Expression of the retinoblastoma family of tumor suppressors during murine embryonic orofacial development
Authors: Leezer JL , Hackmiller RC , Greene RM , Pisano MM 
Published: March 2003

Structured AbstractAuthors –Leezer JL, Hackmiller RC, Greene RM, Pisano MM.Objective –To elucidate the role of the retinoblastoma (Rb) family of tumor suppressors and growth regulators in transforming growth factorβ(TGFβ)‐mediated embryonic palatal growth and morphogenesis.Design –The spatio‐temporal expression patterns of the RB1, RB2/p130, and p107 tumor suppressor genes, their gene products (pRb, p130 and p107) and phosphoforms were examined in the developing murine secondary palate utilizing reverse transcriptase polymerase chain reaction (RT‐PCR) and immunoblot/immunolocalization analyses with phospho‐specific antibodies.Results –The RB1, RB2/p130, and p107 tumor suppressor genes and their gene products (pRb, p130, and p107) were differentially expressed in embryonic palatal tissue during the critical period of secondary palate development [gestational days (GD) 12–14]. Both hyper‐ (115 kDa) and hypo‐phosphorylated (110 kDa) forms of pRb were expressed, with a notable transient decrease in expression on GD 13. Functional (hypo‐phosphorylated) forms of pRB predominated during the critical period of palatogenesis. As opposed to pRb expression, p130 expression was transiently elevated on GD 13 in the embryonic palate, and functional (hypo‐phosphorylated) forms were expressed at exceedingly low levels. p107 levels gradually declined over the course of palatogenesis. This diminution in expression, however, was accompanied by a transition to more functional (hypo‐phosphorylated) forms of the p107 protein. When compared with Rb expression patterns in the whole embryo/fetus, each of the three Rb proteins exhibited unique and specific temporal patterns of expression in the developing palate. RT‐PCR analyses of RB1, RB2/p130, and p107 mRNA expression in embryonic palatal tissue revealed patterns of expression which paralleled steady‐state protein levels of pRb, p130, and p107 in the developing tissue. Immunolocalization of the Rb proteins demonstrated ubiquitous expression of pRb, p130, and p107 in embryonic palate mesenchyme and epithelium during GD 12–14 of development with intense nuclear staining of the Rbs in palate epithelial cells on days 12 and 13 of gestation.Conclusion –Changing patterns of pRb, p130, and p107 protein expression and phosphorylation were evident in the embryonic secondary palate during the course of palatal ontogenesis. As the function of all three Rb proteins is regulated primarily through their phosphorylation, this suggests significant variation in Rb protein functionality during the course of palate development. Additional evidence from our laboratory (manuscript in preparation) indicates that the TGFβs, key regulators of palatal growth and morphogenesis, are able to differentially modulate phosphorylation of the Rb proteins in this developing tissue, and hence the TGFβs may regulate the function(s) of the Rb proteins during palatal ontogenesis. Although functionality of the Rb proteins is known to be regulated primarily through post‐translational modification (i.e. phopsphorylation), comparison of RB1, RB2/p130, and p107 steady‐state mRNA levels with pRb, p130, and p107 steady‐state protein levels in the developing palate levels suggests a significant degree of regulation at the transcriptional level. Differential patterns of Rb expression and phosphorylation in the developing palate suggest that pRb, p107, and p130 may each play unique roles in various aspects of growth, morphogenesis and cellular differentiation during palatal ontogenesis. Studies directed at elucidating the precise cellular role(s) of pRb, p107, and p130 during palate development, and at TGFβregulation of Rb expression/phosphorylation are ongoing in our laboratory.