The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pronephros morphogenesis and left-right establishment in zebrafish

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease. Among 12 reported Nphp gene products, Inv/Nphp2, Nphp3 and Nek8/Nphp9 are localized to the proximal segment in the primary cilium. However, the functional relationships are unknown. This study focused on phenotype analysis of nek8 knockdown embryos and the genetic relationship between nek8 and inv in zebrafish. Knockdown of nek8 produced both pronephric cysts and abnormal cardiac looping. Simultaneous knockdown of nek8 and inv synergistically increased the incidence of these defects. Interestingly, nek8 mRNA rescued inv morphant phenotypes, although inv mRNA could not rescue nek8 morphant phenotypes. These results suggest that Nek8 acts downstream of Inv function.     

A role of the cryptic gene in the correct establishment of the left-right axis

During vertebrate embryogenesis, a left-right axis is established. The heart, associated vessels and inner organs adopt asymmetric spatial arrangements and morphologies. Secreted growth factors of the TGF-beta family, including nodal, lefty-1 and lefty-2, play crucial roles in establishing left-right asymmetries [1] [2] [3]. In zebrafish, nodal signalling requires the presence of one-eyed pinhead (oep), a member of the EGF-CFC family of membrane-associated proteins [4]. We have generated a mutant allele of cryptic, a mouse EGF-CFC gene [5]. Homozygous cryptic mutants developed to birth, but the majority died during the first week of life because of complex cardiac malformations such as malpositioning of the great arteries, and atrial-ventricular septal defects. Moreover, laterality defects, including right isomerism of the lungs, right or left positioning of the stomach and splenic hypoplasia were observed. Nodal gene expression in the node was initiated in cryptic mutant mice, but neither nodal, lefty-2 nor Pitx2 were expressed in the left lateral plate mesoderm. The laterality defects observed in cryptic(-/-) mice resemble those of mice lacking the type IIB activin receptor or the homeobox-containing factor Pitx2 [6] [7] [8] [9], and are reminiscent of the human asplenic syndrome [10]. Our results provide genetic evidence for a role of cryptic in the signalling cascade that determines left-right asymmetry.     

A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection

Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1, a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea, which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1-dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe.     

Establishing a left-right axis in the embryo

Vertebrates exhibit evolutionarily conserved asymmetries in the pattern of internal organ placement that are essential for their normal physiological function. Left-right asymmetries in organ situs are dependent upon the formation of an intact left-right axis during embryogenesis. Recently many of the molecular components involved in the initiation and maintenance of the left-right axis have been described. These molecules and their function in promoting left-right asymmetries are reviewed.     

FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development

FGF receptor 2 isoform IIIb (FGFR2b), originally discovered as a receptor for FGF7, is known to be an important receptor in vertebrate morphogenesis, because FGFR2b null mice exhibit agenesis or dysgenesis of various organs, which undergo budding and branching morphogenesis. Since FGF7 null mice do not exhibit marked defects in organogenesis, it has been considered that other FGF(s) than FGF7 might function as a major ligand for FGFR2b during organogenesis. One of the candidate ligands is FGF10, because FGF10 binds to FGFR2b with high affinity and the formation of the limb and lung is arrested in FGF10 null mice as found in FGFR2b-deficient mice. Previous analyses of FGF10 null mice revealed that FGF10 is required for limb and lung development. To elucidate the role of FGF10 in wide-range organogenesis, we further analyzed the phenotypes of the FGF10 knockout mice. We found diverse phenotypes closely related to those for FGFR2b-deficient mice, which includes the absence of thyroid, pituitary, and salivary glands, while minor defects were observed in the formation of teeth, kidneys, hair follicles, and digestive organs. These results suggest that FGF10 acts as a major ligand for FGFR2b in mouse multi-organ development.     

sprouty4 acts in vivo as a feedback-induced antagonist of FGF signaling in zebrafish

In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, we have isolated a zebrafish sprouty4 gene, based on its extensive similarities with the expression patterns of both fgf8 and fgf3. Through gain- and loss-of-function experiments, we demonstrate that Fgf8 and Fgf3 act in vivo to induce the expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses, Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, an opposite phenotype to the dorsalisation induced by overexpression of Fgf8 or Fgf3. Conversely we have shown that inhibition of Spry4 function through injection of antisense morpholino oligonucleotide leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, we show that Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, our analysis reveals that signaling through FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the regulation of bone morphogenetic protein (BMP) expression.     

Depletion of FGF acts as a lateral inhibitory factor in lung branching morphogenesis in vitro

Previous studies have shown that the interaction of positive and inhibitory signals plays a crucial role during lung branching morphogenesis. We found that in mesenchyme-free conditions, the lung epithelium exerted a lateral inhibitory effect on the neighbouring epithelium via depletion of fibroblast growth factor 1 (FGF1). Contrary to previous suggestions, bone morphogenetic protein 4 could not substitute for the inhibitory effect. Based on of this observation, we used a reaction-diffusion model of the substrate-depletion type to represent the initial phase of in vitro branching morphogenesis of lung epithelium, with depletion of FGF playing the role of lateral inhibitor. The model was able to account for the effects of the FGF1 concentration, extracellular matrix degradation and different subtypes of FGF on morphogenesis of the lung bud epithelia. These results suggest that the depletion of FGF may be a key regulatory component in initial phase of branching morphogenesis of the lung bud epithelium in vitro.     

BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo

A model of left-right axis formation in the chick involves inhibition of bone morphogenetic proteins by the antagonist Car as a mechanism of upregulating Nodal in the left lateral plate mesoderm. By contrast, expression of CFC, a competence factor, which is absolutely required for Nodal signaling in the lateral plate mesoderm is dependent on a functional BMP signaling pathway. We have therefore investigated the relationship between BMP and Nodal in further detail. We implanted BMP2 and Noggin-expressing cells into the left lateral plate and paraxial mesoderm and observed a strong upregulation of Nodal and its target genes Pitx2 and Nkx3.2. In addition Cfc, the Nodal type II receptor ActrIIa and Snr were found to depend on BMP signaling for their expression. Comparison of the expression domains of Nodal, Bmp2, Car and Cfc revealed co-expression of Nodal, Cfc and Bmp2, while Car and Nodal only partially overlapped. Ectopic application of BMP2, Nodal, and Car as well as combinations of this signaling molecules to the right lateral plate mesoderm revealed that BMP2 and Car need to synergize in order to specify left identity. We propose a novel model of left-right axis formation, which involves BMP as a positive regulator of Nodal signaling in the chick embryo.     

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.     

Insights into the establishment of left-right asymmetries in vertebrates

The body-plan of vertebrates, while exteriorly essentially symmetric along its medio-lateral plane, displays numerous left-right differences in the disposition and placement of internal organs. Such left-right asymmetries, established during embryogenesis, are controlled by complex epigenetic and genetic cascades that impart laterality information to the different embryo structures and organ primordia. A key and evolutionarily conserved feature of these information cascades among vertebrate embryos is the left-sided transfer of information from the node to the lateral plate mesoderm during early somitogenesis stages. We review here recent evidence concerning the mechanisms that regulate the laterality of such transfer. Furthermore, we propose a model of left-right axis specification that underscores the role of the node as an integrator of laterality information and the evolutionary conservation of the mechanisms that convey such information to and from the node.     

Unveiling the establishment of left-right asymmetry in the chick embryo

Vertebrates display striking left-right asymmetries in the placement of internal organs, which are concealed by a seemingly bilaterally symmetric body plan. The establishment of asymmetries about the left-right axis occurs early during embryo development and requires the concerted and sequential action of several epigenetic, genetic and cellular mechanisms. Experiments in the chick embryo model have contributed crucially to our current understanding of such mechanisms and are reviewed here. Particular emphasis is given to the elucidation of a genetic network that conveys left-right information from Hensen's node to the organ primordia, characterized to a significant degree of detail in the chick embryo. We also point out a number of early and late events in the determination of left-right asymmetries that are currently poorly understood and for whose study the chick embryo model presents several advantages. We anticipate that the availability of the chick genome sequence will be combined with multidisciplinary approaches from experimental embryology, biophysics, live-cell imaging, and mathematical modeling to boost up our knowledge of left-right organ asymmetry in the near future.     

Signalling in plant embryos during the establishment of the polar axis.

In brown algae fertilization takes place free from surrounding tissue layers. The cytoskeleton and transmembrane links to the cell wall are involved in establishing and stabilizing the polar axis and in determining the fate of cells in the early embryo. In seed plants, the egg cell and zygote exhibit apical basal polarity. Mutant studies suggest that axes of polarity of the early embryo depend on signalling between the apical and basal compartments, possibly involving auxin. Development of somatic cells into plant embryos involves extracellular matrix-derived arabinogalactan proteins. This suggests a role for the cell wall in plant embryogenesis.     

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral–aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral–aboral gene regulatory network. A model for oral–aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Lefty's function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral–aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.     

Ectodermal syndecan-2 mediates left-right axis formation in migrating mesoderm as a cell-nonautonomous Vg1 cofactor.

Heparan sulfate proteoglycans expressed on the Xenopus animal cap ectoderm have been implicated in transmitting left-right information to heart and gut primordia. We report here that syndecan-2 functions in the ectoderm to mediate cardiac and visceral situs, upstream of known asymmetrically expressed genes but independently of its ability to mediate fibronectin fibrillogenesis. Left-right development is dependent on a distinct subset of glycosaminoglycan attachment sites on syndecan-2. A novel in vivo approach with enterokinase demonstrates that syndecan-2 functions in left-right patterning during early gastrulation. We describe a cell-nonautonomous role for ectodermal syndecan-2 in transmitting left-right information to migrating mesoderm. The results further suggest that this function may be related to the transduction of Vg1-related signals.     

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.     

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

Previous studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. Here we show that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, we analyzed the role of Dlp in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, our results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila.