Maternal nodal and zebrafish embryogenesis

In fish and amphibians, the dorsal axis is specified by the asymmetric localization of maternally provided components of the Wnt signalling pathway. Gore et al. suggest that the Nodal signal Squint (Sqt) is required as a maternally provided dorsal determinant in zebrafish. Here we test their proposal and show that the maternal activities of sqt and the related Nodal gene cyclops (cyc) are not required for dorsoventral patterning.     

The Wnt/calcium pathway activates NF-AT and promotes ventral cell fate in Xenopus embryos

It is thought that inositol-1,4,5-trisphosphate (Ins(1,4,5)P(3))-Ca(2+) signalling has a function in dorsoventral axis formation in Xenopus embryos; however, the immediate target of free Ca(2+) is unclear. The secreted Wnt protein family comprises two functional groups, the canonical Wnt and Wnt/Ca(2+) pathways. The Wnt/Ca(2+) pathway interferes with the canonical Wnt pathway, but the underlying molecular mechanism is poorly understood. Here, we cloned the complementary DNA coding for the Xenopus homologue of nuclear factor of activated T cells (XNF-AT). A gain-of-function, calcineurin-independent active XNF-AT mutation (CA XNF-AT) inhibited anterior development of the primary axis, as well as Xwnt-8-induced ectopic dorsal axis development in embryos. A loss-of-function, dominant negative XNF-AT mutation (DN XNF-AT) induced ectopic dorsal axis formation and expression of the canonical Wnt signalling target molecules siamois and Xnr3 (ref. 4). Xwnt-5A induced translocation of XNF-AT from the cytosol to the nucleus. These data indicate that XNF-AT functions as a downstream target of the Wnt/Ca(2+) and Ins(1,4,5)P(3)-Ca(2+) pathways, and has an essential role in mediating ventral signals in the Xenopus embryo through suppression of the canonical Wnt pathway.     

Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis

Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of beta-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.     

Planar cell polarity signalling controls cell division orientation during zebrafish gastrulation

Oriented cell division is an integral part of pattern development in processes ranging from asymmetric segregation of cell-fate determinants to the shaping of tissues. Despite proposals that it has an important function in tissue elongation, the mechanisms regulating division orientation have been little studied outside of the invertebrates Caenorhabditis elegans and Drosophila melanogaster. Here, we have analysed mitotic divisions during zebrafish gastrulation using in vivo confocal imaging and found that cells in dorsal tissues preferentially divide along the animal-vegetal axis of the embryo. Establishment of this animal-vegetal polarity requires the Wnt pathway components Silberblick/Wnt11, Dishevelled and Strabismus. Our findings demonstrate an important role for non-canonical Wnt signalling in oriented cell division during zebrafish gastrulation, and indicate that oriented cell division is a driving force for axis elongation. Furthermore, we propose that non-canonical Wnt signalling has a conserved role in vertebrate axis elongation, orienting both cell intercalation and mitotic division.     

Wnt-Ryk signalling mediates medial-lateral retinotectal topographic mapping

Computational modelling has suggested that at least two counteracting forces are required for establishing topographic maps. Ephrin-family proteins are required for both anterior-posterior and medial-lateral topographic mapping, but the opposing forces have not been well characterized. Wnt-family proteins are recently discovered axon guidance cues. We find that Wnt3 is expressed in a medial-lateral decreasing gradient in chick optic tectum and mouse superior colliculus. Retinal ganglion cell (RGC) axons from different dorsal-ventral positions showed graded and biphasic response to Wnt3 in a concentration-dependent manner. Wnt3 repulsion is mediated by Ryk, expressed in a ventral-to-dorsal decreasing gradient, whereas attraction of dorsal axons at lower Wnt3 concentrations is mediated by Frizzled(s). Overexpression of Wnt3 in the lateral tectum repelled the termination zones of dorsal RGC axons in vivo. Expression of a dominant-negative Ryk in dorsal RGC axons caused a medial shift of the termination zones, promoting medially directed interstitial branches and eliminating laterally directed branches. Therefore, a classical morphogen, Wnt3, acting as an axon guidance molecule, plays a role in retinotectal mapping along the medial-lateral axis, counterbalancing the medial-directed EphrinB1-EphB activity.     

Wnt信号通路与干细胞定向分化

Wnt信号通路在胚胎发育过程中参与背腹轴的形成、细胞极性的建立以及决定细胞命运.利用干细胞定向分化模型可在体外培养条件下探索Wnt信号通路在哺乳动物早期胚胎发育过程中的分子机理,了解其信号网络对干细胞定向分化中各个事件的调控机制.本文通过综合近期Wnt信号通路的研究,阐述经典Wnt信号通路(Canonical Wnt signaling pathway)在干细胞定向分化中的作用.     

bicoid RNA localization requires specific binding of an endosomal sorting complex

bicoid messenger RNA localizes to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first localized cytoplasmic determinant to be identified, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that the mRNA is recognized by a complex of many redundant proteins, each of which binds to the localization element in the 3' untranslated region (UTR) with little or no specificity. Indeed, the only known RNA-binding protein that co-localizes with bicoid mRNA is Staufen, which binds non-specifically to double-stranded RNA in vitro. Here we show that mutants in all subunits of the ESCRT-II complex (VPS22, VPS25 and VPS36) abolish the final Staufen-dependent step in bicoid mRNA localization. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm. However, the role of ESCRT-II in bicoid localization seems to be independent of endosomal sorting, because mutations in ESCRT-I and III components do not affect the targeting of bicoid mRNA. Instead, VPS36 functions by binding directly and specifically to stem-loop V of the bicoid 3' UTR through its amino-terminal GLUE domain, making it the first example of a sequence-specific RNA-binding protein that recognizes the bicoid localization signal. Furthermore, VPS36 localizes to the anterior of the oocyte in a bicoid-mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This function of ESCRT-II as an RNA-binding complex is conserved in vertebrates and may clarify some of its roles that are independent of endosomal sorting.     

豌豆cop1 cDNA的克隆、序列分析及其在大肠杆菌中的表达

以拟南芥cop1 cDNA为探针,从豌豆(Pisum sativum) cDNA文库中克隆到了豌豆cop1 cDNA。序列分析表明,它全长为2863bp,其中包括604bp 5′非编码区、243bp 3′非编码区和2016bp编码区,编码672个氨基酸。在大肠杆菌中实现了豌豆cop1基因的高效表达。对拟南芥、豌豆和番茄3种植物cop1的序列同源性比较表明,cop1可能是一种进化上很保守的蛋白质。     

Wnt proteins are lipid-modified and can act as stem cell growth factors

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.     

The molecular nature of the zebrafish tail organizer

Based on grafting experiments, Mangold and Spemann showed the dorsal blastopore lip of an amphibian gastrula to be able to induce a secondary body axis. The equivalent of this organizer region has been identified in different vertebrates including teleosts. However, whereas the graft can induce ectopic head and trunk, endogenous and ectopic axes fuse in the posterior part of the body, raising the question of whether a distinct organizer region is necessary for tail development. Here we reveal, by isochronic and heterochronic transplantation, the existence of a tail organizer deriving from the ventral margin of the zebrafish embryo, which is independent of the dorsal Spemann organizer. Loss-of-function experiments reveal that bone morphogenetic protein (BMP), Nodal and Wnt8 signalling pathways are required for tail development. Moreover, stimulation of naive cells by a combination of BMP, Nodal and Wnt8 mimics the tail-organizing activity of the ventral margin and induces surrounding tissues to become tail. In contrast to induction of the vertebrate head, known to result from the triple inhibition of BMP, Nodal and Wnt, here we show that induction of the tail results from the triple stimulation of BMP, Nodal and Wnt8 signalling pathways.     

Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).     

Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3' untranslated region.

In the Caenorhabditis elegans hermaphrodite germ line, sperm and then oocytes are made from a common pool of germ-cell precursors. The decision to differentiate as a sperm or an oocyte is regulated by the sex-determining gene, fem-3. Expression of fem-3 in the hermaphrodite germ line directs spermatogenesis and must be negatively regulated to allow the switch to oogenesis. In adult hermaphrodites (which are producing oocytes), most fem-3 RNA is found in the germ line, consistent with both the requirement for fem-3 in hermaphrodite spermatogenesis and the maternal effects of fem-3 on embryonic sex determination. Whereas loss-of-function mutants in fem-3 produce only oocytes, hermaphrodites carrying any of nine fem-3 gain-of-function (gf) mutations make none; instead sperm are produced continuously and in vast excess over wild-type amounts. Genetic analyses suggest that fem-3(gf) mutations have escaped a negative control required for the switch to oogenesis. Here we report that all nine fem-3(gf) mutants carry sequence alterations in the fem-3 3' untranslated region (3' UTR). There is no increase in the steady-state level of fem-3(gf) RNA over wild-type, but there is an increase in the polyadenylation of fem-3(gf) RNA that is coincident with the unregulated fem-3 activity. Results of a titration experiment support the hypothesis that a regulatory factor may bind the fem-3 3' UTR. We speculate that fem-3 RNA is regulated through its 3' UTR by binding a factor that inhibits translation, and discuss the idea that this control may be part of a more general regulation of maternal RNAs.