Relative expression levels of selected angiogenesis related molecules during the course of CAM development.

<p>The expression levels, however, did not show any statistically significant differences among various time points (single factor ANOVA). <b>a</b>: Vascular endothelial growth factor receptor 2 (VEGFR2), the vessel endothelial marker, reaches a peak at E11, and then decreases steadily, being least at E20. <b>b:</b> There is an increase in the expression levels of HIF1α at E11, a sharp decrease towards E15 with a peak at E20. <b>c:</b> VEGFA peaks at E11 and again at E20. Its lowest expression levels are at E15 <b>d:</b> The stromal derived factor 1 (SDF-1) expression levels reach a peak at E11 and decline towards E15.</p

Bar graphs showing the variation in body mass (g), CAM volume (cm³) and body mass standardized CAM volume (cm³g^-1).

<p><b>a</b>: Notice that body mass increases steadily between E8-E13, sharply between E13-E15 and then steadily between E15-E20, marking the three growth phases. <b>b</b>: The CAM volume increases fast between E8-E13, steadily between E13-E15 and then declined between E18-E20, the latter was thus a phase of regression. The phase of rapid CAM growth precedes that of embryonic growth. On Scheffé's test (p ≤0.05) CAM volume at E8 and E11 were significantly lower than at all the other subsequent days (asterisks). <b>c:</b> Body-mass standardized CAM volume decreased gradually through the entire period.</p

Growth phases and percentage changes in body mass and CAM volume in the developing chick embryo.

<p>Growth phases and percentage changes in body mass and CAM volume in the developing chick embryo.</p

Bar graphs showing the proportions (a) and absolute volumes (b) of the CAM components.

<p><b>a:</b> The mesoderm remained the largest component during the entire growth period at about 70%, declining slightly at E18. The chorion was between 10–18%, being highest at E18 and decreasing to about 10% at E20. The allantois remained at around 10%, peaked at E18 to above this value and then declined to about 8% by E20. The proportion of the large vessels appeared to remain steady at about 4–5% of the CAM. b: In absolute terms, the values of the components followed closely the trend of the proportions. The highest value for the mesoderm was at E15 while the chorion and allantois peaked at E18. Volume of chorion at E8 was significantly lower than all the other time points except E11. The volume at E11 was lower than at E15 and E18. Similarly, chorionic volume was significantly lower at E13 and E20 than at E18. Allantoic volume at E15 and E18 was significantly higher than at both E8 an E11while mesenchymal volume remained significantly higher in the ages E13-E20 than at E8 and E11. Large vessels at E15 and E18 had significantly greater volumes than at E8 and E11.</p

Logarithmic line graphs showing growth rates of CAM (a) and the various coarse components of the CAM (B).

<p><b>a:</b> The CAM grew rapidly in phase I (E8-E13), moderately at phase II (E13-18) and was undergoing regression in phase III (E18-E20). CAM volume was significantly correlated to body mass (P = 0.01). CAM growth was strongly positively related to body mass increase up to E18, but it had a strong negative correlation until time of hatching. <b>b:</b> Logarithmic line graphs showing growth regression analysis of the coarse CAM components. The mesoderm was the fastest growing component while the large vessels were the slowest. Both the chorion and allantois grew at the same rate. When viewed on individual basis at various growth phases, all components were growing fastest in phase I and except the mesoderm, they were regressing in phase III. All components were significantly correlated to body mass during the entire period (P ≤ 0.05). The chorion, allantois and vessels were regressing in phase III (showed a strong negative correlation with body mass).</p

TEM micrographs showing the changing structure of the chorionic layers.

<p><b>a and b</b>: The chorion at E8 is thin with large capillaries (Ca) separated by undifferentiated epithelial cells (arrowhead in a). The mesenchyme (Me) is also shown. By E11 there is recruitment of cells from the subchorionic layer of the mesenchyme (Me). Such cells (white arrow) are loosely attached to primary cells of the chorion (black arrow in b). At these stages the various cell types of the chorionic layer are not differentiated. <b>c and d</b>: The typical chorionic cells such as the villous cavity (VC) cells and basal (BC) cells become recognizable by E12 and by E15 (d) such cells are well differentiated. The chorionic capillaries (Ca) remain on the external aspect of the epithelium. <b>e and f</b>: By E18 the first signs of degeneration are evident in the VC, with loss of microvilli although some cells in the basal layer show some mitotic activity (arrowhead). Clear signs of cell degeneration at E20 include dissolution of basal cells (BC) and crumbling of VC cells.</p

TEM micrographs showing the changing ultrastructure of the allantoic layer between E8 and E20.

<p>Note that in all cases the microvilli project into the allantoic lumen (asterisks) and this is important in identifying the mesenchyme (Me) in cases where mesenchymal cells are not evident. <b>a and b</b>: The allantois at E8 consists of a single layer of squamous fibroblast like cells (f) with numerous filopodia (white arrowheads) extending into the mesenchyme and short microvilli (black arrowheads) towards the allantoic lumen. The mesodermal layer has loose connective tissue and fibroblasts (white arrows). Some cells in the allantois show mitotic figures (black arrow in B). <b>c and d:</b> Thickening of the allantois is notable by E12 where it starts to recruit cells to form a two cell layer separated by enormous intercellular spaces (arrow in c). The first few granules start to appear in the outer layer of the allantois (arrowhead in c). A mesenchymal cell (Mc) is closely aligned with the inner layer of the epithelium. By E15, a two-cell layer is well accomplished and formation of the third layer begins (see layers 1, 2, 3 in d). Notice the granules in the outer layer of the allantois (arrowheads) are well developed. The basal border looks irregular and the basement membrane is amorphous (black arrows) and there are cells close to the epithelium (white arrow). <b>e and f:</b> The allantois has 3–4 well-formed cell layers at E18 and the basement membrane (black arrows) is neatly formed. The outer cell layer has abundant glycogen granules (arrowheads) and short microvilli. By E20, the layers of the allantois look shrunken and the glycogen granules (arrowheads in f) are depleted.</p

Semithin micrographs showing the distribution of cells within the various layers of the developing CAM at E11 and E18.

<p><b>a and b:</b> Both the chorion (white arrows) and the allantois (black arrows) are relatively thin early during development and there are numerous cells concentrated in the subepithelial layer of the chorion (white arrowheads) and also in the subepthelial layer of the allantois (black arrowheads). Note both the chorion and the allantois are of irregular thickness. In the middle part of the mesenchymal layer (asterisks), cells are few. <b>c and d:</b> At E18 the CAM has reached maturity and the chorion (white arrows) and the allantois (black arrows) are both 3–4 times thicker, have an almost uniform thickness and their basal aspects are delineated by a prominent basal membrane (white and black arrowheads respectively). Distribution of cells in the mesenchymal layer (asterisk) is almost uniform.</p