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

Lipinski RJ, Bijlsma MF, Gipp JJ, Podhaizer DJ, Bushman W - BMC Cell Biol. (2008)

Bottom Line: 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.This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli- 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA. lipinski@surgery.wisc.edu

ABSTRACT

Background: Hedgehog (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 mice.

Results: Here, 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 cells demonstrated reduced target gene induction while Gli3 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.

Conclusion: This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli- 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.

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Related in: MedlinePlus

Gli- iMEF morphology in monolayer cell culture. Indicated iMEFs were grown to confluence in monolayer culture and imaged at 40× magnification.
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Figure 1: Gli- iMEF morphology in monolayer cell culture. Indicated iMEFs were grown to confluence in monolayer culture and imaged at 40× magnification.

Mentions: Gli3+/+ (WT), Gli1-/-, Gli2-/-, Gli3-/-, Gli1-/-2-/-, and Gli2-/-3-/- primary MEFs were propagated by described 3T3 protocols for spontaneous immortalization [14]. Each non-clonal immortalized cell line demonstrated a fibroblast-like morphological appearance in monolayer culture although individual lines exhibited subtle morphological differences (Figure 1). Each iMEF line was determined to be tetraploid by flow cytometry analysis (data not shown).


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

Lipinski RJ, Bijlsma MF, Gipp JJ, Podhaizer DJ, Bushman W - BMC Cell Biol. (2008)

Gli- iMEF morphology in monolayer cell culture. Indicated iMEFs were grown to confluence in monolayer culture and imaged at 40× magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542994&req=5

Figure 1: Gli- iMEF morphology in monolayer cell culture. Indicated iMEFs were grown to confluence in monolayer culture and imaged at 40× magnification.
Mentions: Gli3+/+ (WT), Gli1-/-, Gli2-/-, Gli3-/-, Gli1-/-2-/-, and Gli2-/-3-/- primary MEFs were propagated by described 3T3 protocols for spontaneous immortalization [14]. Each non-clonal immortalized cell line demonstrated a fibroblast-like morphological appearance in monolayer culture although individual lines exhibited subtle morphological differences (Figure 1). Each iMEF line was determined to be tetraploid by flow cytometry analysis (data not shown).

Bottom Line: 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.This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli- 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA. lipinski@surgery.wisc.edu

ABSTRACT

Background: Hedgehog (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 mice.

Results: Here, 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 cells demonstrated reduced target gene induction while Gli3 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.

Conclusion: This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli- 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.

Show MeSH
Related in: MedlinePlus