Igor B. Dawid, PhD, Head, Section on Developmental Biology
Reiko Toyama, PhD, Staff Scientist
Sung-Kook Hong, PhD, Research Fellow
Emil Aamar, PhD, Visiting Fellow
Sunit Dutta, PhD, Visiting Fellow 1
Hyunju Ro, PhD, Visiting Fellow 1
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, Student 2
Natalia Machuca, Student 1
The laboratory is engaged in studies of the molecular-genetic mechanisms of early vertebrate development, using the frog Xenopus laevis and the zebrafish Danio rerio as experimental systems.
Application of DNA microarray technology to Xenopus development
Zhao, Tanegashima, Rebbert, Dawid
Developmental processes are associated with large-scale changes in gene expression. These changes are the result of developmental events, but the changes also drive the specification and subsequent differentiation of cells and tissues in the embryo. DNA microarray technology has provided an opportunity to analyze these changes in gene expression on a large scale. The Xenopus embryo has long been a premier model system for studying vertebrate development, and we have carried out a project that applied microarray technology to this system. We compared the RNA populations of ectodermal explants induced with activin with control explants, and we compared RNAs in different regions of the gastrula embryo obtained by microdissection. Selected genes whose expression differs in different samples are undergoing study for their function in embryogenesis.
WGEF activates Rho in the WNT-PCP pathway and controls convergent extension during Xenopus gastrulation
Tanegashima, Zhao, Dawid
The Wnt-PCP pathway regulates cell polarity and convergent extension movements during axis formation in Xenopus. Given that Rho and Rac are activated by Wnt stimulation in the embryo, we predicted the involvement of at least one 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 that 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 co-localized with F-actin in dorsal cells of the Xenopus gastrula while in 293T cells overexpression of WGEF activated RhoA but not Rac1 or Cdc42. 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. The results indicate that WGEF is a component of the Wnt-PCP pathway that constitutes the previously missing link connecting Rho activation with signal transduction through Dishevelled.
XLRIG3, a novel organizer-specific gene, modulates RTK signaling during early embryonic development
Zhao, Tanegashima, Dawid
By comparing RNA from Spemann-Mangold organizer explants with RNA from other regions of the early gastrula embryo, the microarray analysis described above identified XLRIG3, a member of the family of leucine-rich repeats and immunoglobulin-like domains. 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 in complex dorsal structures during tailbud stages. Blocking BMP signaling by either Chordin or truncated BMP receptor induces XLRIG3 while Wnt, nodal, and FGF signaling have little effect on XLRIG3's expression in animal cap explants. Overexpression of XLRIG3 severely affects mesodermal patterning, induces tail protrusions, and inhibits the expression of other organizer-specific genes such as gsc and Chordin. Knockdown of XLRIG3 by an antisense morpholino impairs brain and neural crest formation. XLRIG3 affects signaling by certain receptor tyrosine kinases (RTKs), such as the FGF and EGF receptors, in a complex way. XLRIG3 can physically interact with all four members of the ErbB family, which includes the EGR receptor, as well as with the Xenopus FGF receptor FGFR1. Our studies indicate that XLRIG3 is a transmembrane modulator of RTK signaling with diverse roles during early embryonic development.
A zebrafish mutant that impairs ribosomal RNA maturation
Toyama, Dawid; in collaboration with Azuma
A majority of the mutants studied in zebrafish developmental genetics to date affect patterning in the embryo, but it also seems interesting to apply the power of genetic analysis to basic cellular processes. Such an opportunity arose through the analysis of a mutant named bap28, which affects the brain and leads to malformations and widespread apoptosis by 24 hours of development. Positional cloning showed that the gene responsible for this mutation encodes a large protein similar to the human protein BAP28, which had not been characterized. However, the apparent orthologue in yeast, called UTP10, is known to be a component of a nucleolar ribonucleoprotein complex involved in the maturation of 18S ribosomal RNA (rRNA). Thus, we studied rRNA processing in normal and mutant zebrafish. As the mechanism of rRNA processing in zebrafish was unknown, we generated a basic outline of the processing pathway, which is similar but not identical to that in Xenopus. We found that bap28 mutant embryos are deficient in their ability to accumulate 18S rRNA precursors, thereby supporting the view that the mutation affects rRNA processing. By manipulating the expression levels of p53, we could show that the apoptotic pathway triggered by the bap28 mutation involves p53. It is notable that the mutation affects a discrete region of the embryo, even though it disrupts a basic cellular process.
A mutation in the Mediator component TRAP230 affects zebrafish development
Hong, Dawid; in collaboration with Hukriede, Weinstein
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 TRAP230/MED12 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. Homozygous kto mutant zebrafish embryos show defects in brain, neural crest, and kidney development and die about six days after fertilization. In the affected tissues, differentiation is initiated and many cell type-specific genes are expressed, but there is a failure of morphogenesis and failure to complete differentiation. The results suggest that critical targets of TRAP230 function may include proteins important for cell mobility, cell sorting, and tissue assembly.
Pineal-specific gene expression in zebrafish
Toyama, Machuca, Dawid; in collaboration with Gothilf, Klein
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 of the 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 close-to-perfect specificity in the pineal gland. The genomic control regions responsible for the specificity have been analyzed and defined. Using such a control region, we are currently attempting to generate a transgenic zebrafish line in which fluorescence of the pineal gland will reflect the day-night cycle of the underlying gene expression. Further, we used the existing transgenic zebrafish line to guide dissection of the pineal gland from adult and larval zebrafish, thereby permitting analysis of global transcription patterns at different stages of development (with the DNA microarray method). Extensive results from these experiments are currently undergoing analysis and evaluation, although it is already clear that we can identify numerous genes that are specifically expressed in the pineal as compared with the rest of the brain. Moreover, the genes change their expression levels within the pineal gland during development and show changes of expression between day and night. The results should aid in understanding the development and function of this important organ.
Zebrafish Barx1 is required for pharyngeal arch patterning
Sperber, Dawid
The Barx1 transcription factor has previously been shown to modulate cellular adhesion molecules and participate in specification of tooth type. To determine the role of barx1 in zebrafish head formation, we examined its 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, the pharyngeal arches, the anterior aspect of the pectoral fin buds, and the gut wall. In addition, transient expression is observed in the posterior lateral line ganglia and developing tail. During early development of the pharyngeal arches (24-32 hours post-fertilization), embryos microinjected with barx1 morpholinos appear normal as indicated by examination of fli1-gfp transgenic fish and neural crest markers that include crestin and dlx2a. By 2.5 days post-fertilization, barx1 morpholino-injected embryos exhibit developmental delay as 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 exhibit small and dysmorphic arch cartilage elements, and expression of chondrogenic markers such as dlx2a and col2a1 is perturbed. These results suggest a role for barx1 at early stages of chondrogenesis within zebrafish pharyngeal arches.
Analysis of heme biosynthesis in zebrafish
Dawid; in collaboration with Kawahara
A collaboration continued with Atsuo Kawahara to characterize genes with a role in hematopoiesis in zebrafish. We isolated the gene encoding coproporphyrinogen oxidase (CPO), the sixth heme synthesis enzyme, from zebrafish. This gene is predominantly expressed in the intermediate cell mass (ICM), the major site of primitive hematopoiesis in zebrafish. Knockdown of zebrafish CPO using antisense morpholinos (CPO-MO) led to a significant suppression of hemoglobin production without apparent reduction of blood cells. Injection of human CPO RNA, but not a mutant CPO RNA, which is similar to a mutant responsible for a hereditary coproporphyria (HCP), restored hemoglobin production in the CPO-MO-injected embryos. The function of CPO in heme biosynthesis is apparently conserved between zebrafish and human, suggesting that CPO-MO-injected zebrafish embryos might be a useful in vivo assay system to measure the biological activity of human CPO mutations.
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 Joined the laboratory during reporting period.
2 Left the laboratory during reporting period.
COLLABORATORS
Mizuki Azuma, PhD, Experimental Transplantation and Immunology Branch, NCI, Bethesda, MD
Yoav Gothilf, PhD, Tel Aviv University, Tel Aviv, Israel
Neil Hukriede, PhD, University of Pittsburgh, Pittsburgh, PA
Atsuo Kawahara, PhD, University of Kyoto, Kyoto, Japan
David Klein, PhD, Laboratory of Developmental Neurobiology, NICHD, Bethesda, MD
Brant Weinstein, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda, MD
For further information, contact idawid@nih.gov.