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.     

Tubular frameworks guiding orderly bone formation in the antler of the red deer (Cervus elaphus)

Deer antler is a bony tissue which re-grows every year after shedding. Growth speed and material properties of this tissue are truly remarkable, making it an interesting model for bone regeneration. Surprisingly, not much is known about the ultrastructure of the calcified tissues and the temporal sequence of their development during antler growth. We use a combination of imaging tools based on light and electron microscopy to characterize antler tissue at various stages of development. We observe that mineralized cartilage is first transformed into a bone framework with low degree of collagen fibril ordering at the micron level. This framework has a honeycomb-like appearance with the cylindrical pores oriented along the main antler axis. Later, this tissue is filled with primary osteons, whose collagen fibrils are mainly oriented along the pores, thus improving the antler's mechanical properties. This strongly suggests that to achieve very fast organ growth it is advantageous to have a longitudinal porous framework as an intermediate step in bone formation. The example of antler shows that geometric features of this framework are crucial, and a tubular geometry with a diameter in the order of hundred micrometers seems to be a good solution for fast framework-mediated bone formation.     

Tissue interactions and antlerogenesis: new findings revealed by a xenograft approach.

Tissue interactions play a pivotal role in organogenesis. Here we describe a xenograft approach to investigate how heterotypic tissue interactions control antler formation in deer. Deciduous antlers grow from the apices of permanent protuberances, called pedicles. Histogenesis of pedicles depends on the antlerogenic periosteum (AP). Pedicles and growing antlers are made up of interior osseocartilage (a mixture of bone and cartilaginous tissue) and exterior skin. In a previous study we hypothesised that pedicle growth may result from mechanical interactions between the interior and exterior components whereas antler generation from a pedicle would involve molecules communicating between the interior and exterior components. To test this hypothesis, we subcutaneously transplanted AP of red deer (Cervus elaphus), either alone or with future pedicle skin, onto nude mice. The results showed that under the nude mouse skin, subcutaneously xenografted AP alone not only could form pedicle-shaped protuberances but also could differentiate into well-organised pedicle-like structures. The overlying mouse skin accommodated the expansion of the grafted AP by initial mechanical stretching and subsequent formation of new skin. Nude mouse skin was not capable of participating in antler tissue formation. However, grafted deer skin together with AP may have successfully rescued this failure after wounding, which highlights the necessity of the specificity of the overlying skin for antler tissue generation. Therefore, we conclude that it is the interaction between the antlerogenic tissue and the overlying skin that results in antlerogenesis: reciprocal mechanical interactions cause pedicle formation, whereas reciprocal instructive interactions induce first antler generation.     

Deer antler regeneration: A stem cell‐based epimorphic process

Full regeneration of deer antlers, a bona fide epimorphic process in mammals, is in defiance of the general rule of nature. Revealing the mechanism underlying this unique exception would place us in a better position to promote organ regeneration in humans. Antler regeneration takes place in yearly cycles from its pedicle, a permanent protuberance on the frontal bone. Both growing antlers and pedicles consist of internal (cartilage and bone) and external components (skin, blood vessels, and nerves). Recent studies have demonstrated that the regeneration of both internal and external components relies on the presence of pedicle periosteum (PP). PP cells express key embryonic stem cell markers (Oct4, Nanog, and SOX2) and are multipotent, so are termed antler stem cells. Now it is clear that proliferation and differentiation of PP cells directly forms internal antler components; however, how PP initiates and maintains the regeneration of external antler components is thus far not known. Based on the direct as well as indirect evidence that is presented in this review, I put forward the following hypothesis to address this issue. The full regenerative ability of external antler tissue components is achieved through PP‐derived chemical induction and PP‐derived mechanical stimulation: the former triggers the regeneration of these external components, whereas the latter drives their rapid elongation. Eventual identification of the putative PP‐derived chemical factors would open up a new avenue for devising effective therapies for lesions involving each of these tissue components, be they traumatic, degenerative, or linked to developmental (genetic) anomalies. (Part C) 96:51–62, 2012. © 2012 Wiley Periodicals, Inc.     

Drosophila egg chamber elongation

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.     

Drosophila egg chamber elongation: Insights into how tissues and organs are shaped

As tissues and organs are formed, they acquire a specific shape that plays an integral role in their ability to function properly. A relatively simple system that has been used to examine how tissues and organs are shaped is the formation of an elongated Drosophila egg. While it has been known for some time that Drosophila egg elongation requires interactions between a polarized intracellular basal actin network and a polarized extracellular network of basal lamina proteins, how these interactions contribute to egg elongation remained unclear. Recent studies using live imaging have revealed two novel processes, global tissue rotation and oscillating basal actomyosin contractions, which have provided significant insight into how the two polarized protein networks cooperate to produce an elongated egg. This review summarizes the proteins involved in Drosophila egg elongation and how this recent work has contributed to our current understanding of how egg elongation is achieved.     

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.     

Deer antler – A novel model for studying organ regeneration in mammals

Deer antler is the only mammalian organ that can fully grow back once lost from its pedicle – the base from which it grows. Therefore, antlers probably offer the most pertinent model for studying organ regeneration in mammals. This paper reviews our current understanding of the mechanisms underlying regeneration of antlers, and provides insights into the possible use for human regenerative medicine. Based on the definition, antler renewal belongs to a special type of regeneration termed epimorphic. However, histological examination failed to detect dedifferentiation of any cell type on the pedicle stump and the formation of a blastema, which are hallmark features of classic epimorphic regeneration. Instead, antler regeneration is achieved through the recruitment, proliferation and differentiation of the single cell type in the pedicle periosteum (PP). The PP cells are the direct derivatives of cells resident in the antlerogenic periosteum (AP), a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body. Both the AP and PP cells express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro and, therefore, they are termed antler stem cells, and antler regeneration is a stem cell-based epimorphic regeneration. Comparisons between the healing process on the stumps from an amputated mouse limb and early regeneration of antlers suggest that the stump of a mouse limb cannot regenerate because of the limited potential of periosteal cells in long bones to proliferate. If we can impart a greater potential of these periosteal cells to proliferate, we might at least be able to partially regenerate limbs lost from humans. Taken together, a greater understanding of the mechanisms that regulate the regeneration of antlers may provide a valuable insight to aid the field of regenerative medicine.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Improbable appendages: Deer antler renewal as a unique case of mammalian regeneration

Deer antlers are periodically replaced cranial appendages that develop from permanent outgrowths of the frontal bones known as pedicles. Antler re-growth is a unique regenerative event in mammals which in general are unable to replace bony appendages. Recent evidence suggests that antler regeneration is a stem cell-based process that depends on the activation of stem cells located in the pedicle periosteum which are presumed to be neural crest-derived. It has been demonstrated that several developmental pathways are involved in antler regeneration that are also known to play a role in the control of skeletal development and regeneration in other vertebrates. However, in contrast to most other natural examples of regeneration of complete body structures, antler regeneration apparently neither depends on a functional nerve supply nor involves a direct contact between wound epithelium and mesenchymal tissue. Antlers thus demonstrate that regeneration of a large bony appendage in a mammal can be achieved by a process that differs in certain aspects from epimorphic regeneration in lower vertebrates.     

Antler development was inhibited or stimulated by cryosurgery to periosteum or skin in a central antlerogenic region respectively

Antler development is triggered by interactions between antler stem cells resident in the antlerogenic periosteum (AP) and the niche cells in the upper portion of overlying skin mediated by diffusible molecules. These interactive cell populations are interposed by the lower portion of the skin and the subcutaneous loose connective tissue (SLCT). It is known that mechanical deletion of just the central AP (having an area equivalent to the size of a pedicle base) by cutting through the skin and SLCT effectively stimulates the marginal AP to initiate antler development. This study was designed to investigate whether the SLCT layer plays a role in antler development by acting as a physical barrier. The results showed that the marginal AP failed to give rise to an antler after the central AP was cryosurgically destroyed with the preservation of the collagen structure of the SLCT. Furthermore, antler development was significantly advanced when the collagen structures of the skin and SLCT layers were substantially attenuated by repeated sprays with liquid nitrogen while keeping the central AP intact. Therefore, we conclude that the interposing SLCT layer acts as a physical barrier between antler stem cells and the niche cell types, and that timing of antler development is primarily controlled by the permeability of the SLCT layer to the putative interactive diffusible molecules.     

PLU1 histone demethylase decreases the expression of KAT5 and enhances the invasive activity of the cells

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle-vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.     

Effects of Auxin and Kinetin on the Development and Differentiation of Potato Tissue Cultured in vitro

The present investigation deals with the influence of auxin and kinetin on cultures of potato tuber tissue, and the effect of the size of the explants on the organ formation. Exogenous application of auxin is indispensable for initiating callus formation of the tissue, but kinetin is not necessarily required. Kinetin serves to maintain the callus development, indicating that the stimulation of callus growth due to exogenous auxin would presumably be mediated by the addition of kinetin to the medium. A relatively low concentration of auxin in the medium is required for root formation on the culture. In this case, IAA is markedly superior to any other auxin in root forming activity. NAA is somewhat less effective than IAA, and 2,4-D shows no stimulation on root formation. The growth and organ formation of potato tissue are remarkably affected By the variation in sizes of the explants. Spontaneous formation of a sprout was observed on the large explant in the medium without auxin. Small explants form only callus or roots in the presence of auxin and kinetin irrespective of combination or concentration. It seems likely that a sufficient amount of stimulus for initiation of bud formation may be contained in the large sized explant itself.     

Gene expression of axon growth promoting factors in the deer antler

The annual regeneration cycle of deer (Cervidae, Artiodactyla) antlers represents a unique model of epimorphic regeneration and rapid growth in adult mammals. Regenerating antlers are innervated by trigeminal sensory axons growing through the velvet, the modified form of skin that envelopes the antler, at elongation velocities that reach one centimetre per day in the common deer (Cervus elaphus). Several axon growth promoters like NT-3, NGF or IGF-1 have been described in the antler. To increase the knowledge on the axon growth environment, we have combined different gene-expression techniques to identify and characterize the expression of promoting molecules not previously described in the antler velvet. Cross-species microarray analyses of deer samples on human arrays allowed us to build up a list of 90 extracellular or membrane molecules involved in axon growth that were potentially being expressed in the antler. Fifteen of these genes were analysed using PCR and sequencing techniques to confirm their expression in the velvet and to compare it with the expression in other antler and skin samples. Expression of 8 axon growth promoters was confirmed in the velvet, 5 of them not previously described in the antler. In conclusion, our work shows that antler velvet provides growing axons with a variety of promoters of axon growth, sharing many of them with deer's normal and pedicle skin.     

Effects of insulin-like growth factor 1 and testosterone on the proliferation of antlerogenic cells in vitro.

Androgen hormones and growth factors are implicated in pedicle formation and antler transformation in deer. The potential to form a pedicle and an antler is only found in the antlerogenic periosteum (AP) overlying the presumptive antler growth region. Histological studies (Li and Suttie, '94) showed that AP consists of an inner cellular layer and an outer fibrous layer. Pedicle and antler are mainly derived from the cellular layer cells of the AP. Ossification takes place in four stages: intramembranous (IMO), transitional (OPC), pedicle endochondral (pECO) and antler endochondral (aECO). However, the precise mechanism whereby androgen hormones and growth factors control pedicle and antler formation is unknown. The aim of this study was to use cell culture techniques to investigate how testosterone and IGF1 affects the proliferation of antlerogenic cells from the four ossification stages of pedicle/antler in vitro. The results showed that in serum-free medium IGF1 stimulated the proliferation of antlerogenic cells from all four ossification stages in a dose-dependent manner. In contrast, testosterone alone did not show any mitogenic effects on these antlerogenic cells. However, in the presence of IGF1, testosterone increased proliferation of the antlerogenic cells from the IMO and the OPC stages (pedicle tissue), and reduced proliferation of the antlerogenic cells from transformation point (TP) and aECO stages (antler tissue). Therefore, the results from the present in vitro study support the in vivo findings that androgen hormones stimulate pedicle formation but inhibit antler growth. The change in the mitogenic effects of testosterone on antlerogenic cells from positive to negative occurs approximately at the change in ossification type from OPC to pECO. Therefore, these results reinforce the hypothesis that the transformation from a pedicle to an antler takes place at the time when the ossification type changes from OPC to pECO rather than at the time when the pedicle grows to its full species-specific height.     

Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth.

Our study explores the mechanisms behind neurite galvanotropism. Using phase, differential interference contrast and ratiometric fluorescence microscopy, we reveal four responses of N1E-115 mouse neuroblastoma cells to 0.1-1.0 mV/microns uniform DC electric fields: cathode-directed neurite initiation and elongation, cathode-biased growth cone filopodial protrusions, transient cathode-localized calcium increases, and persistent cathode-localized membrane depolarizations. These newly demonstrated events are temporally and spatially correlated, suggesting that they are causally related. The calcium increases are prevented by calcium channel blockers and by the removal of extracellular calcium. We therefore propose that the observed field-induced membrane depolarizations activate voltage-dependent calcium channels, resulting in cathode-localized calcium influx. This, in turn, may initiate the observed cathode-biased growth cone filopodial protrusions, followed by the cathode-directed neurite elongation.     

Pericycle cell proliferation and lateral root initiation in Arabidopsis

In contrast with other cells generated by the root apical meristem in Arabidopsis, pericycle cells adjacent to the protoxylem poles of the vascular cylinder continue to cycle without interruption during passage through the elongation and differentiation zones. However, only some of the dividing pericycle cells are committed to the asymmetric, formative divisions that give rise to lateral root primordia (LRPs). This was demonstrated by direct observation and mapping of mitotic figures, cell-length measurements, and the histochemical analysis of a cyclin-GUS fusion protein in pericycle cells. The estimated duration of a pericycle cell cycle in the root apical meristem was similar to the interval between cell displacement from the meristem and the initiation of LRP formation. Developmentally controlled LRP initiation occurs early, 3 to 8 mm from the root tip. Thus the first growth control point in lateral root formation is defined by the initiation of primordia in stochastic patterns by cells passing through the elongation and young differentiation zones, up to where lateral roots begin to emerge from the primary root. Therefore, the first growth control point is not restricted to a narrow developmental window. We propose that late LRP initiation is developmentally unrelated to the root apical meristem and is operated by a second growth control point that can be activated by environmental cues. The observation that pericycle cells divide and lateral root primordia form without intervening mitotic quiescence suggests that lateral organ formation in roots and shoots might not be as fundamentally different as previously thought.     

Compound genetic ablation of nidogen 1 and 2 causes basement membrane defects and perinatal lethality in mice

Nidogen 1 and 2 are basement membrane glycoproteins, and previous biochemical and functional studies indicate that they may play a crucial role in basement membrane assembly. While they show a divergent expression pattern in certain adult tissues, both have a similar distribution during development. Gene knockout studies in mice demonstrated that the loss of either isoform has no effect on basement membrane formation and organ development, suggesting complementary functions. Here, we show that this is indeed the case. Deficiency of both nidogens in mice resulted in perinatal lethality. Nidogen 1 and 2 do not appear to be crucial in establishing tissue architecture during organ development; instead, they are essential for late stages of lung development and for maintenance and/or integrity of cardiac tissue. These organ defects are not compatible with postnatal survival. Ultrastructural analysis suggests that the phenotypes directly result from basement membrane changes. However, despite the ubiquitous presence of nidogens in basement membranes, defects do not occur in all tissues or in all basement membranes, suggesting a varying spectrum of roles for nidogens in the basement membrane.     

Exploring the mechanisms regulating regeneration of deer antlers

Deer antlers are the only mammalian appendages capable of repeated rounds of regeneration; every year they are shed and regrow from a blastema into large branched structures of cartilage and bone that are used for fighting and display. Longitudinal growth is by a process of modified endochondral ossification and in some species this can exceed 2 cm per day, representing the fastest rate of organ growth in the animal kingdom. However, despite their value as a unique model of mammalian regeneration the underlying mechanisms remain poorly understood. We review what is currently known about the local and systemic regulation of antler regeneration and some of the many unsolved questions of antler physiology are discussed. Molecules that we have identified as having potentially important local roles in antlers include parathyroid hormone-related peptide and retinoic acid (RA). Both are present in the blastema and in the rapidly growing antler where they regulate the differentiation of chondrocytes, osteoblasts and osteoclasts in vitro. Recent studies have shown that blockade of RA signalling can alter cellular differentiation in the blastema in vivo. The trigger that regulates the expression of these local signals is likely to be changing levels of sex steroids because the process of antler regeneration is linked to the reproductive cycle. The natural assumption has been that the most important hormone is testosterone, however, at a cellular level oestrogen may be a more significant regulator. Our data suggest that exogenous oestrogen acts as a 'brake', inhibiting the proliferation of progenitor cells in the antler tip while stimulating their differentiation, thus inhibiting continued growth. Deciphering the mechanism(s) by which sex steroids regulate cell-cycle progression and cellular differentiation in antlers may help to address why regeneration is limited in other mammalian tissues.     

Tissue collection methods for antler research

The rapid growth of deer antlers makes them potentially excellent models for studying tissue regeneration. In order to facilitate this, we have developed and refined antler tissue sampling methods through years of antler research. In the study, antler tissues were divided into three main groups: antler stem tissue, antler blastema and antler growth centre. For sampling stem tissue, entire initial antlerogenic periosteum (around 22 mm in diameter) could be readily peeled off from the underlying bone using a pair of rat-toothed forceps after delineating the boundary. Apical and peripheral periosteum/ perichondrium of pedicle and antler could only be peeled off intact when they were cut into 4 quadrants and 0.5 cm-wide strips respectively. Antler blastema included blastema per se, and potentiated and dormant periostea. Blastema per se was sampled after it was divided into 4 quadrants using a disposable microtome blade. Potentiated and dormant periostea were collected following the same method used for sampling peripheral periosteum of pedicle and antler. The antler growth centre was divided with a scalpel into 5 layers according to distinctive morphological markers. The apical skin layer could be further separated into dermis and epidermis using enzyme digestion for the study of tissue interaction. We believe that the application of modern techniques coupled with the tissue collection methods reported here will greatly facilitate the establishment of these valuable models.