Denegre JM et al. (1997), Localized maternal proteins in Xenopus revealed...

XB-ART-15566

Dev Biol 1997 Dec 15;1922:446-54. doi: 10.1006/dbio.1997.8773.

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Localized maternal proteins in Xenopus revealed by subtractive immunization.

Denegre JM , Ludwig ER , Mowry KL .

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It has long been appreciated that the localization of cytoplasmic determinants in the egg can provide the foundation for patterning in the embryo. Differences in cell fate among the early blastomeres are thus a consequence of asymmetric distributions of informational molecules prior to fertilization. The frog egg has a single axis of asymmetry present prior to fertilization, the animal/vegetal axis, and the localization of developmental information appears to be polarized along this axis. Such developmental information can be localized as either RNA or protein; localized RNAs are well documented in the Xenopus oocyte, and some are thought to play roles in axial patterning. While it is apparent that not all of the localized maternal components are RNAs, much less is known about maternal proteins that might be localized in the egg. In the present study, we have taken a novel approach to identify localized maternal proteins within the Xenopus egg. Using a subtractive immunization strategy, we have generated monoclonal antibodies which recognize antigens that are restricted to the vegetal cortex of fertilized eggs. Analysis of biogenesis during oogenesis reveals two distinct patterns of localization to the cortex. At least three of these localized antigens are proteins, and these localized proteins could represent maternal determinants with roles in patterning.

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	FIG. 1. Immunosuppression reduces the immune response to nonlocalized antigens. Shown are vegetal cortical views of whole-mount preparations of fertilized eggs, stained with (A) preimmune serum; (B) serum from a mouse that was immunized with animal hemisphere extract, and immunosuppressed; and (C) serum from a mouse that was immunized with animal hemisphere extract, no immunosuppression. The photographs were taken on a Zeiss IM inverted microscope using a 401 objective.
	FIG. 2. Vegetally restricted antigens are revealed by staining of fertilized eggs with MAbs. (A) D4-3F7, animal view. (B) D4-3F7, vegetal view. (C) D4-4G8, animal view. (D) D4-4G8, vegetal view. (E) D5-3B3, animal view. (F) D5-3B3, vegetal view. (G) D5-5G7, animal view. (H) D5-5G7, vegetal view. All eggs were albinos, cleared for optical sectioning, and the views are 15 mm from the plasma membrane. The scale bars represent 20 mm, and the images were obtained using a Zeiss confocal microscope.
	FIG. 3. Colocalization of vegetal antigens with germ plasm. Shown are vegetal views of fertilized eggs (A–F) or an eight-cell embryo (G– I) labeled with MitoTracker and stained with the following MAbs: (A–C) D5-3B3, (B–F) D4-4G8, and (G–I) D5-5G7. MAb staining is shown as green, germ plasm is shown in red, and regions of colocalization appear as yellow. All eggs and embryos were cleared for optical sectioning, and the images were obtained using a Zeiss confocal microscope. The scale bars represent 20 mm.
	FIG. 4. Two distinct patterns of biogenesis during early oogenesis. Oocytes were stained with the indicated MAbs and imaged using a Zeiss confocal microscope. Scale bars for all panels represent 50 mm. (A, B) Serial sections of a stage I oocyte, stained with D5-5G7. (C) Stage I oocyte stained with D5-3B3. (D) Stage II oocyte stained with D5-3B3. (E) Stage I oocyte stained with D4-4G8. (F) Stage III oocyte stained with D4-4G8.
	FIG. 5. Protein antigens are revealed by Western blot analysis. Egg extracts were subjected to SDS–PAGE, transferred to nitrocellulose filters, and probed with D4-4G8 (lane 1), D4-3F7, (lane 2), or D5-5G7 (lane 3). The positions of the proteins recognized by the MAbs stainare indicated by arrowheads, and the sizes of molecular weight standards are shown at the right.