Molecular fingerprinting of TGFß‐treated embryonic maxillary mesenchymal cells

Abstract

The transforming growth factor‐ß (TGFß) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGFß on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGFß in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGFß action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's Atlas^TM Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGFß‐treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane‐based cDNA array (1178 genes) was performed with ³²P‐labeled cDNA probes synthesized from RNA isolated from either TGFß‐treated or vehicle‐treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImage^TM analysis in order to determine differences in gene expression between control and TGFß‐treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up‐regulated, while those of 8 other genes were down‐regulated, by a factor of twofold or greater in response to TGFß. Affected genes could be grouped into three general functional categories: transcription factors and general DNA‐binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta‐actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in response to TGFß treatment. Gene expression profiling results were verified by Real‐Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGFß responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGFß in orchestrating craniofacial ontogeny.