In Drosophila, the germ layers are established by the gradient of nuclear Dorsal protein, with the ventral-most cells expressing the highest levels of Dorsal. In response, these cells express mesoderm-specific transcription factors such as twist and snail.
Isolated animal cap cells from an early blastula developed into ectoderm, while isolated vegetal cells developed into endoderm. When animal and vegetal cells were combined, only ectoderm and endoderm formed -- no mesoderm formed.
When animal cap and vegetal cells from a late blastula were combined, all cell types (ectoderm, mesoderm, and endoderm) formed. Thus, the embyo becomes competent to form mesoderm during the transition from early to late blastula.
Marking animal and vegetal cells with dyes revealed that mesoderm arose from animal cells but only formed in the presence of vegetal cells. Thus, mesoderm was induced in animal cells by secreted signals from vegetal cells.
The egg possesses cytoplasmic determinants required for the formation of endoderm and mesoderm. These determinants are vegetally localized prior to fertilization. Two key determinants are Veg-T (transcription factor) and Vg-1 (secreted factor). This correlates to amphibian embryo fate maps, as the mesoderm arises from the marginal zone.
|Mesoderm||Marginal||Vegetal secretions induce animal cells into mesoderm.|
The Nieuwkoop Center is responsible for mesodermal cells' dorsal-ventral identity. When Nieuwkoop removed the marginal zone and recombined animal and vegetal caps, the animal cells overlying vegetal cells became dorsal mesoderm (notochord and somites). Later researchers combined the animal cap with different vegetal blastomeres. Dorsal mesoderm always resulted, except with highly ventral vegetal blastomeres which induced ventral mesoderm (ie, blood cells). Thus, vegetal secretions induce mesoderm in overlying animal cells, and the Nieuwkoop Center ventralizes overlying mesoderm.
Identifying D/V Axis Determinants in Xenopus
Differential screens reveal gene expression at a time and place.
Observing Xenopus homologs of mammalian genes for activity.
Injecting cloned mRNAs to test for ability to induce a new axis.
Determining Their Function
Once identified, the role of such genes has been assessed by injecting into one cell of the two-cell embryo the corresponding mRNA, antisense DNA oligonucleotides or DNA construct containing a dominant mutation. One then asks whether this treatment either causes or blocks axis formation/dorsalization.
Transplantation experiments suggested that vegetal cells uniformly express a mesoderm-inducing signal prior to activation of the zygotic genome. Thus, researchers sought a maternal mRNA localized to the vegetal region of the Xenopus egg. Vg1, a secreted TGF-β, was identified due to its vegetally localized mRNA in the unfertilized egg. Furthermore, injection of activated Vg1 mRNA can induce mesoderm.
The maternal mRNA VegT is localized to the vegetal region of the Xenopus egg. VegT encodes a transcription factor required for endoderm formation and induction of animal cells into mesoderm. Depletion of VegT mRNA by antisense oligos causes the vegetal region to form into mesoderm and ectoderm, instead of endoderm. Also, Nieuwkoop experiments show that vegetal cells depleted of VegT are unable to induce animal caps to form mesoderm.
There must also be signals that differentiate the dorsal from the ventral side of the embryo at an early stage. We have already seen that β-catenin becomes localized on the ventral side of the embryo. However, β-catenin is a transcription factor, not a secreted factor, so it must be activating the expression of genes that cause the induced mesoderm to adopt dorsal fates.
We now know that the Nieuwkoop center establishes a gradient of Xnr, which stands for Xenopus nodal related molecules (TGF-β-like cell signaling proteins). There are five of these molecules in Xenopus, and they appear to act in a similar, overlapping fashion. Highest levels of expression are seen on the dorsal side of the embryo, and lower levels are seen on the ventral side. Thus, a key activity of the Nieuwkoop center is to increase the level of expression of Xnr molecules on the dorsal side of the embryo. Both loss-of-function and gain-of-function experiments (using injection into early blastomeres followed by Nieuwkoop assays (culturing of explants) indicate that the Xnrs are both necessary and sufficient for induction of both dorsal and ventral mesoderm at the blastula stage.
Consistent with the model, Xnr1 is expressed in a dorsal (high) to ventral (low) gradient in the early embryo, and different levels of Xnr have different effects on induction of ventral versus dorsal mesoderm. In conclusion, the recent data support a new model in which VegT plus Vg1 at the vegetal pole (provided by localized deposition of maternal mRNA), plus the dorsal accumulation of b-catenin (as a result of cortical rotation) set up, in the prospective endoderm, a zygotic dorsal to ventral gradient of Xnr activity. High levels of Xnr induce dorsal mesoderm, and low levels induce ventral mesoderm.