Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines

Jerry J Gipp,David J Podhaizer,Wade Bushman,Robert J Lipinski,Maarten F Bijlsma

doi:10.1186/1471-2121-9-49

Jerry J Gipp, David J Podhaizer + Show 3 more

Open Access

https://doi.org/10.1186/1471-2121-9-49

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Abstract

BackgroundHedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice.ResultsHere, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/- iMEFs was severely reduced while Gli2-/-3-/- iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/- iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.ConclusionThis study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

Highlights

Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence supporting a role in healing and repair processes and tumorigenesis
We showed previously that mouse embryonic fibroblasts (MEFs) from Gli null mice provide a tractable cell-based system in which to quantitatively examine the regulation of Hh target gene expression by the Gli transcription factors [11]
We describe the generation of immortalized Gli null MEFs and characterize their transcriptional and migratory response to Hh ligand stimulation

Summary

Introduction

Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. Null mouse models have been critical in determining the role of Hh signaling in the growth and morphogenesis of tissues and organs These models have proved valuable in gleaning the function of individual Hh signal mediators in pathway regulation. Gli2-/- mice exhibit neural tube defects and demonstrate diminished Hh target gene expression in several tissues [5,6,7]. This supports findings from cell-based assays [8] that Gli functions as a critical target gene activator. Increased target gene expression in tissues derived from Gli null mice relative to tissues from wild type mice [9,10] suggests that Gli functions to repress transcription

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