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1 Rochester Institute of Technology, Rochester, NY
2 St. Mary’s College of Maryland, St. Mary’s City, MD
3 Marine Biological Laboratory, Woods Hole, MA
* Corresponding author: kcrawford{at}smcm.edu
You can observe a lot by watching.
—Yogi Berra
Nearly all of what we know of cephalopod development has been drawn from live or fixed embryos collected from naturally laid jelly capsules (1, 2, 3). Many aspects of development—including pronuclear migration and cleavage (4), cytoplasmic and cellular movements (5), differentiation and organogenesis—would be better understood and more effectively compared to other embryos if examined in the living state. To begin this work, we have used time-lapse video microscopy to record the development of in vitro-fertilized squid embryos, Loligo pealeii, from early cleavage through the formation of the external yolk syncytial layer (YSL) and the early phase of epiboly. Adding the dimension of time to our analysis revealed new and intriguing elements in the development of this organism.
In vitro-fertilized squid embryos were prepared (6) and oriented for imaging in depressions made in 0.2% agarose (Sigma)-lined plastic petri dishes (Falcon) filled with Millipore (0.22 µm) filtered seawater. Dishes of embryos were placed on a universal transmitted light illuminator with an adjustable reflector that allowed for bright field or oblique illumination, or a combination of the two. To minimize heat transfer, a KL 1500 constant-color-temperature fiber-optic source with an infrared filter was used to illuminate the specimen. A Zeiss Stemi SV 11 stereomicroscope with a 1.6x Planapochromat lens was used for time-lapse imaging. An intermediate mount was placed between the lens and microscope body to align the light path with the center of the front lens, right eyepiece, and camera. A computer-controlled Axiovision software program was used for image acquisition from an MRc5 Zeiss digital camera set to optimal resolution (2584 x 1936). Digital images were collected at 5- or 7-min intervals at 21 °C for 2–12.5 h. Throughout these periods, embryos appeared to cleave and develop normally.
Ten separate time-lapse sessions were carried out and images from two are presented. In the first session, images were collected at 5-min intervals, from first cleavage through blastoderm formation; three of these images are presented in Fig. 1a–c. The first image was taken 8 h post-fertilization (hpf) after the fourth cleavage (Fig. 1a). Each cleavage furrow is numbered in the order of its occurrence, and the polar bodies (pb and arrow) are visible resting in the first cleavage furrow. The larger blastomeres, formed by the unequal third cleavage furrow characteristic of cephalopod embryos (1), identify the future anterior midline of the embryo. The second image (Fig. 1b) was taken 9.6 hpf at sixth cleavage (arrowhead). This cleavage separates the central blastomeres from the outer syncytial layer of the embryo, which is continuous with the yolk cell. The final image (Fig. 1c) was taken 16.25 hpf and reveals a well-defined blastoderm surrounded by radiating clusters of cells, the outermost of which are continuous with the yolk cell. The boundary created at sixth cleavage (arrowhead) remains well preserved.
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In teleosts, the YSL is critical to patterning and development (7, 8), and time-lapse video analysis has played an important role in our understanding of this model system (9). As in squid, the YSL separates the yolk from the embryo and regulates the transfer of all nutrients and factors present within the yolk to the developing embryo. Formation of this important layer involves the collapse of blastomeres at the boundary between the developing blastoderm and the yolk cell cytoplasm. That cephalopods form their YSL through similar developmental mechanisms to those of teleosts exemplifies the fundamental similarities that exist between embryos faced with similar structural constraints and highlights the importance and need for further comparative study.
This work was made possible by support from a Faculty Development Grant and the Aldom-Plansoen Distinguished Endowed Professorship in Contemporary Studies to K.C. from St. Mary’s College of Maryland. K.C. is most grateful to Bill Eckberg, Howard University, who graciously provided laboratory space, collaborative guidance, and digital imaging assistance. We thank Rudi Rottenfusser, MBL Zeiss representative, for allowing his summer intern (P.H.W.) to participate in this work. Finally, K.C. wishes to thank J.P. Trinkaus and his many students for reminding us to "watch."
Literature Cited
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