Effects of U0126 and fibroblast growth factor on gene expression profile in Ciona intestinalis embryos as revealed by microarray analysis

Fibroblast growth factor (FGF) induces the notochord and mesenchyme in ascidian embryos, via extracellular signal-regulated kinase (ERK) that belongs to the mitogen-activated protein kinase (MAPK) family. A cDNA microarray analysis was carried out to identify genes affected by an inhibitor of MAPK/ERK kinase (MEK), U0126, in embryos of the ascidian Ciona intestinalis. Data obtained from the microarray and in situ hybridization suggest that the majority of genes are downregulated by U0126 treatment. Genes that were downregulated in U0126-treated embryos included Ci-Bra and Ci-Twist-like1 that are master regulatory genes of notochord and mesenchyme differentiation, respectively. The plasminogen mRNA was downregulated by U0126 in presumptive endoderm cells. This suggests that a MEK-mediated extracellular signal is necessary for gene expression in tissues whose specification does not depend on cell-to-cell interaction. Among 85 cDNA clusters that were not affected by U0126, 30 showed mitochondria-like mRNA localization in the nerve cord/muscle lineage blastomeres in the equatorial region. The expression level and asymmetric distribution of these mRNA were independent of MEK signaling.     

Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis

Summary: Mouse embryos homozygous for the allele eed^{l7Rn5‐3354SB} of the Polycomb Group gene embryonic ectoderm development (eed) display a gastrulation defect in which epiblast cells move through the streak and form extraembryonic mesoderm derivatives at the expense of development of the embryo proper. Here we demonstrate that homozygous mutant ES cells have the capacity to differentiate embryonic cell types both in vitro as embryoid bodies and in vivo as chimeric embryos. In chimeric embryos, eed mutant cells can respond to wild‐type signals and participate in normal gastrulation movements. These results indicate a non–cell‐autonomous function for eed. Evidence of mutant cell exclusion from the forebrain and segregation within somites, however, suggests that eed has cell‐autonomous roles in aspects of organogenesis. A requirement for eed in the epiblast during embryonic development is supported by the fact that high‐contribution chimeras could not be rescued by a wild‐type extraembryonic environment. genesis 31:142–146, 2001. © 2001 Wiley‐Liss, Inc.     

SEUSS and SEUSS‐LIKE 2 coordinate auxin distribution and KNOXI activity during embryogenesis

In Arabidopsis, SEUSS (SEU) and SEUSS‐LIKE 2 (SLK2) are components of the LEUNIG (LUG) repressor complex that coordinates various aspects of post‐embryonic development. The complex also plays a critical role during embryogenesis, as seu slk2 double mutants have small, narrow cotyledons and lack a shoot apical meristem (SAM). Here we show that seu slk2 double mutant embryos exhibit delayed cotyledon outgrowth and that this is associated with altered PIN‐FORMED1 (PIN1) expression and localisation during the early stages of embryogenesis. These observations suggest that SEU and SLK2 promote the transition to bilateral symmetry by modulating auxin distribution in the embryonic shoot. This study also shows that loss of SAM formation in seu slk2 mutants is associated with reduced expression of the class I KNOX (KNOXI) genes SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS and KNAT2. Furthermore, elevating STM expression in seu slk2 mutant embryos was sufficient to restore SAM formation but not post‐embryonic activity, while both SAM formation and activity were rescued when SLK2 expression was restored in either the cotyledons or boundary regions. These results demonstrate that SEU and SLK2 function redundantly to promote embryonic shoot development and likely act through a non‐cell autonomous pathway to promote KNOXI activity.     

Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects

The mesodermal region in Drosophila is determined by a maternally derived morphogenetic gradient system which specifies the different cell fates along the dorsoventral axis, including the prospective mesodermal cells at the ventral side of the embryo. There are at least two zygotic target genes, twist and snail, which are required for mesoderm formation in Drosophila. To analyze whether a similar mode of mesoderm specification might also apply to short germ band insect embryos, we have cloned twist and snail- related gene fragments from the flour beetle Tri-bolium and have analyzed their expression pattern. Both genes are expressed in a ventral stripe at early blastoderm stage, which is restricted to the region of the developing germ rudiment. The cells expressing the two genes are those that invaginate during gastrulation, indicating that the early stages of mesoderm specification are indeed very similar between the two species. Interestingly, both genes are also expressed during germband extension in a subregion of the growth zone of the embryo which forms the mesodermal cells. This suggests that the expression of the two genes is required for mesoderm formation both at early blastoderm stage and during germband elongation until the end of the segmental growth process. © 1994 Wiley-Liss, Inc.     

Regulatory functions of ROS dynamics via glutathione metabolism and glutathione peroxidase activity in developing rice zygote

Reactive oxygen species (ROS) play essential roles in plant development and environmental stress responses. In this study, ROS dynamics, the glutathione redox status, the expression and subcellular localization of glutathione peroxidases (GPXs), and the effects of inhibitors of ROS-mediated metabolism were investigated along with fertilization and early zygotic embryogenesis in rice (Oryza sativa). Zygotes and early embryos exhibited developmental arrest upon inhibition of ROS production. Egg cells accumulated high ROS levels, and, after fertilization, intracellular ROS levels progressively declined in zygotes in which de novo expression of GPX1 and 3 was observed through upregulation of the genes. In addition to inhibition of GPX activity, depletion of glutathione impeded early embryonic development and led to failure of the zygote to appropriately decrease H₂O₂ levels. Moreover, through monitoring of the glutathione redox status, the developing zygotes exhibited a progressive glutathione oxidation, which became extremely delayed under inhibited GPX activity. Our results provide insights into the importance of ROS dynamics, GPX antioxidant activity, and glutathione redox metabolism during zygotic/embryonic development.     

Cascade effect of cardiac myogenesis gene expression during cardiac looping in <Emphasis Type="Italic">tbx5</Emphasis> knockdown zebrafish embryos

Zebrafish tbx5 expresses in the heart, pectoral fins and eyes of zebrafish during embryonic development. In zebrafish, injection of tbx5 morpholino antisense RNA caused changes of heart conformation, defect of heart looping, pericardium effusion, dropsy of ventral position and decreased heart rate. We suggested that cardiac myogenesis genes might be responsible for this phenomenon. Morpholino antisense RNA which against the initiation site of tbx5 gene was designed in order to knockdown the expression of tbx5, and the results were analyzed by whole-mount in situ hybridization and quantitative real-time PCR. Expression of cardiac myogenesis genes amhc, vmhc and cmlc2 were expressed constantly at the early embryonic development and reached its highest rate right before cardiac looping initiated. These cardiac myogenesis genes showed insufficient expressions within different heart defect embryos. Moreover, vmhc showed ectopic expression in addition to heart looping defect in heart defective embryos at 36 hpf. Our data suggests that the heart failure caused by the knockdown of tbx5 gene might result from the down-regulation of cardiac myogenesis genes. Jen Her Lu and Jenn Kan Lu contributed equally to this work.     

Relation of Nodal expression to the specification of the dorsal‐ventral axis and tissue patterning in the starfish Patiria pectinifera

In this study, we attempted to reveal fundamental aspects of starfish embryogenesis, particularly embryonic axis specification or determination, in Patiria pectinifera. We first cloned PpNodal, which is known to play an important role in the specification of the embryonic axis in a wide range of animals, and studied its expression profile. PpNodal expression was first detected at the mid‐blastula stage and showed a single peak around the onset of gastrulation. These features of Nodal expression were shifted to later stages by several hours, compared with those of sea urchin embryos. After the gastrulation started, the expression level became gradually lowered up to the early bipinnaria stage, while the expression level became drastically lowered in sea urchin embryos during gastrulation. The localized Nodal expression in the presumptive oral region was not observed in starfish embryos, unlike in sea urchin embryos. Furthermore, SB431542, an inhibitor of Nodal receptor, did not affect the formation of the DV axis, although it caused the loss of left‐right asymmetry. In contrast to this, SB525334, a specific inhibitor of TGF‐beta receptor, caused the complete loss of the DV axis. Thus, the usage of signaling molecules during early embryogenesis likely varies among echinoderm classes.     

Isolation and characterization of beta-catenin downstream genes in early embryos of the ascidian Ciona savignyi

Nuclear localization of beta-catenin is most likely the first step of embryonic axis formation or embryonic cell specification in a wide variety of animal groups. Therefore, the elucidation of beta-catenin target genes is a key research subject in understanding the molecular mechanisms of the early embryogenesis of animals. In Ciona savignyi embryos, nuclear accumulation of beta-catenin is the first step of endodermal cell specification. Previous subtractive hybridization screens of mRNAs between beta-catenin-overexpressed embryos and nuclear beta-catenin-depleted embryos have resulted in the identification of beta-catenin downstream genes in Ciona embryos. In the present study, I characterize seven additional beta-catenin downstream genes, Cs-cadherinII, Cs-protocadherin, Cs-Eph, Cs-betaCD1, Cs-netrin, Cs-frizzled3/6, and Cs-lefty/antivin. All of these genes were expressed in vegetal blastomeres between the 16-cell and 110-cell stages, although their spatial and temporal expression patterns were different from one another. In situ hybridizations and real-time PCR revealed that the expression of all of these genes was up-regulated in beta-catenin-overexpressed embryos, and down-regulated in beta-catenin-suppressed embryos. Therefore, the accumulation of beta-catenin in the nuclei of vegetal blastomeres activates various vegetally expressed genes with potentially important functions in the specification of these cells.     

The pleiohomeotic functions as a negative regulator of <Emphasis Type="Italic">Drosophila even-skipped</Emphasis> gene during embryogenesis

Polycomb group (PcG) proteins maintain the spatial expression patterns of genes that are involved in cell-fate specification along the anterior-posterior (A/P) axis. This repression requires cis-acting silencers, which are called PcG response elements (PREs). One of the PcG proteins, Pleiohomeotic (Pho), which has a zinc finger DNA binding protein, plays a critical role in recruiting other PcG proteins to bind to PREs. In this study, we characterized the effects of a pho mutation on embryonic segmentation. pho maternal mutant embryos showed various segmental defects including pair-rule gene mutant patterns. Our results indicated that engrailed and even-skipped genes were misexpressed in pho mutant embryos, which caused embryonic segment defects.