Pitx1 and Tbx4 target apoptotic and cell cycle arrest genes during early hindlimb development

Abstract

Congenital talipes equinovarus (clubfoot) currently affects one in 1000 newborns worldwide and seven in 1000 in the Māori and Polynesian populations. Currently very little is known about the underlying biological changes that can result in clubfoot. Several apoptotic genes have been linked to clubfoot, as well as gene mutation in two hindlimb specific identity genes; T-box 4 (Tbx4) and Paired-like homeodomain transcription factor 1 (Pitx1). The purpose of this study is to address whether apoptotic genes may be regulated by Tbx4 aJamiend Pitx1 and thus be a part of a broader gene network that may be altered in clubfoot patients. Using the mouse embryo as the model organism for limb development, chromatin immunoprecipitation with next generation sequencing (ChIP-Seq) using fore- and hindlimb tissue has revealed Pitx1 and Tbx4 enrichment at putative enhancer regions of apoptotic and cell cycle arrest genes: Cdkn1A, Gadd45b, Igfbp3, Il17b, Rprm, Sesn1, Traf5, Unc5A, Unc5D and Zc3h8. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to identify that Igfbp3 and Il17b expression is significantly upregulated in the forelimb, while Rprm was significantly expressed in the hindlimb. In addition, in situ hybridisation has shown expression of these apoptotic and cell cycle arrest genes to be expressed in different regions along the axis of the developing limb. Pitx1 and Tbx4 may be involved in the morphogenesis of the hindlimb through controlling apoptotic and cell cycle arrest mechanisms.