Functional analysis of a regulatory element controlling spatial and temporal expression of Hoxc8 in mouse development

Elucidation of the genetic basis of transcriptional regulation of Hox genes by the study of their cis-regulatory elements will provide information regarding the establishment of axial specification. To gain insight on the influence of individual enhancer on cis-regulation of Hox genes and developmental patterning, a phylogenetically conserved regulatory sequence, the Hoxc8 early enhancer (EE), is deleted, in vivo, using ES cell technology. Analysis of Hoxc8 expression patterns in the EE knockout embryos at different developmental stages show that the EE is necessary for the correct transcriptional regulation of Hoxc8 expression at both activation and maintenance stages. The EE knockout embryos show temporal delay of Hoxc8 expression at early developmental stages leading to skeletal defects along the A-P axis in the EE knockout mice. These results indicate that the EE is crucial for temporal regulation of Hoxc8 expression and precise temporal expression of Hox genes is critical for establishing regional identities. Comparison between the EE and a Hoxd11 enhancer that is involved in temporal control of Hoxd11 expression suggests a common temporal regulatory system may exist among the Hox clusters. At later developmental stages, the posterior boundaries of Hoxc8 expression domains are not maintained properly, suggesting the EE is involved in maintaining Hoxc8 expression. Therefore, the EE plays different roles in regulating Hoxc8 expression at different developmental stages. The fact that deletion of the EE does not completely eliminate Hoxc8 expression implies the existence of a Hoxc8 regulatory network to some degree independent of the EE. Transgenic analysis of Hoxc8 intergenic region indicates the enhancer elements that can compensate the function of the EE are located outside of the Hoxc8 intergenic region. Finally, the novel skeletal and neurological phenotypes observed in the EE knockout mice suggests possible interaction between Hoxc8 and Hoxb8. Taken together, this study provides direct knowledge of enhancer behavior in the context of the intact gene cluster and illustrates the importance of precise temporal regulation of Hox genes through cis-regulatory elements on pattern formation.