Expression profiling of transforming growth factor beta superfamily genes in developing orofacial tissue

Abstract

Background: Numerous signaling molecules have been shown to participate in the dynamic process of orofacial development. Among these signal mediators, members of the transforming growth factor beta (TGFbeta) superfamily have been shown to play critical roles. Developing orofacial tissue expresses TGFbeta and bone morphogenetic protein (BMP) mRNAs, their protein isoforms and TGFbeta- and BMP-specific receptors. All these molecules display unique temporospatial patterns of expression in embryonic orofacial tissue, suggesting functional roles in orofacial development. For example, the TGFbetas and BMPs regulate maxillary mesenchymal cell proliferation and extracellular matrix synthesis. This is particularly noteworthy in that perturbation of either process results in orofacial clefting. Although the cellular and phenotypic effects of the TGFbeta superfamily of growth factors on embryonic orofacial tissue have been extensively studied, the specific genes that function as effectors of these cytokines in orofacial development have not been well defined.

Methods: In the present study, oligonucleotide-based microarray technology was utilized to provide a comprehensive analysis of the expression of the panoply of genes related to the TGFbeta superfamily, as well as those encoding diverse groups of proteins functionally associated with this superfamily, during orofacial ontogenesis.

Results: Of the 7000 genes whose expression was detected in the developing orofacial region, 249 have been identified that encode proteins related to the TGFbeta superfamily. Expression of some (27) of these genes was temporally regulated. In addition, several candidate genes, whose precise role in orofacial development is still unknown, were also identified. Examples of genes constituting this cluster include: TGFbeta1-induced antiapoptotic factor-1 and -2, TGFbeta-induced factor 2, TGFbeta1 induced transcript-1 and -4, TGFbeta-inducible early growth response 1, follistatin-like 1, follistatin-like 3, transmembrane protein with EGF-like and two follistatin-like domains (Tmeff)-1 and -2, nodal modulator 1, various isoforms of signal transducers and activators of transcription (Stat), notch, and growth and differentiation factors.

Conclusions: Elucidation of the precise physiological roles of these proteins in orofacial ontogenesis should provide unique insights into the intricacies of the TGFbeta superfamily signal transduction pathways utilized during orofacial development.

Figure 1. Photomicrographs of ventral views of the developing orofacial region of a GD-13 mouse embryo

(a) upper and lower lips and jaws (maxilla and mandible); (b) the embryonic oral cavity; the lower half of the photo contains the roof of the oral cavity with the maxillary processes, primary palate and secondary palatal processes; the upper half contains the base/floor of the oral cavity showing the tongue and the mandible; (c) a magnified view of the roof of the oral cavity: note that the upper lip and the primary palate are completely formed, and the developing secondary palatal shelves are derived from the medial aspect of each maxillary process. The region demarcated by the blue line was excised from GD-13 embryos for extraction of total RNA. Corresponding regions were dissected from the developing orofacial region of GD-12 and GD-14 embryos. (UL) upper lip; (LL) lower lip; (Mx) maxilla; (Md) mandible; (P1) primary palate; (P2) secondary palate; (T) tongue.

Figure 2. Heat map illustrating the statistically significant alterations in expression of the TGFß superfamily of genes during murine orofacial development

Each row of the heat map represents a gene, and each column represents a time point in development (gestational day labeled along the bottom). The color saturation represents the level of gene expression. Red indicates an increase in gene expression, whereas blue indicates a decrease. Genes whose expression demonstrated a two-fold or greater increase or decrease are depicted. The list of genes comprising the heat map is listed in Table 2.