Jason O. Brant

A Comparative Analysis of Gene Expression Profiles during Skin Regeneration in Mus and Acomys.

The African spiny mouse (Acomys spp.) can heal full thickness excisional skin wounds in a scar-free manner with regeneration of all dermal components including hair and associated structures. Comparing Acomys scar-free healing from Mus scarring identifies gene expression differences that discriminate these processes. We have performed an extensive comparison of gene expression profiles in response to 8mm full-thickness excisional wounds at days 3, 5, 7 and 14 post-wounding between Acomys and Mus to characterize differences in wound healing, and identify mechanisms involved in scar-free healing. We also identify similarities with scar-free healing observed in fetal wounds. While wounding in Mus elicits a strong inflammatory response, wounding in Acomys produces a moderated immune response and little to no increase in expression for most cytokines and chemokines assayed. We also identified differences in the ECM profiles of the Acomys wounds, which appear to have a collagen profile more similar to fetal wounds, with larger increases in expression of collagen types III and V. In contrast, Mus wounds have very high levels of collagen XII. This data suggests that an overall lack of induction of cytokines and chemokines, coupled with an ECM profile more similar to fetal wounds, may underlie scar-free wound healing in Acomys skin. These data identify candidate genes for further testing in order to elucidate the causal mechanisms of scar-free healing.

Protein or amino acid deprivation differentially regulates the hepatic forkhead box protein A (FOXA) genes through an activating transcription factor‐4–independent pathway

The FOXA (forkhead box A) proteins (FOXA1, FOXA2, and FOXA3) play a critical role in the development of the liver, and they also regulate metabolism in adult hepatic tissue. The liver responds to changes in nutrient availability by initiating a number of stress signaling pathways. The present studies demonstrated that in mouse dams fed a low‐protein diet hepatic expression of FOXA2 and FOXA3 messenger RNA, but not FOXA1, was induced. Conversely, fetal liver did not exhibit this regulation. Amino acid deprivation of HepG2 hepatoma cells also enhanced transcription from the FOXA2 and FOXA3 genes. In contrast, endoplasmic reticulum stress inhibited the expression of FOXA1, only slightly induced FOXA2, and had no effect on FOXA3. The FOXA2 and FOXA3 messenger RNA induction by amino acid deprivation did not require activating transcription factor 4, a critical component of the conventional amino acid response (AAR) pathway, but their induction was partially dependent on CCAAT/enhancer‐binding protein β. Simultaneous knockdown of both FOXA2 and FOXA3 by small interfering RNA did not affect the activation of other amino acid responsive genes, suggesting that the FOXA proteins are not required for the known AAR pathway. Collectively, the results document that the hepatic FOXA family of genes are differentially regulated by amino acid availability. (HEPATOLOGY 2009.)

Angelman syndrome imprinting center encodes a transcriptional promoter

Clusters of imprinted genes are often controlled by an imprinting center that is necessary for allele-specific gene expression and to reprogram parent-of-origin information between generations. An imprinted domain at 15q11–q13 is responsible for both Angelman syndrome (AS) and Prader–Willi syndrome (PWS), two clinically distinct neurodevelopmental disorders. Angelman syndrome arises from the lack of maternal contribution from the locus, whereas Prader–Willi syndrome results from the absence of paternally expressed genes. In some rare cases of PWS and AS, small deletions may lead to incorrect parent-of-origin allele identity. DNA sequences common to these deletions define a bipartite imprinting center for the AS–PWS locus. The PWS–smallest region of deletion overlap (SRO) element of the imprinting center activates expression of genes from the paternal allele. The AS–SRO element generates maternal allele identity by epigenetically inactivating the PWS–SRO in oocytes so that paternal genes are silenced on the future maternal allele. Here we have investigated functional activities of the AS–SRO, the element necessary for maternal allele identity. We find that, in humans, the AS–SRO is an oocyte-specific promoter that generates transcripts that transit the PWS–SRO. Similar upstream promoters were detected in bovine oocytes. This result is consistent with a model in which imprinting centers become DNA methylated and acquire maternal allele identity in oocytes in response to transiting transcription.

Cellular events during scar-free skin regeneration in the spiny mouse, Acomys.

In contrast to the lab mouse, Mus musculus, several species of spiny mouse, Acomys, can regenerate epidermis, dermis, hairs, sebaceous glands with smooth muscle erector pili muscles and skeletal muscle of the panniculus carnonsus after full thickness skin wounding. Here, we have compared the responses of these scarring and nonscarring organisms concentrating on the immune cells and wound cytokines, cell proliferation, and the collagenous components of the wound bed and scar. The blood of Acomys is very neutropenic but there are greater numbers of mast cells in the Acomys wound than the Mus wound. Most importantly there are no F4/80 macrophages in the Acomys wound and many proinflammatory cytokines are either absent or in very low levels which we suggest may be primarily responsible for the excellent regenerative properties of the skin of this species. There is little difference in cell proliferation in the two species either in the epidermis or mesenchymal tissues but the cell density and matrix composition of the wound is very different. In Mus there are 8 collagens which are up‐regulated at least 5‐fold in the wound creating a strongly trichrome‐positive matrix whereas in Acomys there are very few collagens present and the matrix shows only light trichrome staining. The major component of the Mus matrix is collagen XII which is up‐regulated between 10 and 30‐fold after wounding. These results suggest that in the Acomys wound the absence of many cytokines resulting in the lack of macrophages is responsible for the failure to up‐regulate fibrotic collagens, a situation which permits a regenerative response within the skin rather than the generation of a scar.

Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.

Improving efficiencies of locus-specific DNA methylation assessment for bovine in vitro produced embryos.

Characterization of DNA methylation is one assessment of chromatin remodeling in early embryos. Unfortunately, evaluation at specific loci is hindered by their small cell numbers. Our objective was to determine if bisulfite sequencing could be optimized for preimplantation embryos, comparing conversion times, primer design, and DNA amplification methods. Methylation at three loci, SATI, OCT4, and IGF2, was investigated in bovine in vitro produced (IVP) embryos, somatic cells, and no template controls. Bisulfite treatment for 15–16 h gave higher quality DNA than treatment for 18 h. Three step primer design improved bisulfite primer specificity, yielding more PCR product than primers previously reported. Following optimization, methylation data were obtained from as few as 4 cell equivalents. Finally, DNA amplification efficiencies were evaluated using miniprep, TempliPhi, or 96-well glycerol stocks with automated TempliPhi. While TempliPhi was better than standard minipreps, the 96-well format proved most efficient. Preliminary methylation profiles of bovine IVP 2-cell, 8-cell, blastocyst stage embryos and somatic cells were 25, 10, 22, and 74% for SATI and 88, 88, 79, and 88% for OCT4, respectively, suggesting that SATI is demethylated during early embryonic reprogramming, while OCT4 remains hypermethylated. IGF2 methylation was 84, 28, and 84% for bovine IVP 8-cell, blastocyst stage embryos and somatic cells; blastocyst stage embryos exhibited more variability, ranging from 0 to 80%. This new assay will enhance assessment of chromatin remodeling in embryos, and be especially useful for evaluating those produced by assisted reproductive technologies.

Perfect chronic skeletal muscle regeneration in adult spiny mice, Acomys cahirinus

The spiny mouse, Acomys cahirinus, is an adult mammal capable of remarkable feats of scar-free tissue regeneration after damage to several organs including the skin and the heart. Here we investigate the regenerative properties of the skeletal muscle of A. cahirinus tibialis anterior in comparison to the lab mouse, Mus musculus. The A. cahirinus TA showed a similar distribution of myosin heavy chain fibre types and a reduced proportion of oxidative fibres compared to M. musculus. There were differences in the matrix components of the TA with regard to collagen VI and the biomechanical properties. A. cahirinus TA regenerated faster with a more rapid induction of embryonic myosin and higher levels of dystrophin than in M. musculus fibres. There were lower levels of inflammation (NF-kB), fibrosis (TGFβ-1, collagens) and higher levels of the anti-inflammatory cytokine Cxcl12. There was a difference in macrophage profile between the two species. After multiple rounds of muscle regeneration the M. musculus TA failed to regenerate muscle fibres and instead produced a large numbers of adipocytes whereas the A. cahirinus TA regenerated perfectly. This clearly improved regeneration performance can be explained by differing levels of growth factors such as adiponectin between the two species.