TEACHER COPY Answer Key

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1 TEACHER COPY Answer Key Activity #1 Observing Morphological Changes in Embryonic Development Observations: The morphology of the chick embryo is a rapid process directed by several factors, including genetic, temporal-spatial molecular regulation, and the mechanical environment. When conducting an experiment, choosing what to measure is an important, and sometimes difficult, decision. To help with deciding, potential data is classified into different types. One such classification is quantitative vs. qualitative. Q. #1 - Science Speak : What s the difference between quantitative and qualitative observations? Quantitative observations or measured data to which a numeric value can be assigned; this type of data can be analyzed using statistics to determine differences between data sets within a certain range of confidence. Examples: 1.2 mm[length], 3.5 grams[mass], 96.8 F[Temp], etc Qualitative observations or data which depict the quality or characteristics of an object, but cannot be expressed as a numeric value. Qualitative observations are important for understanding trends and general features of the phenomenon of study. Examples: color, scent, reactivity(bubbling, photon transmission), roughness, etc. Note that while qualitative observations cannot be directly quantified, there often are quantifiable measurements that can approximate them. Example: color can be defined in terms of Red-Green-Blue (RGB) spectrum to which an intensity value can be assigned to Red, Green, Blue that mimics the observed color. Q. #2 What are three possible qualitative and quantitative observations we can make using the ex-ovo culture system? Qualitative: color of embryo body/eye, complexity of branching in vascular network, presence of different morphological features(aka. Limbs, wing bud, webbing of limbs, etc) Quantitative: cross-sectional area of embryo, length of limbs, # of vein branches Data Collection: For our study of the effects of retinoic acid(ra) on the developing embryo, we are going to use two morphological parameters to compare between treatments. The first is Tip-to-Tail length, defined as the longest line drawn between any two points in the embryo. As previous research has suggested that excessive vitamin A will stunt development, Tip-to-Tail length will serve a measure of overall embryo growth. Secondly, we will measure eye diameter as retinoic acid is also implicated in eye defect formation. As you can imagine, there are several other possibilities, but these should yield informative results that fit within our resources of both time and equipment. Research resources are an important consideration when designing an experiment. Use ImageJ to measure the Tip-to-Tail length and eye diameter of the RA case study data in the file titled RA_Exp_Data.tif. Process the data as instructed, and answer the data analysis questions. Before taking a measurement, be sure to calibrate the image to 6.25µm/pixel, under Image>Properties.

2 Tip-to-Tail Length, mm Record the tip-to-tail length for each experimental condition in table below. Be sure to calibrate image before taking measurements. Note: record data in millimeters (mm), therefore you will need to convert micrometers (µm) mm Tip-to-Tail Length(mm) Control Sham (1mg/ml) (2mg/ml) N/A N/A Plot tip-to-tail length vs Day for all experimental conditions. Control Sham RA 1mg/ml RA 2mg/ml

3 Eye Diameter, µm Record the eye diameter for each experimental condition in table below. Be sure to calibrate image before taking measurements. Note: record eye diameter data in micrometers (µm) Eye Diameter (µm) Control Sham (1mg/ml) (2mg/ml) N/A N/A Plot eye diameter vs Day for all experimental conditions. Control Sham RA 1mg/ml RA 2mg/ml

4 Data Analysis: After collecting the data, researchers must interpret it to gain insight into the phenomenon under study. Work through the following questions as you analyze the data. Q#1) Compare the Tip-to-Tail length and eye diameter between the control and RA treatments. Did addition of retinoic acid stimulate or stunt growth? While the control and treated embryos had roughly the same starting tip-to-tail length, by day 7 the control tip-to-tail length was 25% greater than the 1mg/ml RA treatment. The 2mg/ml RA treatment embryo died before Day 7. These results suggest that RA stunted and/or terminated the growth of the embryo. Q#2) Approximate the growth rate of each treatment by determining the slope of the Tip-to-Tail curves. Which condition had the fastest growth rate? Which was the slowest? Control 0.39 mm/day, Sham 0.34 mm/day, RA 1mg/ml 0.25 mm/day, RA 2mg/ml 0.2 mm/day The control embryo had the fastest growth rate and the 2mg/ml RA treatment had the slowest. The control growth rate was roughly twice as fast as the 2mg/ml RA treatment. This result agrees with the conclusion in question #1, that RA stunted the embryo s growth. Q#3) The 2mg/ml RA treatment only had data for Day 4-5, while the 1mg/ml RA treatment has data from Day 4-7. What does this suggest about the relative toxicity of the two dosages? The 2 fold increase in RA dosage terminated the embryo 2 days sooner than 1mg/ml RA, suggesting that the effect of RA is immediate and significantly dependant on dosage. This underscores the important of administering correct dosage amounts when treating vitamin A deficiency, as a relatively small difference in dosage has a drastic difference in effect. Q#4) On Day 7, is the eye diameter of the RA 1mg/ml treatment greater or less than the control? Do you see any qualitative differences between the eyes of these two treatments? The 1mg/ml RA treatment eye diameter is over 100 µm smaller than the control at Day 7. The control eye has a slightly darker pigmentation than the RA treatment. The lighter pigmentation of the RA treated embryo could be due to either delayed growth or possibly a direct interaction of RA with pigment related gene expression.

5 Q#5) Are the sham results different from the control? What do the sham results indicate about our method of treatment? The sham results do not appear to be significantly different from the control. This indicates that the defects were not induced by the filter paper, but instead can be wholly attributed to the addition of retinoic acid. Q#6) From our data we see that RA treatment effected the global development of the embryo, yet the treatment was applied locally. Why do you think this is? Even though the retinoic acid is applied over a small area away from the embryo, it was placed on a blood vessel of the vasculature network. This particular placement on a vessel facilitated the spread, and uptake, of RA in the embryo body. This emphasizes the substantial role the circulatory system plays in nutrient consumption and trafficking, demonstrating how_ high local concentrations of harmful agents can have global consequences due to rapid transport throughout developing embryo Q#7) In the last image of both RA treatment the vasculature(blood vessel network) appears to be receding. Why do you think this is happening, and what effect would this have on the developing embryo? Since the entire blood vessel network is receding, as opposed to just around the filter paper, it seems that RA is somehow affecting the embryo s ability to create and maintain the network. A possible explanation is that RA treatment decreases the heart rate of the embryo which 1) reduces blood getting to the vasculature and 2) reduces the blood pressure, which may be a key signal for directing blood vessel formation. [other reasons exist, teachers should give credit for logical and well defended answers] Q#8) Our initial motivation for studying retinoic acid excess in the embryo was to assist vitamin A deficient mothers and infants. How do our results inform corrective therapies for this condition? Our results demonstrated the importance of optimizing vitamin A dosage to the patient, as even small changes in dosage could have lethal consequences. Vitamin A excess had a stunting effect, again underlining importance of finding correct dosage. Upper bound dosages for infants could be approximated from this study s data by scaling according to body weight, or blood volume.