By Levi Clancy for Student Reader on
In Xenopus, the Nieuwkoop Center is the most dorsal and vegetal region.
The Nieuwkoop Center is the dorsal- and vegetal-most cell of the early blastula. It gives rise to the Primary Organizer, which is the dorsal lip of the blastopore (DLB). The Primary Organizer has a dorsalizing effect, and together with the Sperm Entry Point (SEP) gives rise to the dorsal/ventral axis. Dorsalized tissue gives rise to somites and pronephric tubules.
The Primary Organizer is also called the Spemann(-Mangold) Organizer/Node after Spemann and Mangold, whose experiments found that the DLB dorsalizes surrounding tissue, thus forming (along with the SEP) the dorsal-ventral axis. Thus the DLB was named after them.
|Dorsal-ventral axis||The DLB dorsalizes surrounding tissue, thus forming (along with the SEP) the dorsal-ventral axis. In addition to dorsalizing surrounding tissue, the primary organizer: fates overlying ectoderm as neural plate tissue; and is determined to be notochord tissue. Dorsalized tissue gives rise to somites and pronephric tubules.|
|Anterior-posterior axis||By inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk|
The Nieuwkoop center arises in the dorsal vegetal zone due to a gradient of nodal-related proteins (for frogs, Xenopus nodal-related or Xnr).
The primary organizer arises in the dorsal marginal zone via induction by the Nieuwkoop center. The marginal zone involutes at the DLB during gastrulation to give rise to the mesoderm.
Induction By Primary Organizer
The DLB uses induction (interaction with adjacent cells) via secreted diffusible signals. A cell that can be induced is competent; embryonic tissues are only competent during gastrulation.
The use of diffusible substances was proven when dorsal lip tissue and ectoderm were cultured together, but separated by a filter with a 0.5µm pore; the ectoderm was induced into neural tissue, despite no cell processes seen to pass through the filter.
Dr. DeRobertis identified genes expressed only in the organizer via differential screening of Xenopus dorsal lips. Direct purification was ineffective because the hypersensitive ectoderm overlying the chordamesoderm was induced by even unnatural substances. Organizer-specific gene products are divided into two groups:
|Transcription||Three homeodomain proteins: Lim1, Gooseceoid and Xnot. Also, HNF3β.|
|Secreted||The noggin, follistatin, chordin and frzb gene products induce the neural plate by antagonizing the ventralizing and mesodermalizing properties of BMP-4 and Wnt-8. These genes can induce a second axis when their mRNA is injected into an early embryo.|
Notice that this figure (multi-cellular) differs from the one immediately above (single-cell)
in that the dorsal-ventral axis is now horizontal.
A transplanted vegetal dorsal cell (from the DLB) induces a new axis.
A transplanted vegetal dorsal cell does not itself give rise to new dorsal tissues.
A transplanted vegetal dorsal cell restores other cells to correct fates.
The three-signal model accounts for the induction of many tissues, with the Nieuwkoop Center playing a pivotal role.
The first signals are the vegetal maternal mRNAs Veg1 and VegT. The second signal is the dorsal-most protein β-catenin, which accumulates via cortical rotation. This induces formation of the Spemann Organizer and the Nieuwkoop Center. The Spemann Organizer is responsible for the third signal by expressing Noggin, Chordin and Follistatin, which bind and inhibit the ventralizing factors BMP-4 and Frzb.
According to the three-signal model, ventral mesoderm (which gives rise to blood forming cells) is induced by underlying vegetal cells (signal 1); the primary organizer (the most dorsal mesoderm, which gives rise to notochord and somites) is induced by the dorsal-most vegetal blastomeres (Nieuwkoop center) (signal 2); and the dorsal-most marginal cells then interact with the adjacent marginal cells (signal 3) and cause them to become lateral mesoderm (which forms kidney and lateral muscle).
The organizer secretes Chordin and Frizbee, diffusible antagonizers of BMP4 and Wnt-8, to establish a gradient of activity (not concentration) of BMP-4 and Wnt8 activity in the mesoderm. In response to this gradient, zygotic genes are transcribed in a gradient: dorsally near the organizer are goosecoid, pintallavis, HNF-3β, Xnot, Xlim-1; ventrally is Brachyury. Different types of mesoderm form along the dorsal-ventral axis.
1st Signal: Veg1
The first signal (Veg1) is a vegetal-localized maternal mRNA that encodes a TGF-β. Injection of Veg1 mRNA rescues irradiated embryos, and at high levels induces dorsal mesoderm. VegT is another vegetal-localized Xenopus mRNA. VegT encodes a T domain protein required for endoderm formation and transcription of mesoderm-inducing signals. VegT-ablated embryos form mesoderm and ectoderm but not endoderm, and cannot induce animal caps to form mesoderm.
2nd Signal: β-catenin
The second signal (β-catenin) differentiates the dorsal region from the ventral region at an early stage. β-catenin was identified when cortical rotation was noted to cause a high concentration of β-catenin to appear near the Nieuwkoop Center. When activated by Wnt signaling, β-catenin links E-cadherin to the actin cytoskeleton and is a transcription factor. As a result, the Nieuwkoop Center contains higher levels of Veg1, VegT and β-catenin to produce a signal inducing dorsal mesoderm.
Xenopus Nodal-Related Proteins
Xenopus nodal related molecules (Xnr) are five TGF-β-like signals with overlapping and similar function. VegT and TCF/LEF (requiring β-catenin as a cofactor) overlap, establishing a Xnr gradient along the D/V axis. High [Xnr] induces dorsal mesoderm, while low [Xnr] induces ventral mesoderm. Ablating and increasing Xnr activity (using Cerberus, a head inducer and Xnr inhibitor) indicates that Xnr is necessary and sufficient for inducing dorsal and ventral mesoderm at the blastula stage.
Overview So Far
After the 1st and 2nd signals, the maternal mRNAs VegT and Veg1 are vegetal-localized. β-catenin accumulates dorsally via cortical rotation. This causes a perfect storm at the vegetal- and dorsal-most position of Veg1, VegT, TCR/LEF and the cofactor β-catenin. From this vegetal- and dorsal-most position arises a dorsal to ventral gradient of Xnr activity. The Xnr gradient is encoded by the zygotic genome. High [Xnr] induce dorsal mesoderm, and low [Xnr] induce ventral mesoderm.
3rd Signal: Noggin, Chordin & Follistatin
The Spemann Organizer emits the third signal to dorsalize adjacent mesoderm. This signal consists of: Noggin, Chordin and Follistatin, which bind and inhibit the ventralizing growth factors BMP-4 and Frzb. Frzb antagonizes Wnt-8. Noggin was identified when injection of Noggin mRNA dorsalized and partially rescued irradiated embryos. Chordin and Frzb were identified during a differential screen for genes expressed only in the DLB. Injection of either Noggin or Chordin mRNA into a four cell stage embryo induces a second axis. The Spemann Organizer exclusively encodes transcriptional activators for BMP-4 and Wnt-8 antagonists, including: three homeobox genes, Goosecoid, Lim1 and Xnot; the fork head protein, HNF3-β.
Xenopus and Drosophila Homologs
This is similar to the the Drosophila Dpp (BMP-4 homolog) activity morphogen gradient, which is highest at the dorsal region and lowest at the ventral region where Sog (Chordin homolog) binds and inactivates Dpp to allow Dorsal protein expression. BMP-4 is a Dpp homolog and Chordin is a Sog homolog. These Xenopus genes can be interchanged with their Drosophila homologs. However, since the dorsal-ventral axis (as well as the heart location) was inverted in an ancestor of vertebrates, Dpp promotes dorsal formation in Drosophila and its homolog BMP-4 promotes ventral formation in Xenopus (accordingly, the opposite goes for Chordin and Sog). Wnt-8 is a homolog of Drosophila's Wingless.
Methods to identify genes for early Xenopus embryo D/V and A/P patterning include: differential screening (aka subtractive hybridization) to identify genes expressed at specific times and places, as with the egg vegetal pole and early gastrula organizer tissue; testing Xenopus homologs of mammalian cell signaling genes for ability to induce mesoderm in isolated animal caps; and testing cloned mRNAs for ability, when injected, to induce a new axis. After identifying relevant genes, their role was assessed by injecting into the one- or two-cell embryo the corresponding mRNA, antisense DNA olignucloetides, RNAi or dominant negative or active DNA construct of the gene. After this injection, the embryo is examined whether it does or does not form an axis (dorsalize).
Spemann and Mangold's experiment to identify the Nieuwkoop Center's role
|Step 1||Niuewkoop found that marginal zone cells from an early blastula (before 64 cell stage) did not form mesoderm when isolated and cultured. However, marginal zone cells from a blastula after the 64 cell stage did form mesoderm.|
|Step 2||Nieuwkoop removed the marginal zone and recombined dye-marked animal and vegetal caps. Dorsal mesoderm arose from animal cap cells nearest the vegetal cap, and from vegetal cells opposite the SEP.|
|Step 3||Other biologists combined the animal cap with different vegetal blastomere cells. Dorsal vegetal blastomere cells induced dorsal mesoderm; ventral vegetal blastomere cells induced ventral mesoderm. Also, dorsal mesoderm induced ventral mesoderm to become lateral mesoderm. Thus, signaling must be involved in dorsal and central cell fates.|
|Step 4||Gimlich and Gerhart found that fertilized eggs did not form a D/V axis when irradiated at the vegetal pole, but could be restored by a single dyed vegetal- and dorsal-most cell (though this cell was not itself dorsal mesoderm). Thus, this vegetal- and dorsal-most structure must induce other regions to become dorsal mesoderm. This vegetal-most and dorsal-most region first induces the primary organizer (which then induces other tissues) and was called the Nieuwkoop center.|
|Step 5||To determine which part of the embryo acts to organize the mesoderm into dorsal structure and to induce neural tube formation, Spemann and Mangold transplanted the dorsal lip of the blastopore of an early gastrula from a light-colored newt into an early gastrula of a dark-colored newt. The donor tissue formed a second embryonic axis. The notochord of this second embronic axis was composed entirely of graft (donor) tissue, while the neural tube and somites were composed only partly of graft (donor) tissue and the kidney tubules and gut of the new axis were composed entirely of host tissue. Spemann and Mangold concluded that the graft tissue induced a new embryonic axis. This structure is named Primary organizer.|
Dorsal-ventral patterning in Xenopus and Drosophila.
Drosophila Dpp is homologous to BMP-4. Dpp activity is highest at the dorsal region. Drosophila Sog is homologous to Chordin. [Sog] is highest at the ventral region, where it binds and inactivates Dpp. These Drosophila genes can be replaced by their Xenopus homologs, but their activity along the dorsal-ventral axis is inverted. This switching occurred in a vertebrate ancestor. Also, Drosophila Wingless is homologous to Wnt-8.