Distinct Regulatory Elements GovernFgf4Gene Expression in the Mouse Blastocyst, Myotomes, and Developing Limb

AbstractEmbryonic development requires a complex program of events which are directed by a number of signaling molecules whose expression must be rigorously regulated. We previously showed that expression ofFgf4,which plays an important role in postimplantation development and growth and patterning of the limb, is regulated in EC cells by the synergistic interaction of Sox2 and Oct-3 with theFgf4EC cell-specific enhancer. To verify whether this mechanism was also operatingin vivo,and to identify new elements controllingFgf4gene expression in distinct developmental stages, we have analyzed the expression of LacZ reporter plasmids containing different fragments of theFgf4gene in transgenic mouse embryos. Utilizing these transgenic constructs we have been able to recapitulate, for the most part,Fgf4gene expression during embryonic development. We show here that most of thecis-acting regulatory elements determiningFgf4embryonic expression are located in conserved regions within the 3′ UTR of the gene. The EC cell-specific enhancer is required to drive gene expression in the ICM of the blastocyst, and its activity requires the Sox and Oct–proteins binding sites. We were also able to identify specific and distinct enhancer elements that govern postimplantation expression in the somitic myotomes and the limb bud AER. The myotome-specific elements contain binding sites for bHLH myogenic regulatory factors, which appear to be essential for myotome expression. Finally, we present evidence that the very restricted pattern of expression ofFgf4transcripts in the AER results from the combined action of positive and negative regulatory elements located 3′ of theFgf4coding sequences. Thus theFgf4gene relies on multiple and distinct regulatory elements to achieve stage- and tissue-specific embryonic expression