Modulation of TGF-beta and extracellular matrix in retionic acid-induced cleft palate
Degitz, Sigmund Jay, Jr.
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Permalink
https://hdl.handle.net/2142/21213
Description
Title
Modulation of TGF-beta and extracellular matrix in retionic acid-induced cleft palate
Author(s)
Degitz, Sigmund Jay, Jr.
Issue Date
1996
Doctoral Committee Chair(s)
Francis, Bettina M.
Department of Study
Veterinary Biosciences
Discipline
Veterinary Biosciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Health Sciences, Toxicology
Health Sciences, Human Development
Language
eng
Abstract
The mechanism of RA-induced cleft palate was studied using a mouse model in which 70 mg/kg RA was administered to pregnant mice on gestation day 12. This treatment resulted in a greater than 95% incidence of cleft palate. Therefore I could examine morphologic, biochemical, and molecular changes at intervening time points between RA treatment and cleft formation. The mechanism of cleft formation was investigated using a multi-faceted approach to examine RA induced changes in palatal shelf morphology, in composition of the palate extracellular matrix and in the synthesis and expression of TGF-$\beta$s protein and TGF-$\beta$s mRNA during shelf growth and remodeling.
The experimental evidence provided here shows that the palatal shelf mesenchyme is the primary target of RA. Treatment resulted in altered mesenchymal cell organization, reduced hydration, reduced palatal shelf size and delayed palatal shelf elevation. The combination of these factors resulted in failure of palatal shelves to make contact and fuse, leading to cleft formation. Other scientists have suggested that alterations in epithelial differentiation are central to cleft formation, and this possibility was investigated in these experiments. My data indicate that epithelial changes are secondary to mesenchymal changes and are not the cause of cleft formation.
The interactions between RA and TGF-$\beta$s are very complex. RA differentially regulated the mRNA and protein levels of TGF-$\beta$1. Changes in mRNA steady state levels were rapid and transient in nature, indicating a direct mediation by RA. Differential regulation is evident because RA treatment resulted in a decrease in the extracellular form of TGF-$\beta$1 in the absence of change in total TGF-$\beta$1 protein levels or levels of intracellular TGF-$\beta$1. Moreover, the pattern of localization and levels of TGF-$\beta$2 and TGF-$\beta$3 proteins were not dramatically affected, although there was a considerable increase in their mRNA steady state levels. The increases in mRNA steady state levels for TGF-$\beta$2 and TGF-$\beta$3, as for TGF-$\beta$1, were rapid and transient in nature, again arguing for direct mediation by RA.
The morphologic findings of altered mesenchymal organization and delayed elevation of the palatal shelves indicate RA-induced changes in palatal shelf extracellular matrix. The patterns of localization of collagen I, III, and IV, fibronectin, tenascin, laminin and hyaluronate were investigated in normal palate development. Collagen I and collagen IV, laminin, and hyaluronate proved to have spatial-temporal patterns of localization during palatal shelf growth and reorientation. However, using immunohistochemistry, RA-induced changes in these molecules could not be demonstrated.
My data demonstrates that excessive RA targets the palate mesenchyme rather than the epithelium, and that clefts are a consequence of shelves failing to make contact. These data provide evidence for interactions between RA and TGF-$\beta$s, and indicate that RA is capable of differentially regulating TGF-$\beta$s isoforms through processes involving different stages of TGF-$\beta$s synthesis and secretion. Further, changes in TGF-$\beta$ isoforms were observed prior to changes in mesenchyme morphology and must be considered as mediators of RA's effects on mesenchyme development.
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