Cell cycle genes PEDF and CDKN1C in growing deer antlers.

Deer antlers are the only mammalian appendage to display an annual cycle of full regeneration. The growth phase in antler involves the rapid proliferation of several tissues types, including epidermis, dermis, cartilage, bone, blood vessels, and nerves. Antlers thus provide an excellent model to study the developmental regulation of these tissues. We describe here the identification of two genes, pigment epithelium-derived factor (PEDF) and cyclin-dependent kinase inhibitor 1C (CDKN1C), both of which are known to be involved in cell proliferation and differentiation. These genes were identified as the result of screening an expressed sequence tag database derived from a cDNA library enriched for sequences from the growing antler tip. PEDF mRNA was detected in developing skin, cartilage, and bone during endochondral ossification. PEDF mRNA was not detected within endothelial cells that exhibited positive immunoreactivity to a CD146 antibody. CDKN1C mRNA was expressed by only the immature chondrocytes within the precartilage region. These results suggested that PEDF and CDKN1C are important genes involved in cell proliferation and differentiation during antler growth.

Expression of VEGF and pleiotrophin in deer antler.

Deer antlers represent a unique model of mammalian regeneration in that they cast and fully regenerate every year. The deer antler thus provides a fascinating model of both rapid angiogenesis and chondrogenesis and the opportunity to investigate unique growth regulatory processes. One such phenomenon is the presence of vascularized cartilage in the growing antler tip-unlike other cartilage, which is typically avascular. The mechanisms by which blood vessels grow in the cartilage as well as the factors that drive antler extension at approximately 1 cm a day have been hitherto largely unknown. The aim of this study was to determine the expression of VEGF and pleiotrophin within the growing antler tip. We isolated cervine VEGF121 and VEGF165 from deer antler and found that mRNA is produced for VEGF in the precartilage and cartilage regions. By in situ hybridization, we examined whether the VEGF receptors Flt-1 and KDR are present in deer antler and found only KDR mRNA within the endothelial cells of the precartilage region. This finding is compatible with VEGF having an angiogenic effect within antler. Pleiotrophin mRNA was found in the vascular smooth muscle cells of the dermis, thus supporting a possible role in vascular growth. High levels of pleiotrophin mRNA were also detected in the precartilage region with possible implications for both angiogenesis and chondrogenesis. This is the first report of cervine angiogenic growth factors within the growing antler tip.

Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery.

The utilization of a deer antler model to study gene expression in tissues undergoing rapid growth has been hampered by an inability to sample the different tissue types. We report here a standardized procedure to identify different tissue types in growing antler tips and demonstrate that it can help in the classification of expressed sequence tags (ESTs). The procedure was developed using observable morphological markers within the unstained tissue at collection, and was validated by histological assessments and virtual Northern blotting. Four red deer antlers were collected at 60 days of growth and the tips (top 5 cm) were then removed. The following observable markers were identified distoproximally: the dermis (4.86 mm), the subdermal bulge (2.90 mm), the discrete columns (6.50 mm), the transition zone (a mixture of discrete and continuous columns) (3.22 mm), and the continuous columns (8.00 mm). The histological examination showed that these markers corresponded to the dermis, reserve mesenchyme, precartilage, transitional tissue from precartilage to cartilage, and cartilage, respectively. The gene expression studies revealed that these morphologically identified layers were functionally distinct tissue types and had distinct gene expression profiles. We believe that precisely defining these tissue types in growing antler tips will greatly facilitate new discoveries in this exciting field.