XlPOU 2 is a potential regulator of Spemann’s Organizer
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Abstract:
XIPOU 2, a member of the class III POU-domain family, is expressed initially at mid-blastula transition (MBT) and during gastrulation in the entire marginal zone mesoderm, including Spemann's Organizer (the Organizer). To identify potential targets of XIPOU 2, the interaction of XIPOU 2 with other genes co-expressed in the Organizer was examined by microinjecting XIPOU 2's mRNA into the lineage of cells that contributes to the Organizer, head mesenchyme and prechordal plate. XIPOU 2 suppresses the expression of a number of dorsal mesoderm-specific genes, including gsc, Xlim-1, Xotx2, noggin and chordin, but not Xnot. As a consequence of the suppression of dorsal mesoderm gene expression, bone morphogenetic factor-4 (Bmp-4), a potent inducer of ventral mesoderm, is activated in the Organizer. Gsc is a potential target of XIPOU 2. XIPOU 2 is capable of binding a class III POU protein binding site (CATTAAT) that is located within the gsc promoter, in the activin-inducible (distal) element. Furthermore, XIPOU 2 suppresses the activation of the gsc promoter by activin signaling. At the neurula and tailbud stages, dorsoanterior structures are affected: embryos displayed micropthalmia and the loss of the first branchial arch, as detected by the expression of pax-6, Xotx2 and en-2. By examining events downstream from the Wnt and chordin pathways, we determined that XIPOU 2, when overexpressed, acts specifically in the Organizer, downstream from GSK-3beta of the Wnt pathway and upstream from chordin. The interference in dorsalizing events caused by XIPOU 2 was rescued by chordin. Thus, in addition to its direct neuralizing ability, in a different context, XIPOU 2 has the potential to antagonize dorsalizing events in the Organizer.Keywords:
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Chordin
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Chordin
Neurula
Otic vesicle
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The ventro-lateral amphibian mesoderm is the presumptive area of blood cells, lateral plates and mesenchyme. To these mesodermal derivatives primordial germ cells (PGCs) must be added in the urodeles. Mesoderm and PGCs are formed from ectodermal cap of the gastrula under inductive influence of ventral endoderm. The present study was carried out to investigate the evolution of ventro-lateral and PGCs determination in Pleurodeles waltlii. The experiments are performed with mesodermal explant extirpated at successive stages of development, from young gastrula to mid-neurula (st. 8a, 9, 13, 15 and 18) and grafted in the tail-bud ventral or dorsal region. In the larva, the analysis of the prestations formed by the graft according to its age and its location on the host is made in serial sections. In dorsal location, the fate of the ventro-lateral gastrula mesoderm is dorsalized by contact with the host axial structures. This influence is characterized by a large amount of nephretic tubules. The potential presence of blood cells and PGCs in the young gastrula ventro-lateral mesoderm having been shown in other experiments, it seems that these cells are unable to self-differentiation before the end of gastrulation. It is proposed that their final determination is acquired only by a complementary interaction taking place during gastrulation and beginning neurulation.
Germ layer
Lateral plate mesoderm
Neurula
Paraxial mesoderm
Neurulation
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Chordin
Neurula
Decapentaplegic
Notochord
Chordate
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Introduction: Numerous reports have shown that recombinant BMPs have positive effects in several conditions associated with poor bone formation. We hypothesize that by inhibiting BMP antagonists Noggin and Chordin using RNA interference we may upregulate endogenous BMP expression and enhance osteogenesis. Methods: MC3T3-E1 cells were transduced with lentiviruses expressing various shRNAs targeting the mouse Noggin (5 shRNAs) and Chordin (3 shRNAs) genes and 1 negative control. At various time points after infection, levels of RNA expression for Noggin and Chordin were monitored through RT-PCR. Western blotting were performed on the cell extracts and culture media to verify the expression and secretion of Noggin and Chordin proteins. Cell extracts were also analyzed on day 2 and 4 after transduction for alkaline phosphastase activity which is a marker of osteogenic differentiation. DO was performed on the right tibia of 54 wild-type mice using a miniature Illizarov distraction device. Animals were randomized into two major groups according to the time of sacrifice: end of distraction (day 17) and mid-consolidation (day 34). Each major group was sub-divided in 3 subgroups representing the type of injection that was administered at the site of distraction on day 8. For each time point, 9 mice were injected with PBS, 9 with a lentivirus plasmid containing a Non-Target (NT) shRNA and 9 with lentivirus plasmids containing shRNAs targeting Noggin-Chordin. To evaluate the success of the infection a GFP marker was added to lentivirus plasmid targeting Noggin-Chordin. For each subgroup 6 collected samples were studied using Faxitron X-ray, μCT and immunohistochemistry and 3 were studied using Rt-PCR. Results: On non-tranduced MC3T3-E1 cells, the levels of RNA expression of Noggin and Chordin was at its highest level on Day 7. At this timepoint, qRT-PCR analysis showed that the shRNAs were effective in knocking down Noggin and Chordin endogenous mRNA levels down to 10% and 17%, respectively, by the most potent of shRNAs tested compared to control. Western Blot analysis also corroborates that the respective shRNAs were effective in knocking down the Noggin and Chordin proteins. Specific activity of alkaline phosphatase was increased in MC3T3-E1 cells stably expressing Noggin and Chordin shRNA.Concerning our in vivo work, all the surgeries and distraction were successful except for the death of 2 animals secondary to post-operative complications. However, at the time of the injection the majority of the injection content was extravasating out of the injection site. Once the samples collected, the Faxitron X-ray and μCT studies did not show a significant difference of bone volume or in the ratio of bone volume/tissue volume at the distraction site in between the 3 injection subgroups at both time points. The Rt-PCR studies did not show a significant difference in inhibiting Noggin or Chordin mRNA levels in between the 3 subgroups at both time points. Finally, the immunohistochemistry studies were stopped after the GFP marker…
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Follistatin
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Blastula
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Primitive streak
FGF8
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Injection of RNA encoding BMP-4 into the early Xenopus embryo suppresses formation of dorsal and anterior cell types. To understand this phenomenon, it is necessary to know the stage at which BMP-4 acts. In this paper, we present three lines of evidence showing that BMP-4 misexpression has no effect on the initial steps of mesoderm induction, either dorsal or ventral, but instead causes ventralization during gastrulation. Firstly, activation of organizer-specific genes such as goosecoid, Xnot, pintallavis and noggin occurs normally in embryos injected with BMP-4 RNA, but transcript levels are then rapidly down-regulated as gastrulation proceeds. Similarly, BMP-4 does not affect the initial activation of goosecoid by activin in animal caps, but expression then declines precipitously. Secondly, embryos made ventral by injection with BMP-4 RNA cannot be rescued by grafts of Spemann's organizer at gastrula stages. Such embryos therefore differ from those made ventral by UV-irradiation, where the defect occurs early and rescue can be effected by the organizer. Finally, the dorsalizing effects of the organizer, and of the candidate dorsalizing signal noggin, both of which exert their effects during gastrulation, can be counteracted by BMP-4. Together, these experiments demonstrate that BMP-4 can act during gastrulation both to promote ventral mesoderm differentiation and to attenuate dorsalizing signals derived from the organizer.
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Abstract In Xenopus, ectodermal cell fates are determined by antagonistic interaction between the BMP subfamily of TGF-β ligands and the organizer-specific secreted factors (e.g. noggin, chordin and follistatin). Inhibition of BMP function by these factors can convert cells from an epidermal to a neural cell fate. In this study, we report that GDF6, a new member of the Xenopus TGF-β family, can function in antagonistic interaction with neural inducers. GDF6 induces epidermis and inhibits neural tissue in dissociated cells, and this activity is blocked by the presence of noggin. We demonstrate that GDF6 binds directly to the neural inducer noggin. Furthermore, we find that GDF6 and BMP2 can form heterodimers and the process seems to require cotranslation of the proteins in the same cells. In normal embryos, GDF6 and BMP2 are coexpressed in several places, including the edge of the neural plate at early neurula stages, suggesting that GDF6 may synergize with BMPs to regulate patterning of the ectoderm. Our data show for the first time that noggin can bind directly to and inhibit another TGF-β family member: GDF6. In addition, BMP and GDF6 heterodimers may play an important role in vivo to regulate cell fate determination and patterning.
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Neurula
Neural Development
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Neurula
Chordin
Notochord
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BMP signals play important roles in the regulation of diverse events in development and in the adult. In amniotes, like the amphibian Xenopus laevis, BMPs promote ventral specification, while chordin and other BMP inhibitors expressed dorsally in the Spemann's organizer play roles in establishment and/or maintenance of this region as dorsal endomesoderm. The activities of chordin are in turn regulated by the secreted proteolytic enzymes BMP1 and Xolloid. Recently, we and others have identified the protein twisted gastrulation (TSG) as a soluble BMP modulator that functions by modifying chordin activity. Overexpression and genetic analyses in Drosophila, Xenopus and zebrafish together with in vitro biochemical studies suggest that TSG might act as a BMP antagonist; but there is also evidence that TSG may promote BMP signaling. Here we report examination of the in vivo function of TSG in early Xenopus development using a loss-of-function approach. We show that reducing TSG expression using antisense TSG morpholino oligonucleotides (MOs) results in moderate head defects. These defects can be rescued both by a TSG that cannot be inhibited by the MO, and by the BMP antagonists chordin and noggin. Furthermore, while neither the onset of gastrulation nor the expression of marker genes are affected in early gastrulae, dorsal marker gene expression is reduced at the expense of expanded ventral marker gene expression beginning at mid to late gastrula stage. TSG-MO and Chd-MOs also cooperate to strongly repress head formation. Finally, we note that the loss of TSG function results in a shift in tissue responsiveness to the BMP inhibitory function of chordin in both animal caps and the ventral marginal zone, a result that implies that the activity of TSG may be required for chordin to efficiently inhibit BMPs in these developmental contexts. These data, taken together with the biochemistry and overexpression studies, argue that TSG plays an important role in regulating the potency of chordin's BMP inhibitory activity and TSG and chordin act together to regulate the extent of dorsoanterior development of early frog embryos.
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Morpholino
Loss function
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