MOLECULAR GENETICS OF EMBRYOGENESIS IN XENOPUS AND ZEBRAFISH
, Head, Section on Developmental Biology
Reiko Toyama, PhD, Staff Scientist
Sung-Kook Hong, PhD, Visiting Research Fellow
Emil Aamar, PhD, Visiting Fellow
Sunit Dutta, PhD, Visiting Fellow
Hyunju Ro, PhD, Visiting Fellow
Steven Sperber, PhD, Visiting Fellow
Kosuke Tanegashima, PhD, Visiting Fellow
Hui Zhao, PhD, Visiting Fellow
Martha Rebbert, BS, Senior Technician
Mark Rath, MFSc, Technician
Nupur Jhawar, Student1
Using the frog Xenopus laevis and the zebrafish Danio rerio as experimental systems, the laboratory engages in studies of the molecular-genetic mechanisms of early vertebrate development. Recently, we focused on gene discovery through forward genetics, DNA microarray technology, and expression profiling to identify genes that play a role in the regulation of embryonic development. These approaches have led to studies on mechanisms of gastrulation, neural crest specification, and brain patterning during early embryogenesis.
Analysis of gene expression in Xenopus development by DNA microarray technology
Developmental processes are associated with large-scale changes in gene expression. These changes both result from developmental events and drive the specification and subsequent differentiation of cells and tissues in the embryo. DNA microarray technology provides the opportunity to analyze changes in gene expression on a large scale. The Xenopus embryo has long been a premier model system for studying vertebrate development, and we carried out a project to apply microarray technology to this system. We compared RNA populations in different regions of the gastrula embryo obtained by microdissection and in explants treated with different signaling factors. We are studying selected genes, whose expression differs in the various samples, for their function in embryogenesis.
Convergent extension during Xenopus gastrulation is regulated by the WNT-PCP pathway
The Wnt-PCP pathway regulates cell polarity and convergent extension movements during axis formation in Xenopus. With Rho and Rac activated by Wnt stimulation in the embryo, we predicted the involvement of at least one guanine exchange factor (GEF) in Wnt-PCP signaling during convergent extension. Using a microarray-based screen for notochord-enriched genes, we identified a GEF with sequence similarity to human WGEF. We found that Xenopus WGEF is involved in Wnt-regulated convergent extension movements, which are critical for the progress of gastrulation. The expression of WGEF begins at the onset of gastrulation and becomes predominant in the notochord during subsequent stages. WGEF protein colocalized with Dishevelled in cells of the Xenopus gastrula, and overexpression of WGEF activated RhoA—but not Rac1 or Cdc42—in mammalian cells or frog embryos. Depletion of WGEF led to suppression of convergent extension in explants from Xenopus embryos, which could be rescued by Rho activation. WGEF protein can bind to Dishevelled and Daam-1, components of the Wnt-PCP pathway. Epistatic analysis placed WGEF below Wnt and Dishevelled in the pathway. Binding studies identified the Dishevelled-binding domain in WGEF as an autoinhibitory domain, suggesting a mechanism for signal transduction. Our results indicate that WGEF is a component of the Wnt-PCP pathway that connects Rho activation with signal transduction through Dishevelled.
In a related project, we investigated the role of profilin in convergent extension. Profilin is an effector of Daam-1 in the regulation of cytoskeletal changes in response to Wnt-PCP signaling in the Xenopus embryo.
Sato A, Khadka DK, Liu W, Bharti R, Runnels LW, Dawid IB, Habas R. Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation. Development 2006; 133:4219-31.
XLRIG3, a novel organizer-specific gene, is required for the differentiation of neural crest
We identified XLRIG3, a member of the leucine-rich repeats and immunoglobulin-like domains family, within the microarray analysis described above by comparing RNA from different regions of the gastrula embryo. XLRIG3 is a maternal factor whose expression increases during early gastrulation, with a preferential localization in the Spemann-Mangold organizer region. Later, XLRIG3 is expressed in the entire neural plate during neurulation and becomes localized in the developing neural crest during tailbud stages. Knockdown of XLRIG3 by an antisense morpholino impairs neural crest formation, as visualized by several marker genes. XLRIG3 exerts a complex effect on signaling by certain receptor tyrosine kinases (RTKs), such as the FGF and EGF receptors. XLRIG3 represents a novel factor required in neural crest development that may play an additional role in other processes in early development.
A mutation in the Mediator component Med12/TRAP230 in zebrafish
Mediator is a complex of many proteins that mediates the transcriptional effect of sequence-specific factors such as nuclear hormone receptors or homeodomain proteins to RNA polymerase and associated factors. We identified a mutation of the gene encoding the Mediator component MED12/ TRAP230 and showed that it affects the development of several systems in zebrafish embryogenesis. We named the locus kohtalo (kto) after the homologous locus in Drosophila. More recently, we analyzed the role of the kto gene in gut development in collaboration with Didier Stanier. In addition, we demonstrated that the kto gene is required for normal organization of rhombomeres in the hindbrain by processes that are currently under study.
Hong SK, Haldin CE, Lawson ND, Weinstein BM, Dawid IB, Hukriede NA. The zebrafish kohtalo/trap230 gene is required for the development of the brain, neural crest, and pronephric kidney. Proc Natl Acad Sci USA 2005;102:18473-8.
MyoS, a transcription factor involved in muscle development in Xenopus
Using microarray analysis, we identified Myoskeletin as a gene induced by activin in animal cap explants of Xenopus. The gene encodes a protein related to the transcription factor Myocardin. Whereas Myocardin is expressed in the heart and is known to be involved in heart and smooth muscle formation, Myoskeletin is expressed in the somites and in hypaxial muscle precursors as they migrate from the somites during tadpole stages. Myoskeletin is required for hypaxial muscle formation; reduction of its expression through injection of an antisense morpholino oligonucleotide leads to suppression of hypaxial muscle formation. Overexpression experiments in animal caps demonstrated that Myoskeletin is capable of inducing several genes, including skeletal muscle–, cardiac muscle–, and smooth muscle–specific genes. We concluded that Myoskeletin is a somite- and hypaxial muscle–specific member of the Myocardin family that is required for hypaxial muscle formation. Paul Krieg and colleagues have independently identified Myoskeletin and studied its function in the Xenopus embryo.
Zhao H, Rebbert ML, Dawid IB. Myoskeletin, a factor related to Myocardin, is expressed in somites and required for hypaxial muscle formation in Xenopus. Int J Dev Biol 2007;51:315-20.
Pineal-specific gene expression in zebrafish
The role of the pineal gland in biological rhythm is an important topic discussed more fully by David Klein elsewhere in this volume. We are involved in a collaborative study of gene expression in the pineal gland in zebrafish. Taking advantage of the specific expression pattern of the enzyme Aanat2, we generated transgenic zebrafish in which green fluorescent protein (GFP) is expressed with high specificity in the pineal gland, based on the analysis of the genomic control regions responsible for the gene encoding Aanat2. We used the transgenic zebrafish line to guide dissection of the pineal gland from embryonic, larval, and adult zebrafish in order to apply the DNA microarray method to analyzing global transcription patterns at different stages of development. We used RNA from brain from which the pineal gland was removed to compare RNA samples from 3-day, 5-day, and 10-day zebrafish embryos; 3-month young adult zebrafish; and 1- to 2-year mature adult zebrafish. We obtained day and night samples at each time point. The resulting experimental results allowed evaluation of developmental changes in the pineal gland, comparison of differences in gene expression in the pineal gland and the brain, and comparison of diurnal differences. While the analysis of the data set is in progress, it is already clear that several genes are expressed differentially in the pineal and the brain. Several of these differentially expressed genes are under further study.
Ziv L, Levkovitz S, Toyama R, Falcon J, Gothilf Y. Functional development of the zebrafish pineal gland: light-induced expression of period2 is required for onset of the circadian clock. J Neuroendocrinol 2005;17:314-20.
Zebrafish barx1 is required for pharyngeal arch patterning
The Barx1 transcription factor has previously been shown to modulate cellular adhesion molecules and to participate in specification of tooth type. To determine the role of barx1 in zebrafish head formation, we examined the gene’s expression during embryogenesis and performed a functional analysis by microinjecting targeted antisense morpholino oligonucleotides to attenuate its translation. Barx1 is expressed in the cranial neural crest, pharyngeal arches, anterior aspect of pectoral fin buds, and gut wall. By 2.5 days post-fertilization, barx1 morpholino–injected embryos exhibited developmental delay exemplified by poor facial outgrowth and micrognathia. Histological analysis and labeling of cell membranes revealed reductions in differentiation and chondrocyte condensation within the arches. Affected larvae stained with Alcian blue exhibited small and dysmorphic arch cartilage elements, and expression of chondrogenic markers such as dlx2a and col2a1 was perturbed. Signaling factors in the bone morphogenetic protein, fibroblast growth factor, and endothelin families control the expression of barx1, as seen in bead implantation experiments. The results suggest a role for barx1 at early stages of chondrogenesis within the pharyngeal arches during zebrafish development.
Protocadherin 18 plays a role in cell behavior during zebrafish embryogenesis
Protocadherin-18 (Pcdh18) belongs to the d2-Protocadherins, which constitute the largest subgroup within the cadherin superfamily. We isolated a full-length zebrafish cDNA for zebrafish pcdh18, which is expressed in a complex and dynamic pattern in the nervous system from gastrula stages onward, with less expression in mesodermal derivatives. Overexpression of pcdh18 in embryos caused cyclopia, mis-localization of hatching gland tissue, and duplication or splitting of the neural tube. Reduction of Pcdh18 expression by use of a morpholino led to delayed epiboly and shortened axis. Using cell transplantation, we showed that overexpression of pcdh18 causes diminished cell migration and reduced cell protrusions, resulting in a tendency of cells to stay more firmly aggregated, probably owing to increased cell adhesion. We suggest a role for pcdh18 in cell adhesion, migration, and behavior, but not in cell specification, during the gastrula and segmentation stages of development.
Analysis of blood and vascular biogenesis in zebrafish
A collaboration continued with Atsuo Kawahara to characterize genes with a role in hematopoiesis in zebrafish. One project involves the gene encoding the sixth heme synthesis enzyme coproporphyrinogen oxidase (CPO) from zebrafish. Using antisense morpholino analysis, we showed that reduction in CPO leads to a significant suppression of hemoglobin production without apparent reduction of blood cells. Further, we isolated and characterized the genes from zebrafish that encode uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO).
The second project concerns the novel factor VAP (Vascular Associated Protein). The vap gene is predominantly expressed in the intermediate cell mass, the site of primitive erythropoiesis and vasculogenesis, and is subsequently enriched in endothelial cells. Knockdown of VAP using antisense morpholinos resulted in decreased numbers of erythrocytes and suppression of hemoglobin production. Further, VAP knockdown caused disorganization of intersegmental vessels. We suggest that VAP plays an important role in the maturation of endothelial and erythroid cells in zebrafish.
Hanaoka R, Dawid IB, Kawahara A. Cloning and expression of zebrafish genes encoding the heme synthesis enzymes uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO). DNA Seq 2007;18:54-60.
Hanaoka R, Katayama S, Dawid IB, Kawahara A. Characterization of the heme synthesis enzyme coproporphyrinogen oxidase (CPO) in zebrafish erythrogenesis. Genes Cells 2006;11:293-303.
1 Left the group during reporting period.
COLLABORATORS
Jonathan Epstein, MS, Unit on Biologic Computation, NICHD, Bethesda, MD
Yoav Gothilf, PhD, Tel Aviv University, Tel Aviv, Israel
Raymond Habas, PhD, Robert Wood Johnson School of Medicine, Piscataway, NJ
Neil Hukriede, PhD, University of Pittsburgh, Pittsburgh, PA
Atsuo Kawahara, PhD, University of Kyoto, Kyoto, Japan
David Klein, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Keiko Ozato, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Didier Stanier, PhD, University of California San Francisco, San Francisco, CA
Brant Weinstein, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
For further information, contact idawid@nih.gov.
