| Term | Yolk | Cleavage | Result | Model Organisms | Notes |
| Alecithal | Little/None | Holoblastic | Blastocyst | Mammal | |
| Centrolecithal | Center | Superficial | Blastoderm | Insect | |
| Isolecithal | Even | Holobastic | Blastula | Urchin, Sand Dollar | |
| Mesolecithal | Uneven | Holoblastic | Blastula | Amphibian | Sometimes known as telolecithal. |
| Telolecithal | Very Uneven | Meroblastic | Blastodisc | Bird, fish, reptile | Sometimes known as extreme telolecithal. |
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| Factor | Overview |
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| CDK | |
| Cyclin | |
| Cytoskeleton | Includes microtubules and microfibers. Microtubules inhibited by colchicine and nocodazole (inhibit chromosome segregation); and microfilaments inhibited by cytochalasin (inhibits cytokinesis). |
In Xenopus, the Nieuwkoop Center is the dorsal- and vegetal-most region. It gives rise to the Primary Organizer (aka Spemann Organizer or Spemann-Mangold Organizer), which is the region known as the dorsal lip of the blastopore (DLB). Spemann and Mangold’s experiments found that 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.
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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:
| Factor | Overview |
| Transcription | Three homeodomain proteins: Lim1, Gooseceoid and Xnot. Also, HNF3β. |
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| 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. |
The primary organizer arises in the dorsal marginal zone via induction by the Nieuwkoop center. 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 marginal zone involutes at the DLB during gastrulation to give rise to the mesoderm.
Notice that this figure (multi-cellular) differs from the one immediately above (single-cell) in that the dorsal-ventral axis is now horizontal.
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.
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 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.
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.
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.
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).
| Step | Overview |
| 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. |
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| 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. |
At the 8-cell stage, the mammalian embryo undergoes compaction where the spherical and loose blastomeres nestle tight together to establish new cell junctions and form a blastocyst. The blastocyst has an outer trophoblast layer (responsible for implantation into the uterine wall) and an inner cell mass placed eccentrically within the blastocoel. The inner cell mass gives rise to the embryonic epiblast (from which the embryo arises), the overlying amniotic ectoderm (separated from the epiblast by the amniotic cavity) and the underlying hypoblast. The mammalian blastocyst is analogous in structure to the yolky bird embryo, without the yolk.
Intrinsic (aka cytoplasmic) and extrinsic determinants coexist, as shown by two (respective) urchin blastula experiments. Removing the progenitor cell of mesoderm from an urchin blastula results in a mesoderm-lacking larva. However, sagittally halving an entire blastula will result in a normal larva. The phenom of extrinsic determinants is known as regulative development.
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