Brett Rosenthal performed the line incisions on several of the germinal cord segments, and Elliott Kim and Robert Gish collected many of the control films for germinal cord retraction and designed and performed many of the amnioserosa incisions. They also provide an accessible system to probe tissue and cell dynamics since the relevant tissue is on the surface of the embryo. For the earliest stage, germinal band retraction, I will focus on understanding the mechanical interactions between the amnioserosa and germinal band that allow them to move together to restore the tail of the developing tissue to the posterior part of the embryo.
In the next phase, dorsal closure, much work has been done to delineate tissue contributions to movement [4,5]. At the tissue level, we use laser microsurgery to investigate the mechanical properties of individual segments of the germline and to create incisions that simultaneously test the necessity of different parts of the amnioserosis after retraction and allow us to separate its biochemical and mechanical contributions. Although laser microsurgery experiments provide us with strong insight into the tissue mechanics of germline retraction and stress distribution in amnioserosa cells during dorsal closure, only in combination with computer simulations can we create a complete model of the physics behind our experimental results.
The next section of this chapter provides an overview of the biological system used to investigate the mechanics of development, namely epithelial cells in Drosophila embryos during germ band retraction and dorsal closure. The next three chapters discuss the results of my work on germ band retraction and dorsal closure.
The System
At the same time, the amnioserosa cells are shortened and widened until the amnioserosa inhabits a teardrop shape on the dorsal surface of the embryo. The amnioserosa and the germinal band maintain their connections throughout retraction except at the caudal end of the germinal band (segments A8 and A9). They worked with one of the u-shaped group, retrograde mutants, where the amnioserosa undergoes premature apoptosis and germ band retraction fails.
Post-germband retraction embryos in (A) wild-type, (B) hindsight mutant, and (C) hindsight mutant with an overexpression of the insulin receptor homologue. The lack of retraction in these embryos suggests that the cells of the amnioserosa are active. In these mutants, the lamellipodia that normally connect the amnioserosa to the caudal end of the germ band are absent.
With the onset of closure, the amnioserosa covers most of the dorsal surface of the embryo in the form of a tear drop (Figure 1.7). Both the amnioserosa and the leading edge of the lateral epidermis contribute to dorsal closure, but neither is absolutely required.
The physics
On the surface of the embryo, two epithelia, the germinal band and the amnioserosa, move dramatically in unison. At the end of withdrawal, the amnioserosa will have a teardrop shape on the dorsal surface of the embryo surrounded by the germinal band [13,53]. Many of the changes in cell shape in the germline are mechanistic. the autonomous but complete unfolding of the germline clearly requires mechanical assistance from the amnioserosa.
Here, we use laser microsurgery to probe the mechanical role of the amnioserosis and germinal cord during retraction. Each cell will also exert a force on the cell below it in the direction of the amnioserosis. In each, the maximum aspect ratio for incisions made in the y direction was consistent with the amnioserosis pulling the germline curve.
Although this leaves the other half of the amnioserosa intact, it was sufficient to stop germinal band retraction (Figure 2.5E, I). This is consistent with the hypothesis that the amnioserosa must pull segments on the germline curve for the germline to disintegrate. This indicates that cell elongation occurs normally in the segment along the germline curve without a complete amnioserosis and in the absence of retraction.
Thus, the same cell-autonomous changes in the germ band curve may depend on the presence of amnioserosis in the segments on the ventral side of the embryo. Of these, the majority attach to the middle of the wound edge (N=4 out of 10), while the rest reattach with equal frequency to the positive y-direction corner of the wound as to the negative y-direction corner (N=3 out of 10 for each ). The segments around the curve of the germ band behave quite separately from the rest of the tissue.
Although most of this tissue responds isotropically to laser-induced wounds, segments in the curve of the germ band show a pronounced anisotropy. Around the curve of the germ band, external tension is dominant, i.e. it determines the direction of cell elongation. These results confirm that the amnioserosa pulls on segments around the curve of the germinal band.
The amnioserosa physically pulls on at least some segments of the germ band, likely causing greater tension toward the amnioserosa. The segments around the germ band curve fall into the red and magenta categories. Many of the changes in the germ band can occur autonomously, but a pulling force from the amnioserosa allows segments to successfully move through the bend in the tissue.
Unlike the rest of the tissue, the segments around the curve of the germ band are more responsive.
