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

Background/Purpose: Scars form as wounds heal in adult organisms. In additi on to disrupting cosmetic appearance, scar tissue can cause significant mor bidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can he al without scarring. Because such inherent physiological mechanisms ultimat ely are under genetic control, a study was initiated to elucidate the diffe rences in gene expression that produce scarless wound healing in the mammal ian fetus but scarring in postnatal wounds. Reverse transcription polymeras e chain reaction (RT-PCR) differential display (DD) was used to detect diff erentially expressed mRNA transcripts in a rabbit model of wound healing. Methods: Adult and 21-day fetal full-thickness rabbit skin specimens from w ounded and unwounded sites were harvested 12 hours postwounding. RNA extrac ted 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 d ifferential expression among the 4 types of specimens: fetal control (unwou nded), fetal wound, adult control, and adult wound. Selected bands distinct ly upregulated or downregulated in feta I wound lanes on the DD gels were e xcised, and the cDNA was extracted, reamplified, cloned into vectors, and s equenced. DD results were confirmed by limiting-dilution RT-PCR using seque nce-specific primers. Results: Differential display (DD) showed 22 amplimers that were significan tly upregulated in all fetal wound samples as compared with little or no ex pression in fetal control, adult control, or adult wound tissues. Conversel y, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcr ipts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct seq uences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was down regulated in fetal wound showed very high sequence homology to part of the human gene for the ri su bunit of the hetero-oligomeric particle CCT (the chaperonin containing T-co mplex polypeptide 1 or TCP-1). An upregulated amplimer showed significant D NA sequence homology to glycophorins A and B. One sequence was identified a s 28S rRNA. The remaining 4 candidate sequences showed no significant homol ogy to known genes, but 1 had high homology to expressed sequence tags of u nknown function. Conclusions: With careful experimental design and proper controls and verif ications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiologic al process such as feta I wound healing. No a priori knowledge of what gene s might be involved, or why, is necessary. This study indicates that downre gulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of w ound healing in the mammalian fetus. Results suggest the hypothesis that do wnregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in pos tnatal wound healing, by preventing the folding of sufficient a-smooth musc le actin to form the stress fibers characteristic of these cells. Copyright (C) 2000 by W.B. Saunders Company.