Influence of Retinoids on Embryonic Chick Intestinal Development J. Orion Rogers and Sheila Shomo Department of Biology Radford University Radford, VA 24142

Dr. Orion Rogers and Sheila Shomo

Objective To ascertain the effects of retinol (Vitamin A) and retinoic acid (RA) on the development of intestines from 14-day-old chicken embryos incubated for 48 hours at 38 o C in culture medium 199 containing either 0.7, 1.3, or 2.8 mM extracellular Ca 2+.

Hypotheses Retinol and retinoic acid (RA) added to cultures of embryonic chick duodena containing altered concentrations of extracellular ionized calcium (Ca 2+ ) will result in an increased number and equal distribution of goblet cells along previllous ridges compared to cultured controls. Previllous ridge height will not increase in the presence of retinoids compared to controls cultured without retinoids.

Hypotheses Retinol and RA will interact with Ca 2+ in the culture medium in a concentration dependent manner. The retinoids will have the smallest effect on differentiation in the lowest concentration of Ca 2+, 0.7mM, and will have the greatest effect in the highest extracellular calcium concentration, 2.8 mM.

Previous Retinoid Research The influence of retinoids on intestinal epithelium has been investigated. It has been known that intestinal goblet cell numbers were decreased in rats that were deficient in vitamin A, i.e. retinol (Manville, 1937), and a report stated that goblet cell numbers in vitamin A deficient rats were decreased by 50% compared to controls (DeLuca et al., 1969).

Previous Retinoid Research (cont.) Additional research has shown that the removal of RA decreased rat intestine goblet cell number by 40%, in 4 days; however, the addition of RA resulted in normal goblet cell numbers within 30 to 48 hours (Olson, et al., 1981). These reports imply that the retinoids may have an effect on embryonic chick intestinal development.

Chicken Embryos Fertile eggs from a White Leghorn layer strain of Gallus domesticus were obtained from the Animal & Poultry Science Department of Virginia Polytechnic Institute and State University. Eggs were incubated at 100 o F for days. Embryos were removed from the eggshell at 14 or 16 days and euthanized by decapitation.

Methods Duodenal loops were explanted, cut into 2- 3 mm long segments, and split longitudinally to expose the mucosa to culture medium. Multiple duodenal segments from 14-day- old embryos were divided among four 25- mL Erlenmeyer flasks of culture medium 199, with each containing one of the four culture treatments.

Culture Treatments 1. Unaltered calcium concentration, 1.3 mM, and retinoid solvent, dimethylsulfoxide (DMSO) or ethanol (EtOH) 2. Altered calcium concentration, 0.7 or 2.8 mM, and retinoid solvent, DMSO or EtOH 3. A retinoid, e.g M RA, in 1.3 mM calcium 4. A retinoid in altered calcium concentration, 0.7 or 2.8 mM.

Methods The Ca 2+ of the medium 199 was adjusted to 0.7 mM or 2.8 mM by the addition of EGTA or CaCl 2, respectively. Flasks were gassed with 95% O 2 / 5% CO 2, stoppered tightly, and incubated for 48 hours at 38 o C. After incubation, duodenal segments were fixed for 3 hours in Carnoy’s fixative and dehydrated for 2 hours in 100% ethanol. Dehydrated tissue was cleared with HemoDe, infiltrated, and embedded in paraffin.

Sectioning Ribbons of paraffin were cut 5  m thick using a microtome.

Staining Goblet Cells Goblet cells were stained by the periodic acid-Schiff (PAS) procedure with fast green as a counterstain. Removal of glycogen was accomplished by a 30 minute pretreatment with 0.5% amylase.

Counting Goblet Cells Goblet cells were counted on previllous ridges that were sectioned symmetrically from top to bottom. Counts were made on every third section of specimen until 100 previllous ridges were counted. Previllous ridge height measurements were made from base to tip on every tenth symmetrically cut ridge until a total of 30 ridges had been measured. Results were expressed as an average height in  m.

Schedule of Experiments 1 week of 14-days-old uncultured embryos 1 week of 16-days-old uncultured embryos 3 weeks of Retinoic Acid mM Ca 2+ 3 weeks of Retinoic Acid mM Ca 2+ 3 weeks of Retinol mM Ca 2+ 3 weeks of Retinol mM Ca 2+ Summary: 14 experiments over 14 weeks with 48 embryos from 14 dozen eggs

Time involved in culture experiments One experiment consists of 3 embryos and 12 culture flasks Dissection of embryos - 2 hours Fixation through embedding - 8 hours Sectioning embedded tissue - 3 hours Staining slides - 4 hours Counting goblet cells and measuring previllous ridges - 6 hours Data analysis -1 hour Time involved in one experiment = 24 hours

Results after 10 weeks of experiments Total eggs set = 134 Unfertile eggs = 63 (47.0%) Dead embryos = 19 (14.2%) Deformed embryos = 4 (3.0%) Living embryos used = 36 (26.9%) Living embryos unused = 12 (9.0%)

Figure 1. Beak length Beak length results indicates that 14-day-old embryos (n = 29) are Hamburger-Hamilton stage 40 embryo, i.e. 14-day, and 16-day-old embryos (n = 7) are Hamburger-Hamilton stage 42 embryos, i.e.16-day. * Beak lengths differed significantly (P < ).

Figure 2. Third Toe Length Third toe length results indicate that 14-day-old embryos (n = 29) are Hamburger-Hamilton stage 40 embryos, i.e. 14-day and 16-day-old embryos (n = 7) are Hamburger-Hamilton stage 42 embryos, i.e.16-day. *Third toe lengths differed significantly (P < ).

Figure 3. Goblet Cell Number in Uncultured Tissue Total goblet cell numbers increased significantly by 62% from 14 to 16 days in uncultured duodena. Preliminary Data

Figure 4. Previllous Ridge Height in Uncultured Tissue Previllous ridge height increased significantly (p < ) by 65% from 14 to 16 days in uncultured duodena. Preliminary Data

Figure 5. Goblet Cell Distribution in Uncultured Tissue Goblet cells increased by 16% in the distal half of the previllous ridge in 16 versus 14-day-old duodena, but the difference was not statistically significant. Preliminary Data

Figure 6. Effect of Retinoic Acid on Goblet Cell Number Increasing extracelluar calcium increased goblet cell number. 10  M Retinoic acid had a dramatic and unexpected effect of completely inhibiting previllous ridge growth and goblet cell development. Preliminary Data

Figure 7. Effect of Retinoic Acid on Previllous Ridge Height Increasing extracelluar calcium had no consistent effect on previllous ridge height. 10  M Retinoic acid had a dramatic and unexpected effect of completely inhibiting previllous ridge growth. Preliminary Data

Figure 8. Effect of Retinoic Acid on Goblet Cell Distribution Increasing extracelluar calcium had no consistent effect on goblet cell distribution. 10  M Retinoic acid had a dramatic and unexpected effect of completely inhibiting previllous ridge growth. Preliminary Data

Preliminary Conclusions Both goblet cell number and previllous ridge height increased significantly from 14 to 16 days in uncultured duodena. Retinoic acid had a dramatic and unexpected effect of inhibiting previllous ridge growth and goblet cell development, but had no deleterious effect on tissue morphology. Increasing extracellular calcium resulted in an increase in goblet cell number, but it had no consistent effect on previllous ridge height or goblet cell distribution.

Future Projects Complete histological processing of retinol in 0.7, 1.3 and 2.8 mM Ca 2+ experiments Conduct concentration-response studies of retinoic acid and retinol Conduct time course studies of retinoic acid and retinol Collaborate with colleagues to measure effects of RA and retinol on other indices of differentiation such as enzyme activity

Acknowledgements Many thanks to Dr. Paula Stanley and the Faculty Development Center for the Faculty/Student Collaborative Grant that made this project possible. This grant funded the costs of eggs and consumable supplies. Thanks to the Department of Biology for research space in 318 Reed Hall and a BIOL 491 Research Grant.

Bibliography DeLuca, L., Little, E. P., and Wolf, G. Vitamin A and protein synthesis by rat intestinal mucosa. J. Biol. Chem. 244: , Manville, I. A. The interrelationship of vitamin A and glucuronic acid in mucine metabolism. Science 85: 44-45, Olson, J. A., Rojanapa, W., and Lamb, A. J. Vitamin A and goblet cells in rat intestine. Ann. N. Y. Acad. Sci. 359: 181, 1981.