Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate 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.

The roles of extra-embryonic tissues in early vertebrate body patterning have been extensively studied, yet we know little about their function during later developmental events. Here, we analyze the function of the zebrafish extra-embryonic yolk syncytial layer (YSL) specific transcription factor, Mtx1, and find that it plays an essential role in myocardial migration. Downregulating the function of Mtx1 in the YSL leads to cardia bifida, a phenotype in which the myocardial cells fail to migrate to the midline. Mtx1 in the extra-embryonic YSL appears to regulate the embryonic expression of fibronectin, a gene previously implicated in myocardial migration. We further show dosage-sensitive genetic interactions between mtx1 and fibronectin. Based on these data, we propose that the extra-embryonic YSL regulates myocardial migration, at least in part by influencing fibronectin expression and subsequent assembly of the extracellular matrix in embryonic tissues.

Summary Emerging evidence indicates that paracrine signals from endothelial cells play a role in tissue differentiation and organ formation [1–3]. Here, we identify a novel role for endothelial cells in modulating hepatocyte polarization during liver organogenesis. We find that in zebrafish, the apical domain of the hepatocytes predicts the location of the intrahepatic biliary network. The refinement of hepatocyte polarization coincides with the invasion of endothelial cells into the liver, and these endothelial cells migrate along the maturing basal surface of the hepatocytes. Using genetic, pharmacological, and transplantation experiments, we provide evidence that endothelial cells influence the polarization of the adjacent hepatocytes. This influence of endothelial cells on hepatocytes is mediated at least in part by the cell-surface protein Heart of glass and contributes to the establishment of coordinately aligned hepatocyte apical membranes and evenly spaced intrahepatic conduits.

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishmentSomites are the morphologically distinct segmental units that are transiently formed during early vertebrate development and subsequently give rise to metameric and fundamental structures such as the vertebrae of the axial skeleton, their associated muscles, and tendons. The somites are subdivided from the anterior end of the unsegmented paraxial mesoderm, called the presomitic mesoderm (PSM), and sequentially generated in an anterior to posterior direction in a rhythmic fashion at regular spatiotemporal intervals. The molecular mechanism underlying the periodical formation of somites is coupled to an internal oscillator, referred to as the "segmentation clock", which has been evidenced by the cyclic expression of genes in the PSM (Palmeirim et al. 1997;Maroto and Pourquie 2001). Most genes that exhibit a cyclic expression pattern in the PSM are involved in the Notch signaling pathway . Various hairy/Enhancer of split (Espl)-related basic helix-loop-helix (bHLH) genes (hairy-1, hairy-2, and Hey2 in the chicken; Hes1, Hes5, Hes7, and Hey2 in the mouse; her1 and her7 in the zebrafish) that are transcriptional targets of the Notch signaling are expressed in a dynamic pattern of stripes across the PSM in a posterior to anterior direction. In addition, Lunatic fringe, encoding a glycosyltransferase that modulates the Notch signaling in the chicken and mouse, and the Notch ligand deltaC in the zebrafish also show an oscillatory expression pattern in the PSM. These cyclic genes, as well as other components of the Notch signaling pathway, were shown to be required for the proper somite segmentation in mice ( The oscillating and anteriorly propagating wave of gene expression, which is maintained in the posterior PSM, becomes fixed to cause segmentation in the anterior PSM. Activity gradients of signaling molecules, including fibroblast growth factor (FGF), Wnt, and retinoic acid are proposed to regulate the differentiation of PSM cells along the anteroposterior axis from a state permitting the oscillating gene expression to a state driving the segmentation program (Dubrulle et al. 2001;Sawada et al. 2001;Aulehla et al. 2003;Moreno and Kintner 2004). For instance, FGF signaling is the highest at the posterior end of the PSM with a gradual decrease toward the anterior, suggesting a role for FGF signaling in maintaining the characteristics of the posterior PSM cells (Dubrulle et al. 2001;Sawada et al. 2001). In fact, overexpression of FGF8 in the entire PSM of chick embryos causes an increase in the expression of Brachyury in the posterior PSM and suppresses morphological segmentation (Dubrulle et al. 2001). Furthermore, transient activation or inhibition of FGF signaling results in the formation of smaller or larger somites, respectively. Thus, FGF signaling appears to maintain the posterior characteristics, which allows the cyclic gene expression, and the level of FGF activity regulates the transition of the posterior PSM ...

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1. A method for adapting a standard stereotaxic frame for use with neonatal rats as young as postnatal day 1 (PD 1) was devised, and single-unit extracellular recordings were obtained from neurons in the locus ceruleus (LC) in urethan-anesthetized rats during different stages in development from PD 1 to PD 34. 2. The spontaneous firing pattern of neonatal LC neurons was characterized by long silent periods punctuated by brief epochs of sporadic firing. At PD 7-14, LC neurons exhibited periodic occurrences of irregular firing that lasted for 20-30 s. By PD 20, the pattern and rate of spontaneous activity were virtually indistinguishable from that of adults. 3. Conditioning stimulation of the dorsal noradrenergic bundle (DNB), given 10-200 ms prior to a test stimulus to the DNB, markedly reduced the amplitude of the antidromic action potentials to the test stimulus and sometimes resulted in spike decomposition. This refractoriness of the soma-dendritic membrane of LC cells was significantly attenuated with development and approached adult levels after PD 18. 4. Antidromic responses elicited by DNB stimulation were followed by a phase of inhibition or inhibition-excitation. Postactivation excitation was most prominent in cells that were not spontaneously active, and decreased steadily throughout development, probably because of the steady increase in spontaneous firing rate seen during maturation. 5. Although the conduction velocity of LC axons increased steadily from birth through PD 26, conduction time remained unchanged. 6. Neonatal LC neurons were equally sensitive to noxious and nonnoxious somatosensory stimuli. As development proceeded, LC neurons became less sensitive to innocuous somatosensory stimuli such as air puffs and tactile stimuli while simultaneously becoming more sensitive to noxious stimuli. Auditory and visual stimuli became effective for the first time at PD 14 and PD 12, respectively. 7. These results indicate that the electrical activity of LC neurons in the developing brain is intimately related to input from peripheral sensory sources. Therefore, the influence of the LC on the developing brain may occur predominantly through sensory input.

The medaka is becoming an attractive model organism for the study of vertebrate early development and organogenesis and large-scale mutagenesis projects that are aimed at creating developmentally defective mutants are now being conducted by several groups in Japan. To strengthen the study of medaka developmental genetics, we have conducted a large-scale isolation of ESTs from medaka embryos and developed tools that facilitate mutant analysis. In this study, we have characterized a total of 132,082 sequences from both ends of cloned insert cDNAs from libraries generated at different stages of medaka embryo development. Clustering analysis with 3-prime sequences finally identified a total of 12,429 clusters. As a pilot analysis, 924 clusters were subjected to in situ hybridization to determine the spatial localization of their transcripts. Using EST sequence data generated in the present study, a 60-mer oligonucleotide microarray with 8,091 unigenes (Medaka Microarray 8K) was constructed and tested for its usefulness in expression profiling. Furthermore, we have developed a rapid and reliable mutant mapping system using a set of mapped EST markers (M-marker 2003) that covers the entire medaka genome. These resources will accelerate medaka mutant analyses and make an important contribution to the medaka genome project.

Vertebrate endoderm development has recently become the focus of intense investigation. We have identified a novel sox gene, 226D7, which is important in zebrafish endoderm development. 226D7 was isolated by an in situ hybridization screening for genes expressed in the yolk syncytial layer (YSL) at the blastula stage. 226D7 is expressed mainly in the YSL at this stage and, during gastrulation, its expression is also detected in the forerunner cells and endodermal precursor cells. The expression of 226D7 is positively regulated by Nodal signaling. The knockdown of 226D7 using morpholino antisense oligonucleotides results in a lack of sox17-expressing endodermal precursor cells during gastrulation, and, consequently, lacks endodermal derivatives such as gut tissue. The effect is strictly restricted to the endodermal lineage, while the mesoderm is normally formed, a phenotype that is nearly identical to that of the casanova mutant (Dev. Biol. 215 (1999) 343). We further demonstrate that overexpression of 226D7 increases the number of sox17-expressing endodermal progenitor cells without upregulating the expression of the Nodal genes, cyclops and squint. Region-specific knockdown and overexpression of 226D7 by injection into the YSL suggest that 226D7 in the YSL is not involved in endoderm formation and 226D7 in the endoderm progenitor cells is important for endoderm development. Taken together, our data demonstrate that 226D7 is a downstream target of Nodal signal and a critical transcriptional regulator of early endoderm formation.