Uma Karadge

Defense Date

3-8-2007

Graduation Date

Summer 2007

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Biological Sciences

Committee Chair

Richard P. Elinson

Committee Member

John A. Pollock

Committee Member

Sarah Woodley

Keywords

confocal microscopy, fate mapping, FDA ( Flourescein Dextran amine, microinjections

Abstract

The Puerto Rican tree frog, Eleutherodactylus coqui, is a striking example of a deviation from the indirect mode of development among amphibians. E. coqui does not have an intermediate tadpole stage. It hatches out directly as a free-living froglet, deriving nutrition from the yolky material attached to its intestine. The innermost of the three germ layers, the endoderm, forms the lining of the gut. In the amphibian model Xenopus laevis, the endoderm originates from the vegetal hemisphere. The cells, which are originally present on the vegetal surface of the embryo, end up forming the definitive gut. They, thus, provide nutrition as well as contribute towards the definitive gut. Using fate mapping, I determined the fate of the vegetal yolky cells in E. coqui. When one of the vegetal yolky cells in E. coqui is injected with a lineage tracer, Fluorescein Dextran Amine (FDA), the label ended up only in the yolky endoderm attached to the intestine in the developing embryo. There is no label in any part of the gut or body in all stages of development. The yolky endoderm is gradually used up by the developing embryo and the label disappears completely after all of the yolky endoderm is used. I conclude that the vegetal yolky cells in E. coqui are only nutritional in function. When the yolky endoderm was being utilized, the label appeared in the mesonephric kidneys. This suggests that when the yolk was being used, FDA was picked up by blood and cleared during filtration in kidneys. FDA, which has a molecular weight of 10,000, freely passes the glomerular filtration membrane and enters the nephric tubules. Confocal microscopy resolved that the dye is trapped in the form of aggregates or vesicles in the individual epithelial cells lining the nephric tubules. Some of the FDA entering the tubules is likely cleared in the form of dull fluorescent debris in the cloaca of the frogs. I also carried out fate mapping of the cells situated towards the animal pole (above-AV) and the cells near the equator (below-AV) and found that cells from both of these areas ended up in the definitive gut. They also contributed towards tissues and organs of mesodermal and ectodermal origin. Taking together all these data, I conclude that the vegetal yolky cells in E. coqui provide nutrition only. The gut is derived from cells closer to the animal pole. A more comprehensive fate map and further analysis of the utilization of yolk in E. coqui is required to better understand dye clearance and labeling in the mesonephric kidneys.

Format

PDF

Language

English

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