dullard is necessary for neural development.
