BUFFALO, NEW YORK
Williamsville North High School
State University of New York
LONDON, ONTARIO
University of Western Ontario
Three-dimensional visualization of meiosis I in Ohio43
--Lee, TC, Walden, DB, Cheng, PC

Visualization is interdependent with gene mapping and DNA sequencing, because spatial relationships are important to understand function (Brakenhoff et al., Nature 317:748-749, 1985; Dempsey, The Maize Handbook, Springer-Verlag, 432-441, 1994). The ability to visualize the chromosomes, especially in three-dimensional (3D) space, has great benefits for the better understanding of a gene’s function. For these reasons, a better technique is needed to study the chromosomes in 3D. Studying the stages of meiosis I serves as an excellent opportunity to evaluate the capability of 3D imaging techniques. The loosening of the chromosomes in meiosis I exemplify the advantages of 3D imaging.

Tassel inflorescence of Ohio43 inbred were collected from the field station at the University of Western Ontario in London, Ontario, Canada, during the summer of 1998. Tassels at the proper developmental stages were fixed in 3:1 (95%ETOH: acetic acid) solution and stored in mason jars at room temperature. The anthers were treated with the Feulgen-method and cleared in methyl salicylate (winter green oil).

An Olympus GB200 laser scanning microscope equipped with a PlanApo-60X oil immersion objective (NA=1.4) and high precision piezo stage was used in this study. To observe the fluorescence of Feulgen-stained specimens, the 488nm emission line of Ar ion laser was used as the excitation light and >520nm fluorescent emission was detected. Series of optical sections (1024 x 768, 8 bits) were obtained at an interval of 0.1µm. The resulting images were processed through a deconvolution algorithm (Holmes and Liu, Biomedical Visualization, VCH, 283-327, 1992). AutoDeblur™ software (AutoQuant, Watervliet, New York) running a no-neighbor deconvolution algorithm was executed by an Intel Pentium® II based computer operating at 300MHz with 352MB RAM.

For 3D reconstruction, the raw image was reduced to 512x384 in 8 bits format by a binning method to overcome computer hardware and software limitations. Rendering by maximum intensity volumetric method was performed using the VayTek™ Voxblast software. Stereographs were generated using projections from various vantage points.

To ensure better printing contrast, all images are presented in reverse contrast. A stereoviewer can be used to view the sterograph images. Figure 1 is a stereograph of a premeiotic interphase nucleus. Figure 2 shows the lepotene stage. Figure 3 is in the zygotene stage, showing the chromosome mass aggregated to form a "cap" on the nucleolus. Figure 4 is in the pachytene stage. Notice the attachment of chromosome #6 to the nucleolus (No) through the nucleolus organization region (NOR). This nucleolus attachment provides an important feature for the identification of chromosome #6. Figure 5 represents chromosome structures in late diplotene. Figure 6 represents diakinesis (notice the chiasmata), Figure 7 is anaphase I (notice the anaphase bridges, which are the threads stretching across the metaphase plate, and the spindle fibers are also visible). The decrease in the size of the nucleolus throughout the images of prophase I and its disappearance in Figure 7 of anaphase are also noticeable. Figure 8 is late telophase I.
 


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