Microarray is a powerful tool for analysing gene expression patterns in genome-wide view and has greatly contributed to our understanding of spatiotemporal embryonic development at the molecular level. Members of FGF (fibroblast growth factor) family play important roles in embryogenesis, e.g. in organogenesis, proliferation, differentiation, cell migration, angiogenesis, and wound healing. To dissect spatiotemporally the versatile roles of FGF during embryogenesis, we profiled gene expression in Xenopus embryo explants treated with SU5402, a chemical inhibitor specific to FGF receptor 1 ( FGFR1), by microarray. We identified 38 genes that were down-regulated and 5 that were up-regulated in response to SU5402 treatment from stage 10.5 -11.5 and confirmed their FGF-dependent transcription with RT-PCR analysis and whole-mount in situ hybridization (WISH ). Among the 43 genes, we identified 26 as encoding novel proteins and investigated their spatial expression pattern by WISH. Genes whose expression patterns were similar to FGFR1 were further analysed to test whether any of them represented functional FGF target molecules. Here, we report two interesting genes: one is a component of the canonical Ras-MAPK pathway, similar to mammalian mig6 (mitogen-inducible gene 6) acting in muscle differentiation; the other, similar to GPCR4 (G-protein coupled receptor 4), is a promising candidate for a gastrulation movement regulator. These results demonstrate that our approach is a promising strategy for scanning the genes that are essential for the regulation of a diverse array of developmental processes.

We used cDNA microarray analysis to screen for FGF target genes in Xenopus embryos treated with the FGFR1 inhibitor SU5402, and identified neurotrophin receptor homolog (NRH) as an FGF target. Causing gain of NRH function by NRH mRNA or loss of NRH function using a Morpholino antisense-oligonucleotide (Mo) led to gastrulation defects without affecting mesoderm differentiation. Depletion of NRH by the Mo perturbed the polarization of cells in the dorsal marginal zone (DMZ), thereby inhibiting the intercalation of the cells during convergent extension as well as the filopodia formation on DMZ cells. Deletion analysis showed that the carboxyl-terminal region of NRH, which includes the "death domain," was necessary and sufficient to rescue gastrulation defects and to induce the protrusive cell morphology. Furthermore, we found that the FGF signal was both capable of inducing filopodia in animal cap cells, where they do not normally form, and necessary for filopodia formation in DMZ cells. Finally, we demonstrated that FGF required NRH function to induce normal DMZ cell morphology. This study is the first to identify an in vivo role for FGF in the regulation of cell morphology, and we have linked this function to the control of gastrulation cell movements via NRH.

Gastrulation is a morphogenetic process in which tightly coordinated cell and tissue movements establish the three germ layers (ectoderm, mesoderm, and endoderm) to define the anterior-to-posterior embryonic organization [1]. To elicit this movement, cells modulate membrane protrusions and undergo dynamic cell interactions. Here we report that ankyrin repeats domain protein 5 (xANR5), a novel FGF target gene product, regulates cell-protrusion formation and tissue separation, a process that develops the boundary between the ectoderm and mesoderm [2, 3], during Xenopus gastrulation. Loss of xANR5 function by antisense morpholino oligonucleotide (MO) caused a short trunk and spina bifida without affecting mesodermal gene expressions. xANR5-MO also blocked elongation of activin-treated animal caps (ACs) and tissue separation. The dorsal cells of xANR5-MO-injected embryos exhibited markedly reduced membrane protrusions, which could be restored by coinjecting active Rho. Active Rho also rescued the xANR5-MO-inhibited tissue separation. We further demonstrated that xANR5 interacted physically and functionally with paraxial protocadherin (PAPC), which has known functions in cell-sorting behavior, tissue separation, and gastrulation cell movements [4-6], to regulate early morphogenesis. Our findings reveal for the first time that xANR5 acts through Rho to regulate gastrulation and is an important cytoplasmic partner of PAPC, whose cytoplasmic partner was previously unknown.

Control of the cell surface allows modulation of the cell's biological response, producing practical applications and satisfying scientific interests. Consequently, to meet such goals and interests, diverse approaches were developed in cell surface engineering techniques. Poly(ethylene glycol) (PEG) intermediates were widely employed to modify proteins, enzymes, artificial surfaces, liposomes, and drugs for practical usage. PEGylation was also used for modification of cell surface properties. A method was recently developed for the rapid incorporation of proteins into mammalian cell membranes using lipid-PEG(n) derivatives under physiological conditions. This is a rapid and homogeneous method to incorporate lipid-PEG(n), which was used as a model to study the modification of cellular properties and cell-cell interactions. Because the stability of molecules incorporated into the cell surface shows the usefulness of the anchoring technique, it was also investigated whether potential factors such as time, the concentration of the incorporated lipid-PEG(n), and the type of medium affect this incorporation. At concentrations greater than 10 microM, when dual typed lipid-PEG(n) was incorporated into erythrocytes, antigenic recognition was dramatically attenuated, resulting in the successful development of stealth cells.

A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.T he vertebrate inner ear is a sensory organ responsible for balance and sound detection. Dysfunction of the inner ear is among the most common congenital disorders, affecting at least 1 in 500 births (1) and ∼40% of sensorineural deafness is associated with inner ear malformations (2). However, the study of hearing and balance impairment in humans is limited by the inability to follow inner ear development. Vertebrates share similarities in the sequence of developmental events that form the inner ear: the formation of an otic placode from an ectodermal thickening, morphogenesis to form the otocyst, and regional patterning of the otic vesicle (OV), resulting in the 3D membranous labyrinth (3, 4). Multipotent sensory progenitor cells are induced in the ectoderm surrounding the anterior neural plate, a domain termed the preplacodal region (PPR), and six1 has been characterized as marking this panplacodal domain. Signals from hindbrain and the regional expression of different transcription factors differentiate the PPR into the otic placode. The OV is partitioned by asymmetrical expression of various developmental regulators to pattern subdomains of the developing inner ear.The abilities of Xenopus to elucidate the cellular and molecular aspects of developmental processes position it as a valuable model organism (5-7). Earlier studies of lineage analysis and spatiotemporal expression of transcription factors during inner ear development led to construction of an inner ear fate map in Xenopus and this fate map allows us to interpret gene expression patterns within the context of the anatomy (8, 9). In recent years, genetic and genomic approaches have been developed in Xenopus tropicalis (10-13). To advance our understanding of inner ear development, we have screened N-ethyl-N-nitrosourea (ENU) mutagenized X. tropicalis colonies and recovered an inner ear mutant named eclipse (ecl). The ecl mutant perturbs specificity protei...

Regulating the cell surface modulates the actions of the biological cell response, derives practical applications, and is of scientific interest. On the basis of our previous study using dioleylphosphatidylethanolamine poly(ethylene glycol) with multiple units of ethyleneoxide (DOPE-PEG (n)), we demonstrated the potency of DOPE-PEG (80) as a cell surface modulator. We prepared conjugates of DOPE-PEG (80) and two antagonistic peptides (C1, SGGGCLFNLPWLCG; C26, SGGGCPFSFLPWCG), specifically designed for the inhibitory receptor of natural killer (NK) cells. We confirmed that NK cells exhibited cytotoxicity against DOPE-PEG (80)-peptides-incorporated target cells. We further investigated whether the DOPE-PEG (80)-peptides could affect the cytotoxicity of NK cells in a concentration-dependent manner. C1 peptide showed down-regulation of cytotoxicity at higher concentration, whereas C26 peptide exhibited the saturated cytotoxicity of NK cells at the same concentration. These results suggest that DOPE-PEG (80) can achieve the role of a cell surface modulator without inhibiting the action of conjugated molecules, despite their relatively small size.