title: RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.
abstract: OBJECTIVE
  Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing.
  
  
  METHODS
  Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers.
  
  
  RESULTS
  Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function.
  
  
  CONCLUSIONS
  With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.
authors.affiliation: Department of Pathology, Center for Genomic Sciences, University of Pittsburgh School of Medicine, PA, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Darden
authors.forename: F Z
authors.initials: FZ
authors.lastname: Hu
authors.forename: M D
authors.initials: MD
authors.lastname: Ehrlich
authors.forename: M C
authors.initials: MC
authors.lastname: Gorry
authors.forename: D
authors.initials: D
authors.lastname: Dressman
authors.forename: H S
authors.initials: HS
authors.lastname: Li
authors.forename: D C
authors.initials: DC
authors.lastname: Whitcomb
authors.forename: P A
authors.initials: PA
authors.lastname: Hebda
authors.forename: J E
authors.initials: JE
authors.lastname: Dohar
authors.forename: G D
authors.initials: GD
authors.lastname: Ehrlich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018833:Chaperonins / Q000235:genetics*; D002921:Cicatrix / Q000235:genetics* / Q000517:prevention & control; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D006021:Glycophorins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D014945:Wound Healing / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D006021:Glycophorins; D012333:RNA, Messenger; D018833:Chaperonins