Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% (=617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential -regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization.

During limb development, fibroblast growth factors (Fgfs) govern proximal–distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximal–distal and anterior–posterior axes by maintaining Sonic hedgehog (Shh) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER) that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application), while prolonged exposure (24 h) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER.

Chitosan polymers (Cs), from which microparticles (CsM) may be precipitated to deliver various intracellular payloads, are generally considered biologically inert. We examined the impact of cell culture conditions on CsM size and the effect of chitosan on CD59 expression in primary human smooth muscle cells. We found that particle concentration and incubation time in biological buffers augmented particle size. Between pH 7.0 and pH 7.5, CsM size increased abruptly. We utilized CsM containing a plasmid with a gene for CD59 (pCsM) to transfect cells. Both CD59 mRNA and the number of CD59-positive cells were increased after pCsM treatment. Unexpectedly, CsM also augmented the number of CD59-positive cells. Cs alone enhanced CD59 expression more potently than either pCSM or CsM. This observation strongly suggests that chitosan is in fact bioactive and that chitosan-only controls should be included to avoid misattributing the activity of the delivery agent with that of the payload.

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.

Fibroblast growth factors (FGFs) and Sonic hedgehog (SHH) are morphogens known to be integrally involved in limb outgrowth/development. Published reports assert that for SHH to be expressed, FGFs have to be secreted from the apical ectodermal ridge (AER); however, not much is known about the molecular mechanisms underlying this regulation. SHH expression can be induced by the application of FGF‐soaked beads in the posterior region of the developing limb bud. DNA microarray analysis, following FGF bead application, revealed that LHX2 (LIM Homeobox 2) was upregulated >5‐fold. LHX2 encodes a transcription factor that is highly conserved across species, is expressed in the distal limb mesenchyme, and implicated in the integration of signaling inputs from the AER (FGFs), ZPA (SHH), and the dorsal ectoderm (WNT7a). Furthermore, LHX2/LHX9 double knockout mice show a marked reduction in SHH expression, confirming a role for LHX2 in regulating the signals that direct limb patterning and growth. Thus, we hypothesize that LHX2 is an intermediate in the FGF‐to‐SHH pathway. In order to test this hypothesis, we implanted FGF‐soaked heparin sulfate beads in embryonic chicken limb buds (Hamburger ‐ Hamilton stage 23), harvested the embryos, and assessed LHX2 expression in response to FGF induction via whole mount In situ hybridization (WMISH). Our results show that LHX2 is rapidly upregulated in response to FGF (<3 hrs). Its upregulation persisted through the experimental time course (24 hrs) and overlaps temporally with the upregulation of SHH in response to FGF. We therefore conclude that LHX2 is a downstream target of FGF and the early induction of LHX2 suggests that its regulation is direct. Further studies are underway to determine whether LHX2 can upregulate SHH and establish LHX2’s role as an intermediate in the FGF to SHH pathway. Grant Funding Source: Institutional

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.

Limb outgrowth is controlled, in part, by fibroblast growth factor (FGF) and sonic hedgehog (Shh). FGF up‐regulation of SHH is essential for coordinated limb pattering; however, not much is known about this mechanism. We recently found that LHX2 is an intermediate in FGF‐mediated SHH up‐regulation. LHX2 is part of a family of genes that are known to integrate signaling events. Thus, we hypothesized that FGF would up‐regulate LHX2 through associated potential cis regulatory modules (PCRM). Multiple PCRMs were found in association with the LHX2 locus. PCRMs were screened using comparative genomics, proximity to the LHX2 promoter, and transcription factor binding sites related to FGF signaling. To exam the role of suspected PCRMs, we electroporated PCRM‐reporter constructs into chick embryos. We found one PCRM with activity in the brain coincident with LHX2 expression, but none of our initial PCRMs exhibited activity in the limbs. We reevaluated the LHX2‐associated PCRMs in light of limb‐specific chromatin activation marks and identified 5 additional PCRMs for analysis. Studies are underway to determine limb‐related PCRM activity of this new cohort. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying the FGF‐SHH regulatory loop.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.