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MOLECULAR BIOLOGY OF SPEMANNâS ORGANIZER AND NEURAL INDUCTION - Lecture 5
Having discussed the early events in mesoderm induction, we now turn to signaling events that take place during gastrulation.
. Dorsalization of mesoderm and neural induction by Spemannâs Organizer during
The âOrganizerâ experiment (Spemann and Mangold, 1924) is the best known experiment in e
gastrulation
mbryology. It has led, more than any other, to the current view that development occurs through a cascade of cell-cell interactions. If the dorsal lip (the site where gastrulation starts) of the blastopore is transplanted to the opposite side of the embryo, it is able to recruit host cells organizing them into a secondary (twinned) body axis containing many histotypes and complex structures.
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The organizer has three main properties: 1) it induces neural tissue on the overlying ectoderm, 2) imparts more dorsal characteristics to the mesoderm of the marginal zone (i.e., âdorsalizes mesodermâ), leading to the formation of somites and trunk muscles, and 3) it induces a secondary gut (âdorsalization of the endodermâ). This small region of the gastrula has been a goldmine for the isolation of new molecules involved in cell signaling. We made organizer-specific libraries and screened them for cDNAs expressed in the organizer (e.g., identification of goosecoid , chordin , cerberus , Frzb-1 ). Other labs used functional injection assays, injecting pools of synthetic mRNAs into the ventral side of embryos and then doing sib-selection until a single gene is identified (e.g., identification of noggin , Siamois , Xtwn , dickkopf ).
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2. Chordin, noggin and follistatin antagonize BMP ventralizing signals.
Microinjection of chordin, noggin or follistatin mRNA will induce secondary axes, rescue UV embryos, dorsalize mesoderm in ventral marginal zone explants and induce CNS differentiation in animal caps.
To our surprise, co-injection of BMP-4 abolished neural induction by chordin, by noggin, and by follistatin, which are secreted proteins of entirely different structures. This antagonism takes place in the extracellular space (as indicated by injecting different blastomeres). Production of the proteins in tissue culture showed that both noggin and chordin bind to BMPs and prevent their binding to BMP receptors. The lack of BMP signaling in turn leads to the dorsalization of mesoderm and neuralization of ectoderm. (The same result can be obtained using a DN-BMPReceptor construct). Thus, the surprising finding was that neural induction and the dorsalization of mesoderm (and also of endoderm) had the same molecular basis: antagonism of ventral BMP signals.
The biggest surprise from these studies on Spemannâs organizer was that patterning by the organizer is effected through secreted antagonists of growth factors:
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Spemannâs organizer is full of inhibitors. Some think that it may have been easier, perhaps, to produce inhibitors during the course of evolution to modulate pathways (âa spanner in the worksâ) than to evolve entirely new signaling pathways. A large number of secreted antagonists have been discovered in various tissues:
3. The Chordino mutation in zebrafish.
A large screen for mutations affecting zebrafish development has been carried out. Two ventralized mutants (less notochord and somites, more blood and small head) were identified. A ventralized mutant is what one would expect from a Spemann organizer mutant.
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Organizer grafts to the ventral side demonstrate that Chordin is required for Spemannâs organizer to induce a CNS.
In control grafts of dorsal lip into the dorsal region, an unexpected cell-autonomous requirement for Chordin was revealed. The embryo prefers to make CNS from cells that express Chordin. Cells injected with Chd-MO remain in the skin ectoderm instead of differenciating into CNS. Why?
5. An early phase of Chordin expression â The Preorganizer
Right after midblastula there is an early phase of Chordin expression in a region called the preorganizer, which gives rise to the CNS later on.
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At gastrula Chd requires Nodal signaling and can be inhibited by Cerberus-short mRNA. The blastula preorganizer expression requires β- Catenin signals since it is blockec by âN-TCF-3.
Injection of β-catenin or GSK-3 mRNA, or treatment with LiCl will induce CNS formation in animal caps (neural differentiation marked by Six3, Otx-2, Rx2a, En2, Krox20, NCAM and Neuro-tubulin. All anterior neural markers require Chordin (and Noggin). Thus β-catenin induces neural tissue through BMP inhibitors such as Chordin and Noggin expressed at the blastula stage.
The preorganizer is required for anterior CNS formation, explaining findings by Embryologists of the 1920âs. Brain tissue can be induced even if the formation of the gastrula organizer is blocked with the inhibitor Cer- Short. These findings help clarify old observations in which planar signals (diffusing in the plane of the ectoderm) versus vertical
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the other repeats in chd. The same is true for R4, indicating that both genes are derived from a common ancestor.
7. Tolloid/Xolloid proteases cleave sog/chd****. Geoffroyâs victory.
Tolloid , and its Xenopus homologue, Xolloid, encodes a secreted zinc metalloprotease that has five protein-protein interaction repeats present in complement C1 subunits and two EGF repeats. The zebrafish tolloid homologue is mutated in the mini-fin dorsalized mutant.
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This proposition was tested by direct biochemical experiments by Piccolo et al. In co- injection experiments Xolloid inhibited the secondary axis-forming ability of chordin but not that of noggin, follistatin or DN-BMPR. Conditioned medium from cells transfected with Xolloid cleaved recombinant chordin , but not noggin , at two sites. Xolloid also cleaves chordin-BMP complexes at the same sites. Cleavage of chordin inactivates its activity and releases active BMP-4 from inactive [Chordin -BMP ] complexes.
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CRs are found in many extracellular matrix proteins such as fibrillar procollagens (the most abundant proteins in the body), thrombospondin, von Willebrand factor, Neuralin-1, Crossveinless-2, Keilin, CTGF (connective tissue growth factor) and other proteins.
In the case of collagen the CR modules bind BMPs and TGFβ. The bound growth factors in principle might also be released by metalloproteases that cut close downstream of the module (as in the case of chordin), providing active signaling factors when required for tissue homeostasis in the adult.
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9. An additional player: Twisted Gastrulation
The Drosophila gene Twisted-gastrulation (dTsg) encodes a secreted protein that is required for the formation of the amnioserosa, the tissue that requires the highest levels of dpp/screw activity. Thus, dTsg functions to promote maximal BMP signaling. Oelgeschläger et al. (2000) noted that xTsg shared some sequence similarity to the part of the CR domains of chordin. This suggested that xTsg might be a BMP-binding protein, which it was.
xTsg binds BMP, but it does not compete with full-length chordin for the binding of BMP. On the contrary, it stimulated it, because xTsg is also a chordin-binding protein capable of forming a stable ternary complex of chordin, BMP and xTsg. However, when the proteolytic product of chordin digestion by Xolloid, CR1, was used the results were very different: the residual BMP binding activity of CR1 was dislodged by xTsg in cross-linking experiments.
The ternary complex is a much better inhibitor of BMP signaling and the proteolytic cleavage of Chordin subsequently provides the molecular switch that permits BMP/xTsg to be released, allowing binding of BMP to its receptor.
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Dkk-1 mRNA inhibited early Xwnt-8 mRNA effects and also late effects caused by injection of plasmid DNA of cytoskeletal actin promoter-Xwnt-8 (pCSKA-Xwnt-8) at the gastrula stage. This effect occurred upstream of Dsh. Since Dkk encodes a secreted protein it ould function as a secreted Wnt antagonist.
In Xenopus antibody injections into the blastocoel caused cyclopia.
sh
How does this head inducer work? By inhibiting canonical Wnt signaling, as described in lecture 2.
11. Head induction II: Frzb-1 antagonizes Xwnt-****.
Another factor secreted by the organizer is Frzb-1 , which antagonizes the ventralizing effects of Xwnt-8 , a gene normally expressed in the lateral and ventral marginal zone. Xwnt- DNA constructs that drive expression of Xwnt-8 at gastrula turn head and notochord structures into muscle, i.e., into more ventral structures. Frzb-1 is a secreted protein containing a domain
nd leads to embryos with enlarged heads and ortened trunks. An anti-BMP co-injected with Frzb-1 induces heads. Cultured cells transfe rzb-1 in the 10-10^ M range. The organizer secretes many other Wnt antagonists of the Frzb.
At a later stage during gastrulation a second secreted frizzled-like protein is secreted by the ventral
similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of Frzb- mRNA blocks expression of XMyoD mRNA a sh cted with a membrane-tethered form of Wnt-1 bind epitope-tagged F
marginal zone. This gene, called sizzled , was discovered by M. Kirschner at Harvard and serves to restrict Xwnt-8 activity to the somite forming region. The marginal zone becomes subdivided by multiple inhibitory factors into regions of distinct cell fates
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The inibition of BMP and Wnt signaling can be sufficient for the formation of head organizer, whereas BMP inhibition is required for trunk organizer. The work on Cerberus suggests that inhibiting Nodal is also an important step for the formation of the head field.
12. Head induction III: Cerberus and head development
Cerberus, a factor with head-inducing activities is a secreted inhibitor that antagonizes simultaenously Nodal, BMP-4 and Xwnt-8.
Cerberus is expressed in the anterior-most endoderm, and provided the first suggestion that anterior endoderm is involved in head induction.
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elgesc
Tw
iehrs, 20 eyns,. Frzb-1 is a , 747-756. Salic creted Xwnt anta velopment 124 ,
Bouwmeester, T., Kim, S.H., Sasai, Y., Lu, B. and De Robertis, E.M. (1996). Cerberus is a head- ind
O hläger, M., LarraĂn, J., Geissert, D. and De Robertis, E.M. (2000). The evolutionary conserved BMP-binding protein Twisted Gastrulation promotes BMP signalling. Nature 405 ,
LarraĂn, J., Oelgeschläger, M., Ketpura, N.I., Reversade, B., Zakin, L. and De Robertis, E.M. isted
, 631-637.
757-763.
(2001). Proteolytic cleavage of Chordin as a switch for the dual activities of gastrulation on BMP. Development 128 , 4439-4447. C. (2001). The Spemann organizer and embryonic head induction. The EMBO J. L., Bouwmeester, T., Kim, S.-H., Piccolo, S. and De Robertis, E.M. (1997) secreted antagonist of Wnt signaling expressed in the Spemann organizer. Cell 88 , A.N., Kroll, K.L., Evans, L.M. and Kirschner, M.W. (1997). Sizzled : a se gonist expressed in the ventral marginal zone of Xenopus embryos. De 4739-4748.
N L
ucing secreted factor expressed in the anterior endoderm of Spemann's organizer. Nature 382 , 595-601. Piccolo, S., Agius, E., Leyns, L., Battacharyya, S., Grunz, H., Bouwmeester, T. and De Robertis, E.M. (1999). Cerberus induces head structures by binding to and inhibiting Nodal, BMP and Wnt signals in the extracellular space. Nature 397 , 707-710. Oelgeschläger, M., Kuroda, H., Reversade, B. and De Robertis, E.M. (2003). Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus laevis. Dev. Cell 4 , 219-230.
