The Effects of UV Irradiation and Tilting on Xenopus laevis Development

Did You Know?!

It is no surprise that UV causes damage to eggs and embryos of Xenopus laevis. However, who would have thought that tilting the embryo could save it from damage? How is it simply tilting the embryo is able to accomplish such a feat? It is not like tilting yourself after a long day on the beach gets rid of the pain and damage. So how exactly is a simple embryo able to accomplish this feat that even large complex organisms cannot?

Background: How does the Egg Form the Dorsal lip?

Xenopus laevis (Source-cep.unt.edu)

A normal egg is symmetrical about an animal-vegetal axis. The symmetry is the result of cytoplasmic reorganization that occurs during the first division. The reorganization occurs by a 30 degree rotation of the egg cortex in relation to the vegetal mass. The clear cortical cytoplasm is shifted up and away from the sperm entry site and toward the dorsal side; whereas, in the animal hemisphere, the pigmented cortex shifts down towards the sperm entry site. The rotation results in the formation of the gray crescent, which is the precursor of the dorsal lip, due to part of the clear vegetal cortex overlying the pigmented marginal zone cytoplasm.  The dorsal lip, which is the fold of blastula showing the dorsal limit of the blastopore during gastrulation and is important for neural development, will form 180 degrees opposite to the sperm entry point. Therefore, the dorsal side forms where the vegetal cortex meets the animal cytoplasm, and the ventral side forms where the animal cortex meets the vegetal cytoplasm (Hardin 1998).

Figure 1-Shows the cytoplasmic reorganization that occurs during the first division (Source-Gerhart, J., Danilchik, M., Doniach, T., Roberts, S., Stewart, R., & Rowning, B. (1989). Cortical rotation of the xenopus egg: consequences for the anteroposterior pattern of embryonic dorsal development. Development, 37-51)

What does UV Irradiation do to Fertilized Eggs?

UV irradiation of the vegetal halves of fertilized eggs of frog embryos results in the halting of the cortical/cytoplasmic rotation that leads to the gray crescent formation. Therefore, with no gray crescent formation, the result is a ventralized embryo, with no dorsal structures. UV irradiation also stops the formation of an array of parallel microtubules associated with the rotation. Because these fertilized eggs can be rescued by tilting them, the UV has to be disabling cytoplasmic rearrangement rather than destroying an essential dorsal determinant.

In the most recent study, it was found that inhibiting the cortical rotation results in the blocking of the dorsal Wnt pathway and of subsequent Spemann organizer formation. Neither goosecoid nor chordin was expressed in UV-treated embryos, showing that there was no organizer during gastrulation (Jansen et al. 2007). In fact, it was found that other than organizer specific morphogenetic movements, the remaining gastrulation movements are normal in UV-treated embryos. The only thing that changes is organizer convergence and extension. In normal embryos, portions of the mesoderm elongates; whereas, in UV-treated embryos, this elongation is greatly reduced (Jansen et al. 2007).

What does UV Irradiation do to Mature Oocytes Eggs?

However, UV irradiation of mature oocytes inhibits dorsoanterior development in a different way. These eggs cannot be rescued by tilting. These eggs do have parallel microtubules and gray crescent formation, but they still fail to develop dorsoanterior structures. The UV targets of mature oocytes have to be different than those in fertilized eggs because these eggs have the array of parallel microtubules and cannot be rescued by tilting. Therefore, it is thought that the target of UV irradiation in mature oocytes would be dorsal determinants. In fact, 70% of the UV is absorbed within 60 micrometers of the egg surface where there are high concentrations of Vgl mRNA and tubulin mRNA (Elinson and Pasceri 1989). Vgl encodes for TGF β-like protein, which induces animal cells to develop dorsal mesoderm and tubulin make microtubules (Elinson and Pasceri 1989). Vgl mRNA is localized in next to the vegetal cortex during prophase I in oocytes, but it is dispersed throughout the vegetal cytoplasm during fertilization (Elinson and Pasceri 1989). It is thought that UV alters Vgl mRNA, thereby preventing it in forming dorsal development.

From Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization by NATHAN MISE and MASAMI WAKAHARA Figure 2: Shows that the second cleavage plane regresses at the pole in an irradiated egg. The fourth cleavage planes, shown by the arrows, are regressed earlier just past the equator. The insert is a normal egg. Figure 3: Irradiated at the 2-4 cell stage. Compared to a normal egg (insert). Figure 4: Shows the median section of X. laevis blastula that was irradiated at the vegetal pole during the 2-4 cell stage. The animal hemisphere is normal, but the vegetal hemisphere is practically uncleaved. The arrow shows the first cleavage. Figure 5: Normal egg for comparison to figure 3. (Source-Züst, B. and Dixon, K. E. (1975). The effect of U.V. irradiation of the vegetal pole of Xenopus laevis eggs on the presumptive primordial germ cells. J. Embryol. Exp. Morphol. 34,209 -220)

Figure 6: UV-F embryos are UV-irradiated after fertilization and UV-O are UV-irradiated as full-grown oocytes. A) internal morphology of normal egg. B) Internal morphology of UV-F embryo. C) Internal morphology of UV-O embryo. D) Normal embryo at stage 12. E) UV-F embryo at stage 12. F) UV-O embryo at stage 12. G) Close up of D. H) Close up of E. I) Close up of F. J) Normal egg at stage 19. K) UV-F embryo at stage 19. L) UV-O embryo at stage 19. Normal embryos at stage 12 have invagination of mesoderm on the dorsal side, but UV-F embryos have radially symmetrical gastrulation. Also, the normal embryo has a well-developed archenteron, but the UV-O embryos have no archenteron space. At stage 19, UV-F and UV-O embryos do not have dorsal axial structures. Also, UV-F embryos have no blastocoels; whereas, UV-O has a large blastocoel. The arrows show the invagination of mesoderm. (Source-MISE, N, and WAKAHARA, M, (1994), Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization, int. J. Dev. Bioi 38: 447-453)

How does tilting save fertilized eggs from UV irradiation?

As stated before, cytoplasmic reorganization during the first division occurs by a 30 degree rotation of the egg cortex in relation to the vegetal mass. By tilting the embryo against gravity, it mimics the normal rotation (Elinson and Pasceri 1989). Because it is the microtubules that are disabled resulting in no cytoplasmic reorganization, by allowing cytoplasmic reorganization by tilting, it allows the formation of the neural crescent and dorsoanterior development. Since cytoplasmic reorganization occurs in the first cell division, tilting can only save UV irradiation before the second division.

How do lithium ions save eggs from UV irradiation?

Lithium ions block the phosphotidylinositol cycle, which is involved in intracellular signaling. Lithium causes animal cells to view a ventral mesodermal signal as a dorsal signal (Cooke et al. 1989). Lithium treated embryos express more goosecoid mRNA. Goosecoid is a homeobox gene, which are genes that encode DNA-binding proteins that are involved in the specification of positional information of the embryo (De Robertis et al. 1991). Goosecoid mRNA induces chordin, which is expressed in the dorsal lip and later throughout the notochord and other organizer-derived tissues. Chordin expression can rescue UV irradiated eggs. It is shown to induce secondary axis formation when injected to ventral vegetal blastomeres.

Figure 7: Shows the effect of UV irradiation at different times. Fertilization occurred at metaphase II. (Source-Elinson and Pasceri, 1989 R.P. Elinson and P. Pasceri, Two UV-sensitive targets in dorsoanterior specification of frog embryos, Development 106 (1989), pp. 511–518)

Experiment Strengths and Weaknesses

MISE, N, and WAKAHARA, M, (1994), Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization, int. J. Dev. Bioi 38: 447-453.

In the case of UV-F embryos (A), vegetal halves of fertilized eggs of Xenopus laevis were simply irradiated with UV before first cleavage and then allowed to develop. In the case of UV-O embryos (B), full-grown oocytes were defolliculated, irradiated with UV. and then allowed to mature with progesterone. UV-irradiated and unirradiated oocytes were stained with different vital dyes and then transferred into a host female previously primed with HCG. After spawning, colored eggs were inseminated with a sperm homogenate (Source- MISE, N, and WAKAHARA, M, (1994), Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization, int. J. Dev. Bioi 38: 447-453).

In this experiment, fertilized eggs and embryos of Xenopus laevis were irradiated with UV and allowed to develop in order to view the dorsoanterior development. The strength of the paper is in the technique. They used UV irradiation procedures that were already tested effectively in previous papers. Also, they limited the duration of UV irradiation to be most effective in obtaining embryos that lacked dorsoanterior structures but also had a low death rate. Also, the embryos were stained with vital dyes that allowed the viewing of blastopore formation and dorsoventral formation. However, the dyes could have been selected better because it was difficult to determine the dorsal lip of the blastopore in some embryos since it was so faint and UV-F embryos do not have a clear dorsoventral polarity in the blastopore. This weakness in the paper is corrected in more recent experiments by using convertible fluorescent protein EosFP instead.

Jansen, Hans J., Wacker, Stephan A., Bardine, Nabila, & Durston, Antony J. (2007). The role of the spemann organizer in anterior–posterior patterning of the trunk. Mechanisms of Development, 124, 668-681.

The strength of this paper is the fact it goes into detail about what happens when embryos are treated with UV radiation. However, the paper does not explain what happens when mature oocytes or how tilting/Li ions could save the embryo from being ventralized. Since these topics are all very similar and related to each other, the paper should have touched base on the results of some of these experiments even if the paper did not redo those exact experiments.

Elinson and Pasceri, 1989 R.P. Elinson and P. Pasceri, Two UV-sensitive targets in dorsoanterior specification of frog embryos, Development 106 (1989), pp. 511–518.

The strengths of this paper are the exact and precise methods carried out and clarity of the explanation of the results. The experimental plan made sense and did not seem to have any damaging flaws. The results of the paper were explained clearly, and the figures in the paper were excellent in simplifying the contents of the results. The main results of the paper could be understood by just looking at one of the figures. There did not seem to be any apparent weaknesses in the paper.

Now You Know…

Bibliography

COOKE, J., SYMES, K. & SMITH, E. J. (1989). Potentiation by the lithium ion of morphogenetic responses to a Xenopus inducing factor. Development 105, 549-558.

De Robertis, E. M., Morita, E. A. and Cho, K. W. Y. (1991). Gradient fields and homeobox genes. Development 112, 669-678.

Elinson and Pasceri, 1989 R.P. Elinson and P. Pasceri, Two UV-sensitive targets in dorsoanterior specification of frog embryos, Development 106 (1989), pp. 511–518.

Gerhart, J., Danilchik, M., Doniach, T., Roberts, S., Stewart, R., & Rowning, B. (1989). Cortical rotation of the xenopus egg: consequences for the anteroposterior pattern of embryonic dorsal development. Development, 37-51.

Hardin, Jeff. (1998, June 24). Initiating the embryonic body plan: polarization of the xenopus embryo. Retrieved from http://people.ucalgary.ca/~browder/polarization.html.

Jansen, Hans J., Wacker, Stephan A., Bardine, Nabila, & Durston, Antony J. (2007). The role of the spemann organizer in anterior–posterior patterning of the trunk. Mechanisms of Development, 124, 668-681.

MISE, N, and WAKAHARA, M, (1994), Dorsoventral polarization and formation of dorsal axial structures in Xenopus laevis: analyses using UV irradiation of the full-grown oocyte and after fertilization, int. J. Dev. Bioi 38: 447-453.

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