INTRODUCTION

In vertebrate limb morphogenesis, wingless-related integration site (Wnt) proteins and fibroblast growth factors (Fgfs) secreted from the apical ectodermal ridge (AER) coordinate proximodistal outgrowth. Fgfs also sustain sonic hedgehog (Shh) in the zone of polarizing activity (ZPA). Shh directs anteroposterior patterning and expansion and regulates AER-, establishing a positive regulatory feedback loop that is vital in sustaining limb outgrowth. The transcription factor LIM homeodomain 2 (Lhx2) is expressed in the distal mesoderm and coordinates AER and ZPA signals that control cellular proliferation, differentiation, and shaping of the developing limb. Yet how Lhx2 is transcriptionally regulated to support such functions has only been partially characterized.

METHODS/RESULTS: We have identified two limb-specific -regulatory modules (CRMs) active within the expression domain in the limb. Chromatin conformation analysis of the locus in mouse embryonic limb bud cells predicted CRMs- promoter interactions. Single-cell RNA-sequencing analysis of limb bud cells revealed co-expression of several Fgf-related and Wnt-related transcripts in -expressing cells. Additionally, disruption of Ets and Tcf/Lef binding sites resulted in loss of reporter-driven CRM activity. Finally, binding of β-catenin to both -associated CRMs supports the associated binding of Tcf/Lef transcription factors.

DISCUSSION

These results suggest a role for Ets and Tcf/Lef transcription factors in the regulation of expression through these limb-specific -associated CRMs. Moreover, these CRMs provide a mechanism for Fgf and Wnt signaling to localize and maintain distal expression during vertebrate limb development.