Cell movement in the lower layer between stages 4 and

In order to trace cell movement in the lower layer, embryos were set up in ring culture

so that the endoderm was now uppermost and directly accessible for labelling. A small

pulse of Dil was applied to the surface of the embryo at a particular point. Control

at the outset. Remaining embryos were cultured further, for up to 6 hours, and the

position of labelled cells recorded under fluorescent light.

5.2.1 Labelling the node at stage 4

In the majority of cases, the pulse of Dil was applied to endoderm cells directly

overlying the node, at stage 4. Control embryos, fixed immediately after labelling,

showed that in about 60% of cases (18/29) the spot of Dil was extremely localised and

well within the shoulders of the node (Fig5.1K,L). In the other embryos (11/29) the

labelled patch was slightly larger and included some surrounding cells, although still

obviously centred on the node. Control embryos were sectioned to ensure that

labelling was confined to the endoderm layer. In addition, some control embryos were

cultured for up to an hour, to confirm that the patch of Dil did not expand by diffusion

or any means other than cell movement.

All other labelled embryos were cultured further, for approximately 6 hours, by which

point they had reached a range of different stages. In those embryos which were now

late stage 4/4+, labelled cells were found scattered in the region anterior to the node

(12/14 embryos; Fig5.1A-D). The labelled cells were no longer in a clump but had

spread and appeared to have mixed with unlabelled cells. However, all labelled cells

had moved anterior to the node, leaving the cells over the node itself blank. Sectioning

confirmed that the labelled cells were in the endoderm layer. In addition, removal of

the anterior endoderm, six hours after labelling over the node, was sufficient to

remove all labelled cells from this region (in 5/5 cases), while some fluorescence

Embryos viewed from the ventral aspect, anterior to the top. Bright field and corresponding fluorescent images are shown for each embryo. A- D Two stage 44- embryos, 6 hours after labelling the node. Labelled cells are scattered in the endoderm anterior to the node. E- H Two stage 5 embryos, 6 hours after labelling the node. Labelled cells are restricted to the midline, but have extended in an AP direction, in a region closely correlating with the emerged axial mesoderm. I J Stage 7 embryo, 6 hours after labelling the node. Labelled cells are found extending from just behind the headfold, back along the midline. K L Stage 4 embryo, immediately after labelling, showing that label is initially extremely localised and well within the shoulders of the node. M- N Stage 5 embryo, 6 hours after labelling posterior to the node. Labelled cells are found in bull’s horns shapes extending from the node, leaving the midline area immediately ahead of the node unlabelled. O- P Stage 44- embryo, 6 hours after labelling half way between the node and the anterior of the embryo. Labelled cells are found right on the anterior boundary of the area

pellucida. Scale bar in A = 300p,m for A, B, E, F; 650p,m for C, D, K- P and 800|Ltm

Some embryos had reached stage 5 after the short incubation. In these embryos,

labelled cells were much less scattered and were instead restricted to the midline

(Fig5.1E-H). They had, however, extended in an anteroposterior direction, and were

seen in a region closely correlating with the emerged prechordal mesoderm and head

process (12/15 embryos). Labelled cells were found overlying the prechordal

mesoderm in all cases, although extending back along the head process to variable

degrees. The cells directly over the node were unlabelled in most cases, and labelling

was never seen posterior to the node.

Finally, the oldest set of embryos had reached the 1-2 somite stage by the time of

fixation. Once again labelled cells were found close to the midline, and had extended

in an AP direction. In 11/15 embryos, labelled cells were found extending from just

behind the headfold back along the midline (Fig5.11,J), while in 3/15 cases the labelled

cells were found more posteriorly, at the level of the somites, although still anterior to

the regressing node. In these latter embryos, the fact that labelled cells were not found

as far anteriorly is likely to be because these embryos were the oldest at the time of

operating, and could have been just beyond stage 4. Therefore, the anterior cells may

have already moved slightly ahead of the node, and so were not labelled.

5.2.2 Labelling anterior to the node at stage 4

The movement of lower layer cells anterior to the node was also analysed using Dil.

Patches of cells in the midline, half way between the stage 4 node and the anterior of

the embryo, were labelled (n=4). Six hours later, these cells were found to be lying

axial mesoderm that had emerged (Fig5.10,P). The group of labelled cells tended to

become elongated along the boundary. Interestingly, cells labelled in this anterior

margin at stage 4 were later found to have moved even further anterior and were lying

over the area opaca.

5.2.3 Labelling just posterior to the node at stage 4

In order to determine whether the above results indicated a global anterior movement

of lower layer cells, pulses of Dil were applied just posterior to the node at stage 4.

Following 6 hours further incubation, all embryos (n=5) had reached stage 5 and

labelled cells were found in and around the node and anterior streak. Interestingly, the

area of labelled cells had extended anteriorly to some extent, but only in more lateral

regions. Thus, labelled cells were found in diagonal lines or bull’s horns shapes

extending from the node, while the midline area immediately ahead of the node was

largely unlabelled (Fig5.1M,N).