The complex morphogenesis of organisms is achieved by consecutive cell-to-cell interactions during development. Recent studies suggest that growth factors play crucial roles in controlling such intercellular communications in a variety of organisms. In addition to secretory factors, transcription factors which act cell-autonomously are thought to be essential for the determination of cell fates. Our main interest is to know how pattern formation and morphogenesis during development is regulated by these growth factors and transcription factors. We address this problem using several model animals, including frog, fly and ascidian, employing embryology, genetics, molecular and cellular biology, and biochemistry. In addition, we have recently introduced genome-wide approaches to elucidate precise genetic program controlling early development.

I. Gastrulation movement regulated by Wnt signaling

Gastrulation is one of the most important processes during morphogenesis of early embryo, involving dynamic cell migration and change in embryo shape. Almost all animals undergo gastrulation to form the gut. In spite of its importance, the mechanism underlying the event has just begun to be studied at molecular level. During Xenopus gastrulation, mesodermal cells migrate to the inside of the embryo and move on the blastocoel roof. One of the important mechanisms for this process is convergent extension. As convergent extension begins, cells are polarized and aligned mediolaterally, followed by the intercalation of these cells. As described above, one of the Wnt signaling pathways, called Wnt/JNK (c-Jun N-terminal kinase) pathway, is shown to be important for the regulation of convergent extension. The pathway is highly conserved among species and initially found to be essential for the establishment of planar cell polarity (PCP) of Drosophila wing hair.

We have previously demonstrated that Xenopus prickle (Xpk), a Xenopus homologue of a Drosophila PCP gene, is an essential component for gastrulation cell movement. Both gain-of-function (GOF) and loss-of-function (LOF) of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrated that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation. To understand the molecular mechanism, we identified proteins which binds to XPK by yeast two-hybrid screening. One of XPK binding protein was found to be a member of the Ste20 kinase family and named as Xenopus prickle-interacting kinase, XPIK. Developmental expression pattern of XPIK is reminiscent of that of XPIK, suggesting functional interaction between XPK and XPIK. GOF and LOF of XPIK resulted in perturbation of gastrulation, leading to shortened or spina bifida embryo. Furthermore, we have found that XPIK is not only sufficient to activate JNK in embryo, but also required for full activation of JNK by Dishevelled. These suggest that XPIK also plays an essential role in connecting extracellular Wnt signal to JNK activation through Dishevelled and XPIK.

In addition, we have been attempting to identify novel regulatory components controlling gastrulation cell movements by an expression cloning method based on morphology of dorsal marginal zone explant (Keller's explant) and of embryo. After 1,500 clones were examined by overexpression in the dorsal region of embryos, approximately 5% of clones were found to perturb normal gastrulation cell movements. Functional relevance of identified genes to cell movements is currently under investigation.

II. Regulation of actin cytoskeletal dynamics during Xenopus gastrulation

Because gastrulation movements are accompanied by dynamic changes in cell polarity, morphology, and motility, it is very likely that actin cytoskeleton is carefully regulated. Thus, we analyzed the regulatory mechanism of actin cytoskeletal dynamics during this process. Among several factors implicated in the regulation of the actin cytoskeleton, we decided to focus on myristoylated alanine-rich C kinase substrate (MARCKS). MARCKS is an actin-binding, membrane-associated protein previously implicated in the regulation of F-actin dynamics. It has been investigated mainly in tissue culture cells and biochemical analyses. MARCKS knock-out mice has been reported to show neural tube defects. Yet, its molecular mechanism has not been elucidated. We demonstrate that MARCKS is essential for gastrulation movements and neural tube closure in Xenopus embryos. Cell biological analyses revealed that MARCKS knock-down with Morpholino oligo (MO) significantly reduced cortical actin formation and caused defects in cell polarity, adhesion, motility and protrusive activity, leading to these developmental defects. We also showed that the Wnt pathway dramatically promoted the formation of lamelipodia- and filopodia-like protrusions and MARCKS is required for this activity. These findings show that the Wnt signaling pathway regulates cortical actin dynamics and MARCKS is requisite for the Wnt function. We conclude that MARCKS is essential for dynamic morphogenetic movements during embryogenesis.

Figure 1 MARCKS is required for convergent extension movements. MARCKS Mo or control Mo, Rhodamine dextran (RhDx), and Venus mRNA were co-injected into one of the two dorsal blastomeres. Venus mRNA alone was injected into the other dorsal blastomere. In the absence of MARCKS Mo, red cells and non-red cells were polarized and intercalated. In contrast, MARCKS Mo-injected cells were not polarized and did not participate in the intercalation.

III. Genetic screening for novel DPP/BMP signaling components utilizing Drosophila model system

Drosophila is one of the ideal model organisms to dissect signal transduction pathway by genetic methods. We have carried out dominant suppressor screening for a transgenic mutant fly that expresses activated DPP/BMP type-I receptors in wing imaginal discs. We isolated 19 suppressor mutants, Suppressor of constitutively activated Dpp singnaling (Scad). Alleles of punt, Mad, shn and dCrebA were found in isolated Scad mutants. Most of the Scad mutants encode a nuclear protein suggesting these molecules regulates DPP signaling at nuclear level.

We now focus to study one of the mutants Scad67. Scad67 was also isolated by a Mexican group and named as tonnalli (tna). We also isolated vertebrate homologs of Scad67/tna, TONAS-1 and TONAS-2. The most characteristic feature of these proteins is the existence of a single SP-RING finger motif in the middle. The SP-RING motif was originally found in the PIAS family SUMO-E3 ligase proteins. We found that TONAS has SUMO-E3 ligase activity and TONAS facilitates specific SUMO-2/3 conjugation to TONAS itself. TONAS also shows strong activity of nuclear body formation in cultured cells. TONAS effectively recruits transcriptional regulators including PML, CBP and P300 to the nuclear body.

It has been shown that the Trithorax group componets are essential factors in ATP-dependent chromatin remodeling complex. Our results suggest a role for Scad67/TNA and TONAS in the connection of the Trithorax/SWI/SNF chromatin remodeling complex to CBP/P300, the relocation of these protein complexes into the nuclear substructure, and the regulation of gene expression.

IV. Brachyury downstream notochord differentiation in the ascidian embryo

Ascidians, urochordates, are one of the three chordate groups, and the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. It contains a notochord, which is a defining characteristic of chordate embryo composed of only 40 cells. To understand the morphogenesis in this simple system, we have focused on a gene, Brachyury, which is known to play an important role in the notochord development. In ascidian, Brachyury is expressed exclusively in the notochord and the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. This gene encodes a sequence-specific activator that contain a T-box DNA-binding domain, and in vertebrates, it is initially expressed throughout the presumptive mesoderm and gradually restricted to the developing notochord and tailbud. The phenotype of the Brachyury mutants in mice and zebrafish revealed that this gene is essential for notochord differentiation. Our goal is to elucidate the down stream pathway of this important gene in ascidian in order to set the stage for understanding not only the formation and function of the notochord but how this important structure has evolved. We conducted the subtractive hybridization screens to identify potential Brachyury target genes that are induced upon Ci-Bra overexpression. Out of 501 independent cDNA clones that were induced, 38 were specifically expressed in notochord cells. We characterized of subcellular-localizations of the 20 GFP fusion gene products in the notochord cell. These products observed after electroporating the embryos at one-cell stage with GFP fusion gene containing notochord specific promoter. They showed various subcellular localizations in the notochord cells of the tadpole tails. In addition to investigate the actual functions of the genes during the notochord formation, functional analyses were performed by injecting of antisence morphollino oligos.

V. Comprehensive analysis of developmentally regulated genes using cDNA microarray

In order to examine the global expression profile during early development of Xenopus laevis, we have collected massive EST sequences from three normalized cDNA libraries of early gastrula, neurula and tailbud stage. To date, more than 100,000 ESTs (~30% of Xenopus laevis ESTs registered in the public database) were produced. Using these cDNA clones, we generated the NIBB 40k cDNA macroarray and 4.6k non-redundant cDNA microarray. With the DNA arrays, we conducted a series of large-scale screening of the genes which expressions are regulated by some transcription factors and signaling factors (Xnr-1, FAST1, FGF etc.). Consequently, we have been able to isolate lots of the candidate genes that encode a variety of signal transduction and transcription regulatory components, and also cytoskeletal components, suggesting dynamic cellular changes in the early development of Xenopus laevis. This also proves DNA array to be an effective screening assay for novel genes which function during early development process of Xenopus laevis.

Adding the EST sequences, the assembled sequences and their annotation information are available through the web at NIBB Xenopus laevis EST database XBD2 (Fig.2). XDB2 also provides the whole mount in situ hybridization images as the spatial expression pattern (Fig.3).

Figure 2 XDB2 (http://xenopus.nibb.ac.jp).

Figure 3 Whole mount in situ hybridization images.