We performed time-lapse imaging of chick embryos cultured in New or EC systems (n=3 each) from early gastrulation to somitogenesis stages (HH4 to HH10) (Fig. 1A). Embryo length was measured from the crown to the end of the primitive streak (C-PS; Fig. 1B, C) and to the pPL (C-pPL; Fig. 1B, D). Measurements were performed in multiple independent frames per developmental stage (n=5 each) for each embryo (total n=6). We found that embryo growth over time displays a similar trend independently of the morphological landmark used (Fig. 1C, D). In fact, both C-PS and C-pPL measurements increase steadily in the embryonic stages analyzed.
To test the reproducibility of the results when the measurements are performed using each landmark, C-PS and C-pPL were measured in a single embryo over time by 3 operators with different years of experience with the chick embryo model (Fig. 1E, F). Figure 1E evidences significant variability in C-PS length measured by distinct users, which can differ up to 0.84 mm (HH5; 2.81 mm average length). This stems from differences in where each operator positions the end of the primitive streak. Importantly, the variability of the C-pPL measurement performed by the independent users is almost negligible (Fig. 1F). This highlights that the contrast between the posterior border of the area pellucida and the area opaca is an easily recognizable landmark for both experienced and inexperienced experimentalists. The pPL landmark thus allows for high precision measurements for embryo length studies.
For pPL to be a valid proxy for the end of the primitive streak, the distance between the two landmarks (PS-pPL) should be approximately constant over time; i.e., any variation in C-pPL length should be exclusively due to a respective variation in C-PS. We found that this is the case, since the natural variations observed in PS-pPL distances over time (Fig. 1G) were neglectable when compared to the progressive increase in C-pPL length (Fig. 1D). These findings evidence that C-pPL variation over time reliably represents C-PS dynamics, thus validating the use of the pPL as a precise and reliable morphological landmark for chick embryo elongation studies.
Finally, when C-PS values were plotted against their respective C-pPL, for all embryos and developmental stages analyzed, a direct proportionality was found represented by the equation [C-PS] = -0.378 + 0.947[C-pPL] with R2 = 0.949 (mean error of estimated C-PS = 0.206±0.132). This means that the absolute value of C-PS length can be directly calculated from C-pPL measurement.