Limb development occurs along three major axes – proximal‐distal, anterior‐posterior, and dorsal‐ventral. Fibroblast growth factors (FGFs) secreted by the apical ectodermal ridge (AER) and sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA) are responsible for proximal‐distal and anterior‐posterior development, respectively, and maintain each other through a positive feedback loop. This reciprocal loop is critical for proper limb development. Recently, we identified LIM homeobox 2 (LHX2) as an intermediate in FGF‐mediated SHH expression. There are over 25 conserved regions of non‐coding DNA associated with the LHX2 gene locus that could serve as regulatory modules and targets of FGF signaling. We hypothesize that FGF regulates LHX2 through at least one of these potential cis regulatory modules (PCRMs). We selected 10 PCRMs with active chromatin marks in the limb and screened for their activity within the LHX2 expression domain (distal mesoderm subjacent to the AER). Each PCRM was inserted into the pTK‐GFP reporter and electroporated into the distal mesoderm of HH20‐23 chicken embryo wing buds. PCRM activity was determined 24 hours later using fluorescence microscopy. We found three of the PCRMs display activity (CRM (−19), CRM (−2), and CRM (−1)) that overlap LHX2 expression in the chicken wing bud. One CRM (−19), approximately 130 kb upstream of the LHX2 locus, is most consistent with the pattern of LHX2. Further studies are underway to determine whether these CRMs interact with the LHX2 promoter and whether FGF regulates their activity.Support or Funding InformationFunded in part by a grant from the LLU Pathology Endowment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IntroductionDuring development, fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER) direct limb outgrowth. FGFs also coordinate limb patterning through cross‐talk with other signaling molecules. The LIM homeobox transcription factor LHX2 is a suspected regulator of patterning cross‐talk, is expressed at the distal tip of the limb bud subjacent to the AER, and is a primary response target of FGF (Figure 1). However, the mechanism by which FGFs upregulate LHX2 is unknown. There are four major FGF signaling pathways through which regulation may occur: Jak/Stat, PLCγ, AKT, and Ras‐related (Figure 2). Based on the suspected roles of each signaling pathway, we hypothesize that FGF mediates LHX2 upregulation through the Ras‐related pathway.MethodsTo determine whether FGFs regulate LHX2 through the Ras‐related pathway, we injected the chemical inhibitor Salirasib (2.5 mM) into the anterior, posterior, and distal tips of the right limb of chick embryos at Hamburger‐Hamilton stage 21–22 (Figure 3). We also examined the potential function of the other Fgf pathways using pathway‐specific inhibitors. The embryos were harvested 24 hours after treatment and assessed for morphology. Whole mount in situ hybridization for LHX2 was then performed (Figure 4).ResultsPreliminary results indicate that inhibition of the Ras‐related pathway caused growth inhibition of the treated limbs in all embryos. In addition, LHX2 expression was markedly decreased in the these limbs. Treatment with a PLCγ‐specific inhibitor did not alter limb growth, while an AKT‐specific inhibitor impaired limb growth and LHX2 expression.ConclusionThese results suggest that the Ras‐related and AKT pathways control Fgf‐mediated LHX2 upregulation, whereas PLCγ does not. Further studies are needed to confirm these findings and to evaluate the Jak/Stat pathway.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fibroblast growth factors (FGFs) and sonic hedgehog (SHH) are mitogens known to affect limb growth and patterning during embryonic development. While FGFs establish the proximal‐distal axis from the apical ectodermal ridge (AER), SHH directs anterior‐posterior patterning from the zone of polarizing activity (ZPA). In addition, FGFs and SHH act in an autoregulatory loop by maintaining each other’s expression to coordinate limb patterning during limb outgrowth. The LIM Homeobox 2 (LHX2) transcription factor, which acts in the progress zone (PZ), was identified as an intermediary in the FGF to SHH arm of the maintenance loop. FGF mediates its action on cells through multiple intracellular signaling pathways including the JAK‐STAT self‐renewal pathway, PKC cell motility pathway, PDK1‐AKT cell survival pathway, and RAS‐associated cell proliferation pathway. We wondered whether one or more of these pathways directed FGF‐mediated upregulation of LHX2 in the FGF‐SHH regulatory loop. To evaluate a specific FGF signaling pathway, we implanted a bead soaked in a selective pathway‐specific inhibitor adjacent to an FGF2‐soaked bead in the posterior margin of Hamburger‐Hamilton stage 23–24 (HH23‐HH24) chicken limbs, a region known to induce LHX2 expression in response to FGF. The embryos were harvested after 4 hours and assayed for LHX2 and SHH expression by whole mount in situ hybridization. Ectopic LHX2 and SHH expression levels were reduced in the presence of the RAS and MEK inhibitors, indicating that FGF requires functional RAS‐associated pathways that utilize MEK to upregulate LHX2 expression.