Date of Award

1994

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Robert P. Thompson

Second Advisor

Stanley Hoffman

Third Advisor

Carwile LeRoy

Fourth Advisor

Edward Krug

Fifth Advisor

Irene Wang

Abstract

Cell adhesion molecules mediate specific cell-cell interactions during embryogenesis and tissue formation. Among cell-cell adhesion molecules (CAMs), we have been particularly interested in the neural cell adhesion molecule (NCAM) which is the prototypical member of the family of Ca++ -independent CAMs. Previous studies of NCAM cDNAs have revealed an alternatively spliced set of small exons (12A, 12B, 12C, 12D) that encode a region in the extracellular portion of the molecule known as the muscle specific domain (MSD). The entire MSD region can be expressed in skeletal muscle, heart, and skin; only exons 12A and 12D have been found in brain. These studies, however, did not reveal which NCAM polypeptides contain the MSD region or the immunohistochemical distribution of these NCAM molecules. To address these questions, we prepared antibodies against the oligopeptides encoded by exons 12A and 12B and by exons 12C and 12D and used these antibodies to study the forms of NCAM containing the MSD region expressed during embryonic chicken heart development. These antibodies recognize certain forms of NCAM found in the heart but do not recognize brain NCAM. In the heart, each of the splice variants of NCAM (ld, sd, and ssd) that differ in their mode of attachment to the plasma membrane or in the size of their cytoplasmic domain is expressed in a form containing and in a form lacking the MSD region. No microheterogeneity is observed in the size of NCAM molecules containing the MSD region, even at the level of CNBr fragments, suggesting that exons 12A-D are expressed as a single unit. We have identified six different isoforms of heart NCAM which exhibit changing temporal and spatial patterns of expression during development: the three forms of NCAM, ld, sd, and ssd, can also be expressed in a form containing the MSD sequence. Depending on the site and the stage of development, the percentage of NCAM molecules containing the MSD region can vary from nearly a to 100 percent. In general, this percentage increases during development. In immunohistochemical studies of hearts from stage 18 embryos, forms of NCAM containing the MSD region colocalized with Z-discs. No other adhesion molecules were found in this distribution at this early stage of development. Studies on isolated cells in vitro demonstrate that the colocalization with Z discs of NCAM molecules containing the MSD region does not depend on cell-cell contact and raise the possibility that this form of NCAM is involved in cell-extracellular matrix interactions. The association of NCAM molecules containing the MSD region with Z discs suggests that this form of NCAM is involved in early myofibrillogenesis.

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