Gastrulation in avian embryos involves highly coordinated morphogenetic movements beginning with the establishment of the primitive streak in the midline. The streak has been shown to form through the large-scale movement of epiblast cells in two counter-rotating streams, which merge at the site of streak formation (Cui et al.,2005; Graeper,1929; Vakaet,1970). As the streak forms, it elongates in both the anterior and posterior directions and prospective mesoderm cells begin to ingress (Lawson and Schoenwolf,2001; Mikawa et al.,2004). Some of the molecular signals driving these processes in amniotes have been characterized and work in mouse and chick embryos implicates both FGF and Wnt pathways (Bertocchini et al.,2004; Chuai et al.,2006; Ciruna and Rossant,2001; Kelly et al.,2004; Stern,2004; Sun et al.,1999; Yang et al.,2002).
These signals ultimately control cell behaviour and have to integrate a variety of cellular responses including cell shape changes, cell polarity and directed migration. Central to this are dynamic changes in cyto-architecture, in particular the actin and microtubule cytoskeleton, which are essential to cell motility. The actin cytoskeleton provides the driving force for migration whereas microtubules are important for establishing direction. In migrating cells, actin filaments polymerize in the protruding front of the cell whereas they contract in the cell body resulting in retraction of the cell's rear. The dynamic organization of the actin cytoskeleton is regulated by small GTPases of the Rho family, in particular Rac1, RhoA, and Cdc42 (Wittmann and Waterman-Storer,2001). In recent years, the interplay between the actin and microtubule cytoskeletons has become better characterized and it seems that microtubules influence actin filament organization by modulating the Rho GTPases (Watanabe et al.,2005). In migrating cells, microtubules form polarized bundles and the centrosome is often repositioned towards the leading edge. Microtubule plus-end capture and anchorage at the cell cortex within the advancing lamellipodium seems to be critical for both of these processes. Microtubule plus-end tracking proteins, also known as +TIPs, such as EB1, CLASPs, and APC, and cortical receptors such as IQGAP1 and Dlg1, appear to be important for establishing cortical contact in migrating cells (Etienne-Manneville et al.,2005; Mimori-Kiyosue et al.,2007; Wittmann and Waterman-Storer, 2005). In addition, a lack of APC seems to cause a decrease in cell migration (Kroboth et al.,2007) and correlates with overall changes in microtubule stability (Akiyama and Kawasaki,2006; Gundersen,2002; Lansbergen et al.,2006; Watanabe et al.,2004,2005). In contrast, non-migrating cells have a radial microtubule array, which is anchored at a centrally located centrosome.
Here we investigated the cytoskeletal organization in gastrulating avian embryos focussing on microtubules and actin. We established a fixation and staining protocol in whole embryos and have examined microtubule arrays in primitive streak stage chick embryos from early to late Hamburger-Hamilton stage 3 (HH3) (Hamburger and Hamilton,1951). We found distinct patterns of cellular organization in different regions of the embryos. Cells in the epiblast contained radial microtubule arrays while the majority of cells in the primitive streak were polarized with microtubule bundles oriented in the direction of migration. In addition, we show that in the primitive streak many cells were arranged in rosette-like structures containing multiple cells with an accumulation of actin in the rosette centre. Using confocal imaging and 3D-reconstruction, we detected tips of cells that were protruding from the centre of rosettes, consistent with cells in the process of ingression.