To measure the effect that thyroid hormone (TH) has on neural stem cells, thyroid hormones in zebrafish were manipulated, fixed, treated with EdU, and imaged. TH levels in zebrafish were altered by adding either, T4, a precursor to the T3 thyroid hormone that is biologically active, or PTU, which blocks the conversion of T4 to T3, therefore significantly decreasing the amount of thyroid hormones available to the body. In the first trial of the experiment we discovered that our drug dosage was too high and would kill the fish, so we lowered the doses in the second trial to 300nM of PTU and .5mM of T4. The 5dpf zebrafish were treated with respective drugs for two days. At 7dpf, the fish were fixed in 4% PFA and the brains were dissected. The dissected brains then went through an EdU Click-it copper-catalyzed cycloaddition reaction where a thiamine analog is added to dividing cells and labels those cells with fluorescent dye. EdU is incorporated into actively dividing cells and is often used to label stem cells in a proliferative state. The brains were then divided into well reacted and poorly reacted and imaged in both bright field and fluorescent field and merged. The representative images of each treatment in well reacted brain are shown in Figure 1. The poorly reacted brain are not shown because the poorly reacted brain could nor be imaged due to time constraints. Because of this, there are no poorly reacted brain EdU+ cell count data for this group.
The EdU positive cells in Lateral Recess and Posterior Recess were then counted and the number was recorded (table 1). The average and standard deviation was calculated for each condition. The result indicated that the average EdU positive cells in Lateral recess in the control is about 35( standard deviation:22), in PTU, 27, and in T4, 44(.7). Meanwhile the EdU positive cells in posterior recess is about 11(7.37) in control 15, in PTU and 13(1.17) in T4 (figure 2, A). The standard deviation was not included due to low sample size. While there are some differences between the different values, due to the low number of samples that are available, no significant conclusion can be reached with this result.
In the data which was gathered by the entire class, the well reacted brain data indicates that in the lateral recess, there is an average of 56(32) EdU+ cells in control, 62(37) in PTU treated cells, and 67(32) in T4 treated cells. This indicates that both PTU and T4 increases the amount of neural stem cell in the lateral recess. In the posterior recess., for controlling the average number of EdU+ cell is about 21(8), in PTU is about 21(5), and in T4 is 24(6) (figure 1, B). This indicates that while PTU does not have an effect on the number of neural stem cells, the addition of T4 increases the number of neural stem cells. However, the data may not be reliable since the standard deviation for the data is high. In the poorly reacted brains, the data indicates that there is an average of 38(21)for control, and 59 for PTU in the lateral region and 18 (7) for control and 23.5 for PTU in the posterior region (Figure 2, C). The standard deviation for PTU treated brains and the average and standard deviation of T4 treated cells were not included due to incomplete data. However, very few things are able to be concluded from the pooly reacted data because it is unknown whether the poor reaction is due to the cells having low neural stem cells or not reing reacted completely to the click it reaction, and there are very small sample size.