CNS Activity of an LHX2‐Associated Cis‐Regulatory Module

Abstract

Limb development can be described along three axes – proximal‐distal, anterior‐posterior, and dorsal‐ventral. Fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) coordinate growth along the proximal‐distal axis, while sonic hedgehog (SHH), secreted from the zone of polarizing activity, directs anterior‐posterior patterning and expansion. AER‐related FGFs and SHH regulate each other in a positive feedback loop. This reciprocal loop is critical for proper limb patterning, however the molecular intermediates involved in this loop are only partially characterized. Previously, we identified LIM homeobox 2 (LHX2) as a downstream target of FGF in the FGF to SHH loop and determined that FGF upregulates LHX2 in the absence of de novo protein synthesis. This suggests that LHX2, expressed in the mesoderm subjacent to the AER, is a primary response target of FGF signaling.Cis‐regulatory modules (CRMs) within noncoding DNA can convey temporal and spatial regulation of an associated gene and are good candidates to convey the FGF‐mediated regulation of LHX2. We identified several potential CRMs (PCRMs) upstream of the LHX2 locus based on sequence conservation, active chromatin marks, and presence of transcription factor binding sites related to FGF signaling. Based on the number of active chromatin marks, we chose four of these PCRMs to investigate. We suspect that FGF regulates LHX2 through one or more of these PCRMs. Thus, we hypothesized that one or more of these PCRMs is active in the LHX2 expression domain during limb development.To test for activity of LHX2 PCRMs we used targeted regional electroporation (TREP) to introduce PCRM‐ptk‐GFP reporter constructs into the distal mesoderm of Hamburger‐Hamilton stage 23 (HH23) chicken limb buds. Transfection efficiency was determined by co‐transfection with a b‐actin promoter‐driven RFP construct. We determined PCRM activity by fluorescence microscopy, following 24 hours of incubation after TREP.None of the four PCRMs tested exhibited activity in the limb. However, subsequent TREP of the reporter construct containing PCRM ‐7 (~ 800 bp conserved region located 215 kb upstream of the LHX2 promoter) into the neural tube of HH10 embryos revealed green fluorescence at the midbrain‐forebrain boundary 24 hours post‐TREP. This activity suggests PCRM ‐7 may play a role in regulating LHX2 expression in the developing forebrain. Further studies are underway to correlate the activity of this CRM with expression of LHX2 or other genes in the topologically associated domain.