Tuesday, August 7, 2012

Keira- Location of cell proliferation in healing larvae (Week 6)

I've been wounding  D.excentricus larva by using a laser to remove one of their arms.

I fixed the healing Dendrasters 1 and 3 days after wounding. I stained them with the phosphohistone antibody (discussed in an earlier post) in order to determine areas of cell proliferation. Were the cells at the tip of the wounded arm dividing rapidly in order to regain the missing length? If that were the case, then we would see a density of dividing cells  in the tip of the wounded arm.

In the larva on the left, fixed one day after wounding, we see dividing cells (yellow) throughout the larval body. Dividing cells are visible almost to the tip of the non-wounded (long) arm. There are few dividing cells in the wounded arm. In the larva on the right, fixed 3 days after wounding, we see a great deal of cell division in the wounded arm, but the density of dividing cells is similar throughout the larval body. No dividing cells are visible in the very tip of the long arm, but cell division is relatively even throughout the rest of the body. We do not see a great density of cell division in the tip of the wounded arm. In order to regrow the arm, cells must be coming from somewhere. It is possible that the wounded arm regrows at the same rate as the normal growth in the rest of the larval body.

I did a similar experiment with the P.miniata larva. I had been bisecting them:

I fixed them after 3 days and 6 days of healing and regrowth. P.miniata larva are preforming considerable repair and regrowth after bisection.  My question about this regrowth was similar to my question about the regrowth of the D.excentricus arms; where are all these cells coming from? I stained the P.miniata larvae with the same phosphohistone antibody.

In the larvae fixed 3 days after bisection, we see cell division spread throughout the larval body. There is some preferential division in the ciliary band of the anterior half, but certainly no noticeable density of division at the wound site (the line of bisection).

In the larvae fixed 6 days after bisection, we see considerable density of cell division at the wound site. The difference between the location of cell division at these stages suggests that the larva must initially recover from the wound before regrowing. Once given time to repair (close) the wound, the larva will start to rebuild its "missing half." Cell division seems to preferentially occur at the wound site starting around 6 days after bisection.

Since the repair that the bisected P.miniata must do is so much greater that the repair required by the D.excentricus arm regrowth, it seems that P.miniata larvae must focus cell divisions at the wound site, while D.excentricus larvae can regrow the arm as a part of their continuing normal growth.

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