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.

Nerve growth factor mRNA expression in the regenerating antler tip of red deer (Cervus elaphus).

Deer antlers are the only mammalian organs that can fully regenerate each year. During their growth phase, antlers of red deer extend at a rate of approximately 10 mm/day, a growth rate matched by the antler nerves. It was demonstrated in a previous study that extracts from deer velvet antler can promote neurite outgrowth from neural explants, suggesting a possible role for Nerve Growth Factor (NGF) in antler innervation. Here we showed using the techniques of Northern blot analysis, denervation, immunohistochemistry and in situ hybridization that NGF mRNA was expressed in the regenerating antler, principally in the smooth muscle of the arteries and arterioles of the growing antler tip. Regenerating axons followed the route of the major blood vessels, located at the interface between the dermis and the reserve mesenchyme of the antler. Denervation experiments suggested a causal relationship exists between NGF mRNA expression in arterial smooth muscle and sensory axons in the antler tip. We hypothesize that NGF expressed in the smooth muscle of the arteries and arterioles promotes and maintains antler angiogenesis and this role positions NGF ahead of axons during antler growth. As a result, NGF can serve a second role, attracting sensory axons into the antler, and thus it can provide a guidance cue to define the nerve track. This would explain the phenomenon whereby re-innervation of the regenerating antler follows vascular ingrowth. The annual growth of deer antler presents a unique opportunity to better understand the factors involved in rapid nerve regeneration.

Antler regeneration: a dependent process of stem tissue primed via interaction with its enveloping skin.

Deer antlers are unique mammalian appendages in that each year they are cast and fully regenerate from permanent bony protuberances, called pedicles. In a previous study, we found that there is a difference in the degree of association between pedicle bone and its enveloping skin: tight at the distal third and loose at the proximal two thirds of a pedicle stump. The distal part has been termed the "potentiated" region, and the proximal part the "dormant" region. In the present study, pedicle stumps were artificially created in yearling sika deer by cutting off the tissue distal to either the potentiated or the dormant region. A piece of impermeable membrane was then inserted into the space between the bone and the skin of each treated pedicle stump, while the control pedicles had the same surgery without membrane insertion. The results showed that the inserted membrane blocked pedicle skin participation in the process of antler regeneration. All three potentiated bony pedicle stumps regenerated skin-less antlers; whereas, one of the three dormant bony pedicle stumps failed to regenerate any antler tissue. The other two dormant stumps eventually regenerated normal antlers; however, this only occurred after loss of the inserted membrane. No antler tissue regenerated from the dormant stumps while the inserted membrane remained in place (up to 55 days). All control pedicle stumps regenerated normal antlers. Therefore, we conclude that it is the pedicle bone, but not pedicle skin, that gives rise to regenerating antlers, and that pedicle bone can acquire the potential to regenerate an antler only when it is primed via interaction with its enveloping skin.

Identification of key tissue type for antler regeneration through pedicle periosteum deletion.

Epimorphic regeneration is the "holy grail" of regenerative medicine. Research aimed at investigating the various models of epimorphic regeneration is essential if a fundamental understanding of the factors underpinning this process are to be established. Deer antlers are the only mammalian appendages that are subject to an annual cycle of epimorphic regeneration. In our previous studies, we have reported that histogenesis of antler regeneration relies on cells resident within the pedicle periosteum (PP). The present study elaborates this finding by means of functional studies involving the deletion of PP. Four yearling and four 2-year-old stags were selected for total PP deletion or partial PP deletion experiments. Of the animals in the total PP deletion group, one showed no signs of antler regeneration throughout the antler growth season. Two showed substantial and one showed marginal delays in antler regeneration (at 34, 20 and 7 days, respectively) compared with the corresponding sham-operated sides. Histological investigation revealed that the delayed antlers were derived from regenerated PP. Unexpectedly, the regenerative capacity of the antler from the total periosteum-deleted pedicles depended on antler length at surgery. Of the four deer that had partial PP deletion, two regenerated antlers exclusively from the left-over PP on the pedicle shafts in the absence of participation from the pedicle bone proper. The combined results from the PP deletion experiments convincingly demonstrate that the cells of the PP are responsible for antler regeneration.

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.

Vascular localization and proliferation in the growing tip of the deer antler.

The process of angiogenesis is of interest because of the significant clinical benefits associated with controlling vascular growth. Within the antler, chondrogenesis and antler elongation are occurring at the rate of 1-2 cm per day and thus blood vessels are growing at this same rapid pace. We demonstrate that the process of angiogenesis in the antler is controlled at various tissue locations. The findings clearly differentiate the spatial location of the stem cells that drive chondrogenesis from the proliferation process driving the angiogenesis. Vessels within the lateral dermis contained BrdU-positive cells, suggesting that these vessels were elongating. Within the precartilage region, proliferating vessels were detected in bundles of complex structure evenly distributed throughout this tissue layer. The support cells within these bundles of vessels were detected by staining with alpha-smooth muscle actin, while the endothelial cells were negative. Additionally, the alpha-smooth muscle actin staining was found in association with the cartilage cells of the antler. The marked proliferation of the vascular associated cells in the precartilage region identified this area as a major region of vascular growth in the antler. We propose that within the precartilage region, the most likely mechanisms to explain the observed vascular morphology are that of vascular extension of the existing vessels and intussusceptive angiogenesis or sprouting to generate the small bundles of vessels. Wiley-Liss, Inc.

Histological examination of antler regeneration in red deer (Cervus elaphus).

Annual antler renewal presents the only case of epimorphic regeneration (de novo formation of a lost appendage distal to the level of amputation) in mammals. Epimorphic regeneration is also referred to as a blastema-based process, as blastema formation at an initial stage is the prerequisite for this type of regeneration. Therefore, antler regeneration has been claimed to take place through initial blastema formation. However, this claim has never been confirmed experimentally. The present study set out to describe systematically the progression of antler regeneration in order to make a direct histological comparison with blastema formation. The results showed that wound healing over a pedicle stump was achieved by ingrowth of full-thickness pedicle skin and resulted in formation of a scar. The growth centers for the antler main beam and brow tine were formed independently at the posterior and anterior corners of the pedicle stump, respectively. The hyperplastic perichondrium surmounting each growth center was directly formed in situ by a single type of tissue: the thickening distal pedicle periosteum, which is the derivative of initial antlerogenic periosteum. Therefore, the cells residing in the pedicle periosteum can be called antler stem cells. Antler stem cells formed each growth center by initially forming bone through intramembranous ossification, then osseocartilage through transitional ossification, and finally cartilage through endochondral ossification. There was an overlap between the establishment of antler growth centers and the completion of wound healing over the pedicle stump. Overall, our results demonstrate that antler regeneration is achieved through general wound healing- and stem cell-based process, rather than through initial blastema formation. Pedicle periosteal cells directly give rise to antlers. Histogenesis of antler regeneration may recapitulate the process of initial antler generation.

Morphological observation of antler regeneration in red deer (Cervus elaphus).

Deer antler offers a unique opportunity to explore how nature solves the problem of mammalian appendage regeneration. Annual antler renewal is an example of epimorphic regeneration, which is known to take place through initial blastema formation. Detailed examination of the early process of antler regeneration, however, has thus far been lacking. Therefore, we conducted morphological observations on antler regeneration from naturally cast and artificially created pedicle/antler stumps. On the naturally cast pedicle stumps, early antler regeneration underwent four distinguishable stages (with the Chinese equivalent names): casting of previous hard antlers (oil lamp bowl), early wound healing (tiger eye), late wound healing and early regeneration (millstone), and formation of main beam and brown tine (small saddle). Overall, no cone-shaped regenerate, a common feature to blastema-based regeneration, was observed. Taken together with the examination on the sagittal plane of each regenerating stage sample, we found that there are considerable overlaps between late-stage wound healing and the establishment of posterior and anterior growth centers. Observation of antler regeneration from the artificially created stumps showed that the regeneration potential of antler remnants was significantly reduced compared with that of pedicle tissue. Interestingly, the distal portion of a pedicle stump had greater regeneration potential than the proximal region, although this differential potential may not be constitutive, but rather caused by whether or not pedicle antlerogenic tissue becomes closely associated with the enveloping skin at the cut plane. Antler formation could take place from the distal peripheral tissues of an antler/pedicle stump, without the obvious participation of the entire central bony portion. Overall, our morphological results do not support the notion that antler regeneration takes place through the initial formation of a blastema; rather, it may be a stem cell-based process.

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.