After getting a sizable portion of ablated embryos fixed in ethanol at the necessary stages (st. 18 and st. 30), in situ hybridization is necessary to determine the gene expression patterns in the embryos. This procedure works by linearizing a large amount of plasmid DNA grown from E. coli cultures. E. coli is used to gather a large amount of DNA for use because they reproduce quickly. After linearizing the DNA, transcription is done in vitro to create antisense RNA that will bind to the location of the RNA for the gene of interest. This RNA is then labeled with fluorescent or colored probes so that the signal can be observed. The genes of interest used in this experiment are XCG-1, Otx2, En2, and Krox20. These are various neural marker genes that are expressed early and later in development. A control group of embryos also undergoes the hybridization procedure as a control to show where normal expression occurs in these embryo stages.
The results of this project include the fact that embryos recover from neural tissue ablations, even ones performed later in gastrulation. This is shown through the gene expression of these embryos, as the gene expression mimics the control embryo gene expression. Future directions of this project include imaging embryos for calcium activity to investigate calcium’s role during the wound healing process and to utilize other marker genes such as Sox2 and NBT to fully investigate the state of recovery post-ablation. Further analysis of this data will also be performed during the semester through the use of paraffin sectioning and imaging to get a slide by slide view of gene expression in experimental embryos.