A single base-pair variation in a limb-specific Lhx9 cis-regulatory region modulates HAND2-associated posterior repression

LIM Homeobox 9 (Lhx9) is a transcription factor involved in vertebrate limb development. Despite conserved anterior expression, its distal expression is species-specific. In chickens, distal expression is restricted to the anterior mesoderm; in mice, it extends into posterior mesoderm. We identified and characterized a limb-specific Lhx9-associated cis-regulatory module, LARR9A. In chicken limb bioassays, chicken LARR9A was active in the distal anterior limb mesoderm, while murine LARR9A was active both anteriorly and posteriorly. HAND2, a posteriorly expressed transcription factor, complements anterior chicken LHX9 expression. Anterior ectopic Hand2 expression in chicken limbs inhibited LHX9 expression and chicken LARR9A activity. We identified an additional HAND2 binding site (E-box 5'C) in chicken LARR9A that was absent in mice. Presence or absence of this E-box is due to a single base-pair variation (A/C) in a core nucleotide. Introduction of the E-box into murine LARR9A excluded activity from the Hand2 domain in both chicken limb bioassays and transgenic mice. Although murine LARR9A exhibited posterior activity in chicken bioassays, it did not in transgenic mice indicating the need for additional posterior regulation to fully explain species-specific Lhx9 expression. Nevertheless, our data suggest that a single base-pair variation in LARR9A is associated with differential Hand2-modulated repression.