New methods for chicken embryo manipulations

Abstract

The capacity to image a growing embryo while simultaneously studying the developmental function of specific molecules provides invaluable information on embryogenesis. However, until recently, this approach was accomplished with difficulty both because of the advanced technology needed and because an easy method of minimizing damage to the embryo was unavailable. Here we present a novel way of adapting the well-known EC culture of whole chick embryos to time-lapse imaging and to functional molecular studies using blocking agents. The novelty of our method stems from the ability to apply blocking agents ex ovo as well as in ovo. We were able to study the function of a set of molecules by culturing developing embryos ex ovo in tissue culture media containing these molecules or by injecting them underneath the live embryo in ovo. The in ovo preparation is particularly valuable since it extends the period of time during which the developmental function of the molecule can be studied and it provides an easy, reproducible method for screening a batch of molecules. These new techniques will prove very helpful in visualizing and understanding the role of specific molecules during embryonic morphogenesis, including blood vessel formation.

Slits affect the timely migration of neural crest cells via robo receptor

“…3B, N=14 embryos) and in 50% of the embryos, the migration of neural crest cells was disrupted morphologically compared to control embryos (data not shown). RoboN-treatment also facilitated neural crest cell migration at the most caudal somites (El-Ghali et al, 2009). These in vivo and ex ovo results were substantiated by culturing neural tubes in the presence or absence of RoboN-conditioned media (Supplementary Fig.…”

“…Chicken embryos were cultured as described previously (El-Ghali et al, 2009). Briefly, embryos are cultured outside of the embryo on a filter ring containing approximately 3ml conditioned culture media (specifically, from 293HEK cells expressing Slit2 or controls) at 37°C in a CO 2 incubator (5%) for 24 hrs until fixation and wholemount staining.…”

Investigating developmental cardiovascular biomechanics and the origins of congenital heart defects

“…OCT is limited by light scattering in embryonic tissue and has a depth of view of 1–2 mm. The speed and non-invasive aspects of confocal microscopy and OCT have been leveraged to enable long-term, time-lapse imaging (Kamei and Weinstein, 2005 ; El-Ghali et al, 2010 ; Kulesa et al, 2010 ; Happel et al, 2011 ).…”

“…Long-term, time-lapse imaging of live embryos, where a single embryo is kept within an imaging platform and followed continuously for a period of hours or days, has been achieved by constructing controllable chambers to maintain physiologic environmental conditions (Orhan et al, 2007 ; Gargesha et al, 2009 ; Kulesa et al, 2010 ; Ma et al, 2010 ; Happel et al, 2011 ; Al Naieb et al, 2012 ). The chick embryo has been studied using long-term, time-lapse techniques combined with both confocal microscopy to track moving cell populations over a period of more than 26 h, in 1.5 min intervals (El-Ghali et al, 2010 ; Kulesa et al, 2010 ) and OCT to measure cardiac function over a 6 h period, in 60 min intervals (Happel et al, 2011 ). A confocal system has been developed for long-term, time-lapse imaging of the zebrafish embryo as well, capable of a 5 day period, in 10 min intervals (Kamei and Weinstein, 2005 ).…”

“…A challenge of these continuous techniques is maintaining the embryo in an exposed and stationary state to facilitate imaging. Shell-less or ex ovo culture systems are often used in long-term studies of chick embryos (Teddy et al, 2005 ; El-Ghali et al, 2010 ; Happel et al, 2011 ), though an in ovo confocal protocol has been proposed (Kulesa et al, 2010 ). In the case of zebrafish embryos, immobile mutants or tricaine anesthetized embryos are used (Kamei and Weinstein, 2005 ).…”