PROCEDURES

Modified from: Kimmel et al., 1995. Developmental Dynamics 203:253-310. Copyright © 1995 Wiley-Liss, Inc. Reprinted only by permission of Wiley-Liss, a subsidiary of John Wiley & Sons, Inc.

Staging

One can determine the approximate developmental stage of a living embryo by examining it with a dissecting stereo-microscope, generally with transmitted (not epi- or incident) illumination and high magnification (about 50x). During the segmentation period the tail elongates, and if the embryo is left within its chorion, the tail eventually curves over the trunk and head so as to obscure the view. When this has happened, one must remove the embryo from its chorion to stage it, either manually with #5 watchmaker's forceps, or by treatment with a proteolytic enzyme (Westerfield, 1994). Late in the pharyngula period the embryo, if removed from the chorion, swims away in response to touch; this can prevented by anesthesia in 0.003% tricaine (3-amino benzoic acid ethyl ester; Sigma Chemical Co.) at pH 7 (Westerfield, 1994). Repeated anesthesia and rinsing appears to slightly but significantly retard subsequent development (see legend to Fig. 2).

Nomarski Optics

A compound microscope equipped with Nomarski diffential interference contrast optics and objectives in the range of 25x to 40x reveals details otherwise not visible in the live preparation, and permits the most critical staging. For instance, Nomarski optics permits the most accurate counts of cells in the blastula and of somites during segmentation. Moreover, 'prim' stages refer to the position of a structure, the primordium of the posterior lateral line, that one must use Nomarski optics to see for the sharpest determination of stages during much of the pharyngula period. The embryo is anesthetised, removed from the chorion, and mounted between bridged coverslips (Westerfield, 1994), for observation and later recovery.

It is always better to stage embryos while they are alive rather than after killing and fixation. For example, staging during the straightening and hatching periods calls for a comparison of sizes of the head and the yolk mass, and differential shrinkage during fixation distorts the normal relationship. Nevertheless, if preservation is good enough, one can fairly reliably stage fixed and whole-mounted embryos (e.g. immunolabeled ones) using other criteria. One cannot easily stage an embryo after it is sectioned.

Photographs

The accompanying photographs are of living embryos, anesthetized for the later stages. The original photographs were made as color slides (Kodak Ektachrome 160T DX), and the black and whites plates are reproduced from internegatives. Sets of copies of the original color slides are available at cost from the authors. The lower magnification views, showing the entire embryo, were made using a Zeiss STEMI stereo dissecting microscope: The embryo is mounted on a depression slide, without an overlying coverslip, in standard embryo medium (Westerfield, 1994) containing 1.5% -3% methyl cellulose to allow positioning as desired. The higher magnification views, showing parts of embryos, were taken with Nomarski optics (generally a Zeiss 40x water-immersion objective) using a Zeiss UEM compound microscope. The embryo was mounted between coverslips, sometimes in 1% agar, for positioning.