Molecular Genetics of Embryogenesis in Xenopus and Zebrafish

Igor B. Dawid, PhD, Head, Section on Developmental Biology
Reiko Toyama, PhD, Staff Scientist
Emil Aamar, PhD, Postdoctoral Fellow
Sunit Dutta, PhD, Postdoctoral Fellow
Sung-Kook Hong, PhD, Postdoctoral Fellow¹
Hyunju Ro, PhD, Postdoctoral Fellow
Steven Sperber, PhD, Postdoctoral Fellow
Kosuke Tanegashima, PhD, Postdoctoral Fellow¹
Hui Zhao, PhD, Postdoctoral Fellow¹
Martha Rebbert, BS, Senior Technician
John Gonzales, MA, MS, Technician

The laboratory uses the frog Xenopus laevis and the zebrafish Danio rerio as experimental systems to study molecular-genetic mechanisms of early vertebrate development. Recently, we focused on gene discovery by forward genetics, DNA microarray technology, and expression profiling in order 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. We are particularly interested in the development of the neural crest and its important derivatives, the pharyngeal arches.

WGEF is a component of the WNT-PCP pathway involved in regulating 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 Wnt stimulation in the embryo activates Rho and Rac, the involvement of at least one GEF² in Wnt-PCP signaling during convergent extension has been predicted. Using a microarray-based screen for notochord-enriched genes, we identified a GEF with sequence similarity to human WGEF³. 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 co-localized with Dishevelled in cells of the Xenopus gastrula, and overexpression of WGEF activated RhoA, but not Rac1 or Cdc42, in mammalian cells and 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 auto-inhibitory 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.

Tanegashima K, Zhao H, Dawid IB. WGEF activates Rho in the Wnt-PCP pathway and controls convergent extension in Xenopus gastrulation. EMBO J 2008;27:606-617.

Xenopus Lrig3, a novel organizer-specific gene, is required for the differentiation of neural crest

Zhao, Tanegashima, Ro, Dawid

We identified Lrig3, 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. We focused on Lrig3, a predicted transmembrane protein that is expressed maternally. Later, Lrig3 is expressed in the entire neural plate during neurulation and becomes localized in the developing neural crest during tailbud stages. Knockdown of lrig3 by an antisense morpholino impairs neural crest formation, as visualized by several marker genes. In the neural crest induction assay involving Chordin plus Wnt3a-injected animal caps, Lrig3 morpholino inhibited expression of slug, sox9, and foxD3 but not of pax3 and zic1. In line with this observation, lrig3 knockdown prevented marker induction bypax3 and zic1, suggesting that Lrig3 acts downstream of these two genes in neural crest formation. Injection of Lrig3 and Wnt3a led to low-level induction of neural crest markers and enhanced induction of Fgf3, Fgf4, and Fgf8 in animal caps, suggesting a positive role for Lrig3 in Wnt signaling. Lrig3 could attenuate Fgf signaling in animal caps, interacted with Fgf receptor 1 in cultured cells, and, according to context, reduced or augmented the induction of neural crest markers by Fgf. We suggest that Lrig3 functions in neural crest formation in Xenopus by modulating the Wnt and Fgf signaling pathways.

Zhao H, Tanegashima K, Ro H, Dawid IB. Lrig3 regulates neural crest formation in Xenopus by modulating Fgf and Wnt signaling pathways. Development 2008;135:1283-1293.

The mych gene is required for neural crest survival during zebrafish development

Hong and Dawid; in collaboration with Tsang

Among Myc family genes, c-Myc plays a role in neural crest specification in Xenopus and in craniofacial development in the mouse. However, there is no information on either the function of other Myc genes in neural crest development or any developmental role of zebrafish Myc genes. We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues, including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain. Thus, Mych is a novel factor required for neural crest cell survival in zebrafish.

Hong SK, Tsang M, Dawid IB. The mych gene is required for neural crest survival during zebrafish development. PLoS One 2008;3:e2029.

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 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, the pharyngeal arches, the anterior aspect of the pectoral fin buds, and the gut wall. By 2.5 days after fertilization, barx1 morpholino–injected embryos exhibit 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 exhibit small and dysmorphic arch cartilage elements, and expression of chondrogenic markers such as dlx2a and col2a1 is perturbed. As seen in bead implantation experiments, the expression of barx1 is controlled by bone morphogenetic protein (BMP). Our results suggest a role for barx1 at early stages of chondrogenesis within the pharyngeal arches during zebrafish development.

Sperber SM, Dawid IB. barx1 is necessary for ectomesenchyme proliferation and osteochondroprogenitor condensation in the zebrafish pharyngeal arches. Dev Biol 2008;321:101-110.

Protocadherin 18 plays a role in cell behavior during zebrafish embryogenesis

Aamar, Dawid

Protocadherin-18 (Pcdh18) belongs to the δ2-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 lesser expression in mesodermal derivatives. Overexpression of pcdh18 in embryos caused cyclopia, mislocation 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 because of increased cell adhesion. We suggest a role for pcdh18 in cell adhesion, migration, and behavior, but not in cell specification, during gastrula and segmentation stages of development.

Aamar E, Dawid IB. Protocadherin-18a has a role in cell adhesion, behavior and migration in zebrafish development. Dev Biol 2008;318:335-346.

Foxj1 and foxj1.2 are expressed in overlapping but distinct patterns in the zebrafish embryo

Aamar, Dawid

The forkhead-related or Fox family of transcription factors play several important roles in development. Foxj1 factors are involved in ciliogenesis in various systems. We isolated two related genes, foxj1 and foxj1.2, from zebrafish and analyzed their patterns through embryogenesis. The patterns suggest functions for the two genes, which we are currently exploring.

Aamar E, Dawid IB. Isolation and expression analysis of foxj1 and foxj1.2 in zebrafish embryos. Int J Dev Biol 2008;52:985-991.

The behavior of the chromatin protein Brd4 in early zebrafish development

Toyama, Rebbert, Dawid; in collaboration with Ozato

Brd4 is a member of the BET subfamily of bromodomain proteins that includes chromatin-modifying proteins and transcriptional regulators. Brd4 plays a role in cell-cycle progression, making it indispensable in mouse embryos and cultured cells. Further, the N-terminal domain of Brd4 participates in a fusion oncogene. Brd4 associates with acetylated histones in chromatin, and the association persists during mitosis, implicating Brd4 in epigenetic memory. We have shown that Brd4 is expressed and localized on mitotic chromosomes in early zebrafish embryos before and after the midblastula transition (MBT), indicating that the Brd4-chromosome association is a conserved property that is maintained even before zygotic transcription. The association of Brd4 with acetylated histones may also be conserved in early embryos, as we found that histones H3 and H4 are already acetylated during pre–MBT stages.

Toyama R, Rebbert ML, Dey A, Ozato K, Dawid IB. Brd4 associates with mitotic chromosomes throughout early zebrafish embryogenesis. Dev Dyn 2008;237:1636-1644.

Pineal-specific gene expression in zebrafish

Toyama, Dawid; in collaboration with Epstein, Klein, Gothilf

The role of the pineal gland in biological rhythm is an important topic that is 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 gene encoding the enzyme Aanat2, we drew on the analysis of the genomic control regions responsible for this gene and generated transgenic zebrafish in which green fluorescent protein (GFP) is expressed with high specificity in the pineal gland. We used the transgenic zebrafish line to guide dissection of the pineal gland from embryonic, larval, and adult zebrafish to analyze, by the DNA microarray method, global transcription patterns at various stages of development. We used RNA from brain from which the pineal gland had been removed for comparison and compared RNA samples from 3-, 5-, and 10-day embryos and 3-month young adult and 1- to 2-year mature adult zebrafish. We obtained day and night samples at each time point at least in triplicate. The resulting experimental findings permitted evaluation of developmental changes in the pineal gland, evaluation of differences in gene expression between the pineal gland and brain, and comparison of diurnal differences. Our results indicate that a large set of genes is preferentially expressed in the pineal gland during development. The data support the known role of the fish pineal as a photoreceptor organ, as genes involved in photoreception and transduction were prominent among the pineal-enriched genes. Frequently, distinct isoforms of certain proteins were expressed in the pineal gland as compared with other organs, e.g., the eye. Developmental changes visualized in these studies suggest that the embryonic pineal gland is enriched for regulatory factors such as transcription factors and signal transducers, whereas adult pineal glands express genes involved in physiological functions such as enzymes, photo-transduction factors, and structural components.

¹Left the group during the reporting period.
²Guanine nucleotide exchange factor.
³weak-similarity GEF

Collaborators

Jonathan Epstein, MS, Scientific Software Support and Bioinformatics Core Facility, NICHD, Bethesda, MD
Yoav Gothilf, PhD, Tel Aviv University, Tel Aviv, Israel
David Klein, PhD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Keiko Ozato, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Michael Tsang, PhD, University of Pittsburgh, Pittsburgh, PA

For further information, contact idawid@nih.gov or visit http://sdb.nichd.nih.gov.