Mammalian development: new trick for an old dog

A recent study has shown that the T-box gene eomesodermin, which was first identified in Xenopus, not only plays a conserved role in vertebrates directing gastrulation, but interestingly in mammals has acquired a new function in the development of the trophoblast.     

Expression pattern of mouse sFRP-1 and mWnt-8 gene during heart morphogenesis

The Wnt genes encode a large family of secreted proteins that play a key role in embryonic development and tissue differentiation in many species (Rijsewijk et al., 1987 and Nusse and Varmus, 1992). Genetic and biochemical studies have suggested that the frizzled proteins are cell surface receptors for Wnts (Vinson et al., 1989, Chan et al., 1992, Bhanot et al., 1996 and Wang et al., 1996). In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified (Finch et al., 1997, Melkonyan et al., 1997 and Rattner et al., 1997). One of these proteins, FrzA, the bovine counterpart of the murine sFRP-1 (93% identity) is involved in vascular cell growth control, binds Wg in vitro and antagonizes Xwnt-8 and hWnt-2 signaling in Xenopus embryos (Xu et al., 1998 and Duplàa et al., 1999). In this study, we report that sFRP-1 is expressed in the heart and in the visceral yolk sac during mouse development, and that sFRP-1 and mWnt-8 display overlapping expression patterns during heart morphogenesis. From 8.5 to 12.5 d.p.c., sFRP-1 is expressed in cardiomyocytes together with mWnt-8 but neither in the pericardium nor in the endocardium; at 17.5 d.p.c., they are no longer present in the heart. In mouse adult tissues, while sFRP-1 is highly detected in the aortic endothelium and media and in cardiomyocytes, mWnt-8 is not detected in these areas. Immunoprecipitation experiments demonstrates that FrzA binds to mWnt-8 in cell culture experiments.     

Expression of Brachyury-like T-box transcription factor, Xbra3 in Xenopus embryo

The Xenopus Brachyury-like Xbra3 gene is a novel T-box gene that is closely associated with Xenopus Brachyury. The expression pattern of Xbra3 during development is similar to that of Xbra. During gastrulation Xbra3 is expressed in the marginal zone, with a gradient of increasing expression from ventral to dorsal. In the early neurula stage Xbra3 is expressed in the notochord and posterior mesoderm, but by the tailbud stage its expression is restricted to the forming tailbud and the posterior portion of the notochord.     

Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.     

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004 [1]; Larsen et al., 2005 [2]; Kweon and Hakansson, 2006 [3]; Cantin et al., 2007 [4]; Collins et al., 2007 [5]). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO₂-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007 [6]). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO₂ and ZrO₂ columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.     

Flik, a chick follistatin-related gene, functions in gastrular dorsalisation/neural induction and in subsequent maintenance of midline Sonic hedgehog signalling.

We have targetted the chick gene Flik with antisense oligodeoxynucleotide treatment at gastrular stages, when it is expressed in organiser-derived structures of the midline (K. Patel et al., 1996, Dev. Biol. 178, 327-342). A specific syndrome of deficient axial patterning and holoprosencephaly is produced. Most aspects of this syndrome can be understood as due to attenuation of dorsalising and neural-inducing signals during gastrulation, followed by failure to maintain the later signals from chordamesoderm/neural midline that pattern the mesodermal and neural cross sections during subsequent stages. Anatomical effects are first apparent at early neurula stages and correspond with what might be expected from a reduced counteraction of the ventralising Bone morphogenetic protein (BMP) pathway at the earlier stages, coupled with inadequate Sonic hedgehog (Shh) signalling subsequently. Delay in the clearing of BMP-4 RNA expression from the presumptive neural region at gastrulation is indeed seen, though chordin RNA expression within organiser derivatives remains normal. Subsequently, specific attenuation of chordamesoderm and neural midline Shh expression is observed. Brief preincubation of stage 4 chick blastoderms in supernatant from Xenopus oocytes that have been injected with Flik RNA prolongs and enhances the competence of their peripheral epiblast to respond to neural inductive signals from grafted Hensen's nodes. This effect specifically mimics that recently observed using microg/ml solutions of recombinant Follistatin (D. J. Connolly et al., 1999, Int. J. Dev. Biol., in press), further suggesting that Flik protein might act in vivo by somehow modulating activity of signalling pathways through BMP or other TGFbeta-related ligands. We discuss the significance of the observations in relation to recent ideas about neural induction, about possible redundancy in gene action, and about subsequent patterning of the axial cross section, suggesting that a Flik function in autocrine/paracrine maintenance of later midline Shh signalling represents a role of the gene separate from that in primary dorsalisation/neural induction.     

A history of stress alters drought calcium signalling pathways in Arabidopsis

Environmental stresses commonly encountered by plants lead to rapid transient elevations in cytosolic free calcium concentration ([Ca2+]cyt) (Bush, 1995; Knight et al., 1991). These cellular calcium (Ca2+) signals lead ultimately to the increased expression of stress-responsive genes, including those encoding proteins of protective function (Knight et al., 1996; Knight et al., 1997). The kinetics and magnitude of the Ca2+ signal, or 'calcium signature', differ between different stimuli and are thought to contribute to the specificity of the end response (Dolmetsch et al., 1997; McAinsh and Hetherington, 1998). We measured [Ca2+]cyt changes during treatment with mannitol (to mimic drought stress) in whole intact seedlings of Arabidopsis thaliana. The responses of plants which were previously exposed to osmotic and oxidative stresses were compared to those of control plants. We show here that osmotic stress-induced Ca2+ responses can be markedly altered by previous encounters with either osmotic or oxidative stress. The nature of the alterations in Ca2+ response depends on the identity and severity of the previous stress: oxidative stress pre-treatment reduced the mannitol-induced [Ca2+]cyt response whereas osmotic stress pretreatment increased the [Ca2+]cyt response. Therefore, our data show that different combinations of environmental stress can produce novel Ca2+ signal outputs. These alterations are accompanied by corresponding changes in the patterns of osmotic stress-induced gene expression and, in the case of osmotic stress pre-treatment, the acquisition of stress-tolerance. This suggests that altered Ca2+ responses encode a 'memory' of previous stress encounters and thus may perhaps be involved in acclimation to environmental stresses.     

Expression pattern of mouse sFRP-1 and mWnt-8 gene during heart morphogenesis

The Wnt genes encode a large family of secreted proteins that play a key role in embryonic development and tissue differentiation in many species (Rijsewijk et al., 1987 and Nusse and Varmus, 1992). Genetic and biochemical studies have suggested that the frizzled proteins are cell surface receptors for Wnts (Vinson et al., 1989, Chan et al., 1992, Bhanot et al., 1996 and Wang et al., 1996). In parallel, a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified (Finch et al., 1997, Melkonyan et al., 1997 and Rattner et al., 1997). One of these proteins, FrzA, the bovine counterpart of the murine sFRP-1 (93% identity) is involved in vascular cell growth control, binds Wg in vitro and antagonizes Xwnt-8 and hWnt-2 signaling in Xenopus embryos (Xu et al., 1998 and Duplàa et al., 1999). In this study, we report that sFRP-1 is expressed in the heart and in the visceral yolk sac during mouse development, and that sFRP-1 and mWnt-8 display overlapping expression patterns during heart morphogenesis. From 8.5 to 12.5 d.p.c., sFRP-1 is expressed in cardiomyocytes together with mWnt-8 but neither in the pericardium nor in the endocardium; at 17.5 d.p.c., they are no longer present in the heart. In mouse adult tissues, while sFRP-1 is highly detected in the aortic endothelium and media and in cardiomyocytes, mWnt-8 is not detected in these areas. Immunoprecipitation experiments demonstrates that FrzA binds to mWnt-8 in cell culture experiments.     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

Expression of the T-box gene Eomesodermin during early mouse development.

We report the expression pattern of a murine homolog of the Xenopus laevis T-box gene Eomesodermin. mEomes expression is first detected in the extra-embryonic ectoderm prior to gastrulation, and persists there until head-fold stages. In the embryo proper, mEomes is expressed throughout the early primitive streak, nascent mesoderm and in the anterior visceral endoderm. Although mEomes expression disappears from the embryo at late-streak stages, a second domain of mEomes expression is observed in the telencephalon beginning around E10.5.