Limites externos

Initial examination of embryonic hearts assayed by TUNEL was performed by light microscopy at X 40 magnification, in order to confirm apoptotic morphology (3.2.1). Throughout the period examined, every region of the heart shows some

apoptotic cell death. However, in the ventricles, the atria and most o f the outflow tract, no obvious trends in PCD were seen without template analysis. In contrast, trends in apoptosis within the endocardial cushions were visible from early E l 2.5 to E l 3.5 on individual TUNEL-assayed sections. These trends are now described in conjunction with descriptions of PCD, using data summation, at E l 0.5 and E l 1.5.

3.2.2.1 PCD in the atrioventricular cushions

From 31 to 48 somites (early E l0.5 to E l 1.5), even templating revealed very little PCD within the atrioventricular cushions (Figures 3.2a-c). By the 46-48 somite stage the mesenchymal cap of the primary atrial septum has fused with the superior and inferior atrioventricular cushions, which themselves appose one another (Figures 3.3a). At 49-51 somites, a focus of cell death becomes visible where the superior

atrioventricular cushion meets the primary atrial septum and the inferior atrioventricular cushion (arrows in Figure 3.2d). These foci of cell death subsequently become so intense as to be visible on individual TUNEL-stained sections (Figure 3.3). At the 49- 51 somite group PCD appears to be higher in the atrioventricular cushions (arrows. Figure 3.3c) than at earlier stages and also compared with neighbouring regions of the rest of the heart. The PCD seen in the cushions at this developmental stage is diffuse and not clearly localised to any particular part of the endocardial cushions. The 49-51 somite group is the one in which the IVS begin fusing with the atrioventricular cushions (Figure 3.3b).

Early in E12.5, at 52-54 somites, the superior and inferior atrioventricular cushions are beginning to fuse with one another. Since the mesenchymal cap of the primary atrial septum has already fused with the superior atrioventricular cushion, a continuum is formed consisting of the atrioventricular cushions, the mesenchymal cap of the primary atrial septum and the interventricular septum. This structure has been previously dubbed the central mesenchymal mass (Webb et a/., 1998a; Section 1.2.6; Figure 3.3f). At this stage, PCD in the atrioventricular cushions appears to be even higher than at the preceding stage and compared with neighbouring cardiac tissues in the same embryos (arrows in Figures 3.3c - compare with Figure 3.3b). Cell death seems to occur mainly at the regions of fusion of the superior atrioventricular cushion with the mesenchymal cap of the primary atrial septum, and the superior

Figure 3.2 Templates showing PCD in the atrioventricular cushions

Transverse sections showing the atrioventricular cushions,

a) At E l0.5, there is very little obvious PCD in the atrioventricular cushions, which have not with one another, the primary atrial septum or the interventricular septum, b), c) There is still only low level PCD within the AV cushions at 43-48 somites, d) At the 49-51 somite stage PCD can be seen to occur between in the superior atrioventricular cushion in the region in which it is contacted by the primary atrial septum and the inferior atrioventricular cushion.

1C = inferior endocardial cushion IVS = interventricular septum LA = left atrium

PAS = primary atrial septum RA = right atrium

s = somites

SC = superior endocardial cushion scale bar = 0.35mm

Figure 3.2

%

Dorsal

Right

Left

Ventral

Figure 3.3 PCD in the atrioventricular cushions

TUNEL analysis performed on transversely sectioned mouse embryos between E11.5 and E l 3.5. TUNEL-positive nuclei are stained brown and indicated with arrows, and the sections are counterstained with methyl green, a) Atrioventricular structures at the 46-48 somite stage, b) PCD in the atrioventricular cushions at the 49-51 somite stage, c) at the 52-54 somite stage, d) at the 55-57 somite stage and e) at E13.5. f) is a

schematic diagram o f the central mesenchymal mass. The dotted lines represent fusion points between mesenchymal structures.

a) Fusion between the mesenchymal cap of the primary atrial septum and the inferior atrioventricular cushion can be seen. Very little PCD is present, b) At the 49-51 somite stage, the junction of the superior and inferior atrioventricular cushions has become more extensive. PCD levels appear to be greater in the atrioventricular cushions, particularly the superior, than previously, c) Tissue continuity at the 52-54 somite stage is even more extensive than in the previous one, and the amount o f PCD appears

greater, being prominent at the region o f fusion between the inferior and superior atrioventricular cushions, and within the mass of the superior atrioventricular cushion. d) At 55-57 somites PCD still occurs mainly at the regions of fusion between the mesenchymal components of the atrioventricular structures and within the mass o f the superior atrioventricular cushion, but the quantity of PCD seems even greater than before, e) At E l 3.5, the PCD appears to have declined in the main mass of cushion tissue and occurs in regions of condensed mesenchyme at the junction o f the superior and inferior endocardial cushions. PCD is also becoming apparent in more peripheral cushion tissue that is forming the atrioventricular valves.

A W = atrioventricular valve cushion IC = inferior endocardial cushion IVS = interventricular septum

MCPAS = mesenchymal cap of the primary atrial septum MCVS = mesenchymal cap of the vestibular spine

PAS = primary atrial septum SC = superior endocardial scale bar = 0.2 mm

Figure 3.3

w #

47 somites

# g ' ' ^

, 4 ^ ^ ' »

r i »

Dorsal

Right

Left

Ventral

atrioventricular cushion with the inferior atrioventricular cushion, and also diffusely within the superior atrioventricular cushion (arrows in Figure 3.3c).

Between 55 and 60 somites, the central mesenchymal mass appears similar to that in the previous somite group (compare Figures 3.3c and d). During this period, PCD in the atrioventricular cushions retains the same distribution as in the 52-54 somite group, at the points of fusion between the superior and inferior atrioventricular

cushions, and the superior endocardial cushion the primary atrial septum and in the mass of the superior cushion (arrows. Figure 3.3d). The PCD intensity is, however, markedly increased compared with that in the previous group (compare Figures 3.3c and d).

At E l 3.5, the atrioventricular region is differentiating into the atrioventricular valves, and the components of the central mesenchymal mass are fused to such a degree that they are difficult to distinguish from one another (Figure 3.3e). PCD still occurs at the junctions of the superior atrioventricular cushion with the mesenchymal cap of the primary atrial septum, and the superior and the inferior atrioventricular cushions, but it is less extensive than previously and is largely limited to regions where tissue

continuity between the structures has not been achieved (arrowheads in Figure 3.3e). In addition, PCD now occurs in the edges of the atrioventricular cushions that are forming the mural leaflets of the atrioventricular valves (arrows in atrioventricular valve. Figure 3.3e).

3.2.2.2 PCD in the outflow tract cushions

Templating of PCD in the outflow tract cushions does not show any significant PCD within their mass during E l 0.5 (arrow. Figure 3.4a) and early E l l . 5 (arrows. Figure 3.4b). At 46-48 somites there is an increase in PCD in the cushions (compare arrows in Figures 3.4b and 3.4c). At this stage, the distal outflow tract cushions have