Meeting Abstract

P1.60  Tuesday, Jan. 4  Pulmonary Development in American alligator, Alligator mississippiensis MINEAR, K.J.*; KURNATH, P; FARMER, C.G.; SCHACHNER, E.; University of Utah, Salt Lake City; University of Utah, Salt Lake City; University of Utah, Salt Lake City; University of Utah, Salt Lake City krystle.minear@utah.edu

We studied the development of the lungs of alligators to gain insight into the evolution of the respiratory system of birds and crocodilians. One hundred and sixty eggs from four different clutches were collected from the Rockefeller Refuge in Grand Chenier, Louisiana. The eggs were estimated at stage 14 in development when collected. They were transported by land to the University of Utah and then incubated at 30˚C under three separate treatments in a mixed clutch design: 12% O2 (hypoxic), 21% O2 (normoxic), and 30% O2 (hyperoxic). Embryos were harvested periodically and fixed using several methods. For analysis by scanning electron microscopy, the trachea was intubated and the lungs fixed with buffered gluteraldehyde. For whole mount imaging using immunohistochemistry with antibodies that bind to alpha actin smooth muscle and e-cadherin, the lungs were fixed using several methods. In younger embryos the entire embryo was fixed in 4% paraformaldehyde (PFA) for 12-16 hours and then dehydrated into 100% MeOH using a stepwise dehydration process. For older embryos the lungs were dissected out in cold, buffered saline and fixed for 1 hour in PFA and then dehydrated or fixed in formalin free IHC Zinc Fixative at 4˚C overnight and then dehydrated. The lungs were stored at -20˚C until they were stained. Our preliminary results indicate there is extensive smooth muscle in the distal bronchi. We have not analyzed sufficient numbers to assess whether or not our oxygen treatments altered bronchial morphogenesis.