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EXPANDED REVIEW OF HISTOGENESIS OF RUMINANT HEADGEAR
2
Here, we enter into a much more detailed review of the state of knowledge of the
3
development of ruminant headgear. Figure citations refer to those in the main paper.
4
Antlers
5
Antlers are unique among mammalian appendages in their ability to completely and
6
periodically regenerate in adults; this fact has prompted more study of antlers than of any other
7
headgear type [1-2], and the process of regeneration has been reviewed extensively [2-4]. For
8
comparison with other ruminant headgear types, our review summarises the state of knowledge
9
of the histogenesis and morphogenesis of primary (first-year) antlers found in red deer (Cervus
10
elaphus) [2, 4-6], fallow deer (Dama dama) [7], white-tailed deer (Odocoileus virginianus) [8],
11
and reindeer (Rangifer tarandus) [9].
12
An antler is bony projection from the lateral crest of the frontal bone and consists of a
13
core of cancellous (spongy) bone surrounded by a sleeve of compact (dense) bone. Externally
14
during growth and prior to rutting, an antler is covered by skin and subcutaneous loose
15
connective tissue (SLCT). Both internal and external components of an antler experience
16
coordinated changes during growth, so a review of antler growth must treat the bone and skin as
17
an integrated structure.
18
Growth is divided into three stages. First, the stage of intramembranous ossification
19
produces the initial bony outgrowth from the lateral crest to form a palpable bump (< 10 mm in
20
height) called a pedicle [4-5]. This pedicle serves as the base for the primary as well as
21
subsequent generations of antlers. As the name of this stage suggests, periosteal deposition of
22
cancellous bone occurs by intramembranous ossification (i.e., ossification directly from a
23
membrane, in this case the periosteum). During this stage, the developing pedicle is covered by
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the same type of skin and SLCT that covers the rest of the skull [4, 6]. The histology of the skin
25
consists of hair follicles, arrector pilli muscles, sweat glands, and mono-lobed sebaceous glands.
26
Although richly vascularised, the SLCT is generally unremarkable [6].
27
Second, the stage of transitional ossification furthers pedicle elongation to the point
28
where it is clearly visible (approximately 25–40 mm in height). As the name of the stage
29
suggests, there is a transformation in the mode of ossification demonstrating that the mode need
30
not be strictly intramembranous or endochondral as described in introductory textbooks. In fact,
31
similar transformations occur in the mandibular condyle [e.g., 10], bone fracture repair [e.g., 11],
32
and solitary osteochondroma formation [12]. In the growing pedicle, the apical-most periosteum
33
partially transforms into perichondrium. Consequently, osteoblasts as well as chondroblasts form
34
in the apical region of the growing pedicle. Apical osteoblasts deposit bony spicules (via
35
intramembranous ossification) that encase clusters of chondroblasts (now chondrocytes) to form
36
bony trabeculae with cores of cartilage [4-5]. Rapid apical growth and the differential ability of
37
the vascular supply to keep pace with pedicle growth is the likely explanation for the clustering
38
of cartilage within bone. Where vascular formation is unable to keep pace with rapid pedicle
39
growth, cartilage is deposited. Conversely, where vascular formation is able to maintain pace
40
with pedicle growth, bone is deposited [5]. In addition to this transitional ossification, typical
41
intramembranous ossification occurs along the sides of the pedicle and progressively thickens the
42
peripheral bony sleeve. Marked changes also occur in the skin and underlying SLCT. The apical-
43
most skin of the pedicle thickens, sweat glands accumulate, and the sebaceous glands enlarge.
44
Compression caused by rapid elongation of the pedicle somewhat flattens the normal undulatory
45
interface between the epidermis and dermis (i.e., the rete apparatus) and compacts the underlying
46
SLCT [6]. The coordinated transformation of the skin/SLCT and periosteum/perichondrium
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suggests the presence of molecular signalling between different tissue types (i.e., heterotypic
48
signalling). In fact, pedicle elongation (and antler formation) can be completely arrested if an
49
impermeable membrane is inserted between the SLCT and the periosteum before the transitional
50
stage of ossification begins [13].
51
Third, the stage of endochondral ossification involves the completion of the pedicle and
52
the establishment of a base from which rapid growth of an antler proceeds. Rapid apical growth
53
exceeds the ability of the rich vascular network to keep pace and causes the apical-most
54
periosteum to completely transform into perichondrium. Elongated columns of cartilage,
55
separated by highly vascularised spaces, are deposited on top of the older osseocartilaginous
56
tissue. As more cartilage is deposited, the oldest and deepest cartilaginous tissues undergo
57
chondroclasia (i.e., the removal of mineralised cartilage by phagocytic cells) and are remodelled
58
into bony tissue much in the same way that endochondral ossification proceeds in long bones.
59
The point at which growth transitions from pedicle to antler is not obvious when looking at the
60
internal components because endochondral ossification characterises both the late apical growth
61
of a pedicle and the entire apical growth of an antler. However, antler skin and pedicle skin differ
62
substantially. Unlike the skin covering a pedicle, the skin covering an antler contains hair
63
follicles that lack arrector pilli muscles and are connected to extremely large bi- to multi-lobar
64
sebaceous glands. This velvet, as the new skin is called, lacks sweat glands and has thickened to
65
the point where the once-undulatory rete apparatus is completely flattened. In addition, the
66
underlying SLCT is flattened into a thin layer, merging almost completely with the
67
periochondrium.
68
69
Induction of pedicle and antler formation is the result of a complex interplay of signalling
molecules. As introduced above, the pedicle originates as an intramembranous outgrowth from
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the lateral crest [5, 14]. The periosteum of the lateral crest is unusual because it contains
71
glycogen-rich, embryonic-like cells [15] that appear predetermined to form lateral crests;
72
transplantation of that periosteum elsewhere on the body induces an ectopic crest-pedicle-antler
73
complex [15]. In addition, the formation of a lateral crest occurs in the absence of any apparent
74
signalling induction from the skin; the presence of an impermeable membrane between the
75
periosteum and skin does not prevent its formation [13].
76
Initiation of pedicle growth from each lateral crest closely coincides with male puberty in
77
most cervids (two exceptions are Hydropotes, which is antler-less, and Rangifer, in which both
78
sexes develop pedicles and antlers at puberty [16]) and an increase in circulating levels of
79
testosterone [17-18]. However, bone growth is more directly regulated by oestradiol, which is
80
converted from testosterone by osteoblasts [19]. Oestradiol suppresses the RANKL-RANK
81
system of bone resorption [20] and is clearly important in antler development: oestradiol is
82
concentrated in antlerogenic and neighbouring tissues [21], promotes pedicle growth in males
83
[22], induces premature mineralisation of antlers and shedding of velvet [23], and regulates the
84
antler cycle in female reindeer [24].
85
In addition to androgenic signals, signalling between the apical periosteum and the skin is
86
required to initiate and modulate the growth of pedicles (and antlers) [13]. Presumably, the close
87
contact between the apical periosteum and skin promotes transit of molecules that are essential to
88
the initiation of pedicle formation and antlerogenesis [13]. For example, pedicle formation and
89
antlerogenesis is completely arrested if an impermeable membrane is placed between the apical
90
periosteum and the skin before the stage of transitional ossification. Once the stage of
91
transitional ossification has begun, however, the impermeable membrane no longer prevents the
92
completion of a pedicle and antlerogenesis, although longitudinal growth is retarded [13].
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Termination of endochondral ossification and longitudinal growth coincides with a large
94
pulse in circulating androgens and the rutting season [4]. Velvet (but not pedicle skin) and most
95
of the antlerogenic tissue are shed, which exposes the bare bone of the antler. The superficial
96
bone tissue likely dies; however, deeper bone tissue remains alive, and continued
97
intramembranous ossification forms lamellar bone within antlers [25]. That a naked antler still
98
contains living bony tissue is likely the reason the antler is not immediately cast; osteoblasts
99
within the antler convert circulating testosterone into oestradiol, which locally suppresses bone
100
resorption [20]. The inhibitory effect of oestradiol on bone resorption weakens with the seasonal
101
decline in testosterone [23] or by the death of osteoblasts in the antler. Consequently, a large
102
number of osteoclasts are recruited. Interestingly, their resorptive activity is not systemic but is
103
limited to the pedicle-antler junction, leading to antler casting. How this focused bone resorption
104
is directed is currently unknown, and future studies to understand the mechanism will be
105
valuable in controlling degenerative bone diseases (e.g., osteoporosis) in other species.
106
Horns
107
Bovid horns are composed of a scabbard-like keratinous sheath covering a bony
108
horncore, neither of which are shed [26-27]. The bony horncore joins seamlessly to the frontal
109
bone via a constricted neck at the base of the horncore. What little is known of early horn
110
development is obscured by inconsistent identification and naming of primordial horn structures.
111
Further confusion has resulted from the inclusion of animals with scurs, also called “loose
112
horns”: incompletely developed horns that have a solid bony core but only a soft tissue
113
connection to the skull. Scurs may arise genetically or via pathology, and they exist in a range of
114
severities. In this review, we wish to establish language to distinguish between three structures of
115
the early horn. In ontogenetic order, these are 1) the soft-tissue anlage, which precedes the
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development of visible horns, 2) the os cornu, a loose, palpable nodule, and 3) the horncore bud,
117
the macroscopic bony bump seamlessly fused to the frontal bone. Future revisions to
118
terminology are likely as additional data become available. More detailed reviews may be found
119
in Janis and Scott [26] and Dove [27].
120
Before birth, above the presumptive horn sites on the frontal bone, the epidermal and
121
dermal components of the future horn acquire their potentials and irreversibly differentiate,
122
evidently precluding post-natal signalling between these tissues [27]. The anlage is positioned
123
within, and differentiates from, both the dermis and SLCT above the periosteum [27], and it
124
retains its connective tissue character. The anlage seems to be the primary inducer of horncore
125
growth, although the mechanism(s) of induction are, to our knowledge, entirely unknown.
126
Despite its name, the relationship of the os cornu to the adult horncore is not at all
127
straightforward. Whether it arises from the anlage, or becomes the horncore bud, or has a
128
separate role (if any) is unclear. Several authors conclude that the os cornu does not exist as a
129
discrete structure (if at all) during normal horn development. Rather, they posit that the os cornu
130
only appears in animals heterozygous for genes controlling horn presence [i.e. in scurred
131
animals; 27]), animals in poor health [28-29], or from manipulation [30], including surgery [27].
132
Nevertheless, many authors proceed to describe early horn development in terms of the os cornu.
133
The os cornu is generally thought to be a palpable nodule that begins in the supra-
134
periosteal tissue. It has been reported to be made of dermis and/or SLCT [27, 31-32] or cartilage
135
[33-34], and may ossify independently in the soft tissue (forming a scur), or after attaching to the
136
frontal bone (forming a normal horn) [27]. Janis and Scott [26] persuasively argue for
137
intramembranous ossification throughout horn development, and Durst [31] and Brandt [32]
138
showed that there is no cartilaginous preformation of the horncore, in contrast to earlier reports
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[24, 25]. Other reports of cartilage in or around the horncore or frontal bone [33-35] are
140
unexplained in this framework.
141
Summarising Dove [27], the os cornu does not ossify prior to fusion to the frontal bone.
142
If there is no SLCT between the os cornu and the periosteum, it fuses through the periosteum to
143
the presumptive horn site. Removing or doubling the periosteum has no effect. Whether
144
embedding and fusion of the os cornu to the frontal bone are simultaneous is unknown, although
145
macroscopic evidence of fusion is very rare. Ganey et al. [36] depicted a bone disc embedded in
146
the frontal bone, separated by a thin layer of connective tissue in a two-thirds term bovine foetus;
147
its genotype for horns was not reported. Janis and Scott [26, 31] stated that the first step involves
148
a connection between the SLCT and the osteoid of the superficial frontal bone, followed by
149
thickening of the osteoid soon after birth. In scurs, it appears that the union between the os cornu
150
and the frontal bone is weak or absent; evidently the os cornu cannot penetrate the supra-
151
periosteal SLCT and ossifies in place, becoming a scur [27]. However, the fate of the os cornu in
152
normal vs. scurred animals has not been directly studied.
153
After induction by the anlage and/or fusion of the os cornu to the frontal bone, the
154
horncore bud begins to develop. By the time the horncore bud is observable, its microstructure
155
differs from the frontal [31]; Dove [27] concluded that the dermal portion of the os cornu
156
becomes the tip of the horncore bud and that the SLCT portion of the os cornu forms the
157
eventual neck of the horn. (The neck has also been called the pedicle; however, we prefer a
158
distinct term to aid discussion and to avoid assumptions of homology with the cervid pedicle.)
159
Further growth of the horncore bone tissue is appositional at both the tip and the surface
160
[26, 31, 37-38]. As horncore growth slows, deposition of compact bone proceeds simultaneously
161
from surface to lumen and base to tip, often continuing into adulthood. Alternatively, depending
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on the clade, sex, and age of an animal, the horncore may be invaded by the frontal sinus [39],
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and the horncores of some taxa may be very thin-walled. Horncores remain living organs
164
throughout life and are actively remodelled to accommodate sheath shape and physiological
165
demands [40]. No evidence of signalling or other interaction between the frontal bones and the
166
horncore periosteum has been reported for any stage of development; unlike antlers, the frontals
167
appear to neither hinder nor accelerate the production of any horn part [27].
168
Nevertheless, there is wide disagreement whether the bovid horncore is apophyseal (i.e.,
169
a direct outgrowth), epiphyseal (i.e., separated at least initially by non-bony tissue), or a
170
combination, with respect to the frontal bone [17, 26-27, 31, 35, 41-43]. Hypotheses about os
171
cornu homology are problematic because of imprecise definitions of the os cornu and the
172
dependence of its structure and fate on genotype and environmental conditions. Resolving these
173
hypotheses will require establishing the relationships among the anlage, os cornu, horn bud,
174
neck, and horncore in normal horns.
175
The skin over the presumptive horn site on the frontal bone likely gains the capacity to
176
form horn tissue (i.e., the keratin sheath) before birth, although normal sheath growth is
177
dependent upon the presence of the os cornu [27]. Horn tissue is continuously produced by the
178
epithelium covering the horncore [26, 31], with newer layers pushing the older layers distally
179
like a stack of keratin cones (Fig. 2b); this layering is more easily detected in temperate species
180
with strong seasonal fluctuations in sheath growth rates [17, 44]. Nevertheless, the sheath tip is
181
usually much thicker proximodistally than the walls are mediolaterally [17], suggesting more
182
rapid production of keratin at the tip. Conventional wisdom that the horn sheath grows only from
183
the proximal end of the horn is therefore unfounded [26]. The horn tissue of juveniles is softer
184
and more fibrous [45] and may “exfoliate” before adulthood [31, 38, 43, 45-47], exposing harder
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and more completely keratinised horn tissue. Distinctive horn shapes are thought to arise by
186
modulating zones of keratin production in the skin surrounding the horn [1, 48], but experimental
187
studies are lacking.
188
The development of polled (hornless) breeds of domesticated bovids has enabled partial
189
identification of the genetic basis of horns. In cattle, the presence or absence of horns (or scurs)
190
is genetically determined by three or four genes and (in scurs) by sex; alleles for horns are
191
recessive [49-50], and heterozygotes may be scurred [21]. In goats, genetic hornlessness is
192
tightly linked to intersexuality and sterility [51]. In cattle, horn-forming tissues have down-
193
regulated genes coding for cadherin junction elements (i.e., cell membrane structures used in
194
cellular adhesion) and epidermal development. Compared to genetically hornless animals, those
195
with scurs have higher expression of genes involved in extracellular matrix remodelling [52].
196
Although no comprehensive picture of signalling pathways triggering or regulating horn
197
development is available for any one species, evidence so far indicates that the sensitivity of
198
horn-producing soft tissues to various exogenous and endogenous molecules changes rapidly
199
after birth. For example, horn development can be fully blocked at Day 2 in calves, but only
200
partially blocked at Day 4 using the same substance [53]. In adult mouflons (Ovis gmelini, a
201
temperate caprine), summer increases in prolactin concentration are positively correlated with
202
horn growth in adults [44, 54]; similarly, in domestic sheep, increased melatonin secretion (from
203
shorter photoperiod in the winter) suppresses prolactin [55-56] and increases gonadotropin
204
secretion [57]. Yet in mouflon lambs, horn growth is insensitive to melatonin [58], and in
205
subadults, melatonin concentration does not correlate with prolactin concentration [44, 54].
206
Plasma testosterone concentrations are inversely correlated with horn growth both seasonally and
207
with age in male mouflons [54, 59]. Across the bovid family, though, the male phenotype is
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associated with increased expression of horns (earlier and faster growth, greater size and
209
symmetry, etc.) [60]. Castration experiments in cattle show that testosterone is important for
210
development of normal male horns, although castrated males do not have female-like horns [61].
211
All this suggests that the relationship among various hormones and horn growth is complex and
212
changes with maturation, but too little is known to infer common signalling pathways among
213
species.
214
Ossicones
215
Giraffes (Giraffa camelopardalis) and okapis (Okapia johnstoni) develop frontoparietal
216
ossicones [62], which share structural and positional characters with the other pecoran headgear
217
types (Main Text Table 1). Giraffes may develop several additional paired or medial skull
218
protuberances that have been called “ossicones”, but these do not experience the complex
219
development of the main frontoparietal ossicones [63], so we do not discuss them. Ossicones are
220
present in both male and female giraffes, but they are not as pronounced in females [62]. Only
221
male okapis have ossicones, and similar sexual dimorphism has been reconstructed in most fossil
222
giraffids [62].
223
The ossicone begins as a separate bony core above the frontoparietal suture in giraffes
224
and above the frontals in okapis [62]. The ossicone was previously thought to originate as a
225
fibrocartilage condensation within the connective tissue above the periosteum [64-65]; however,
226
Ganey et al. [36] showed that it is made primarily of fibrous connective tissue, with some areas
227
of fibrocartilage, and initial ossification is entirely intramembranous, as with the frontals and
228
parietals. Ossicones begin to ossify within a week of birth in the giraffe [65] and remain detached
229
from the skull until sexual maturity [62], primarily growing through bone deposition at the non-
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cartilaginous, dense connective tissue anchor on the skull [36]. This means that, in immature
231
individuals, the ossicones approximate the condition of “loose horns” (scurs) in bovids [35].
232
Upon sexual maturity, the ossicone fuses to the skull and ceases growth at the skull-
233
ossicone interface [62, 65]. Ossicones of giraffes continue to grow after fusion through the slow
234
deposition of lamellar bone (i.e., layered, more mature bone) at the surface [62, 65], in a manner
235
reminiscent to the growth of bovid horncores. In giraffes, the base of the ossicone may be
236
invaded by the frontal sinuses, but never in okapis [65]. Adult male giraffes engage in head-to-
237
side sparring, often callusing the skin at the tips of their skin-covered ossicones [62]. In adult
238
male okapis, the skin retracts from the tips of the ossicones, leaving the bone exposed, often
239
producing necrosis at the skin-bone boundary [65]. The mechanism by which infection is
240
prevented from spreading into the skull is unknown, nor is it known whether this mechanism is
241
also found in cervids, which maintain naked but living antlers for 1–2 months per year [25].
242
The ossicone-like headgear of extinct palaeomerycids could be homologous to the
243
ossicones of extant giraffids [26, 66]; however, no one has specifically investigated the putative
244
histological homologies in the headgear of palaeomerycids, and only extant giraffe ossicones
245
have been studied histologically.
246
Pronghorns
247
Pronghorn antelope (Antilocapra americana) have headgear also called pronghorns. To
248
avoid confusion, we will use the scientific name for the animal and limit ‘pronghorn’ to the
249
structure. The pronghorn horncore (pronghorn core) is bone, with no invasion of the sinuses [67],
250
and maintains a cancellous (spongy) bone interior, unlike the horncores of some bovids [62]. No
251
studies have directly investigated the earliest development of the horncore of A. americana, but
252
Solounias [68] examined the pronghorn cores of 28 newborn A. americana, finding no delayed
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fusion of the pronghorn core. This suggests either an even earlier fusion of an anlage or os cornu
254
than seen in bovid horncores or a cervid-like direct development. There have been no reports of a
255
delayed fusion of the pronghorn core, as seen in the giraffid ossicone or scurred bovids, lending
256
support to the hypothesis of cervid-like development.
257
The keratinous pronghorn sheath of male A. americana sheds and re-grows annually in
258
response to cycles of male hormones [67]. Approximately 30% of females are hornless, and the
259
rest have smaller, irregularly-shed, button-like horns [69]. In a key difference with bovids, there
260
are two centres of cornification on the unbranched, blade-like pronghorn core: a distal site for the
261
main spike and an anterior site for the prong. After the spike and prong are nearly full size, the
262
remainder of the shaft cornifies and elongates, creating a single keratinised sheath surrounding
263
the pronghorn core [46, 67]. Hair from the skin covering the pronghorn core is incorporated into
264
the growing sheath, but the hair is not important structurally, in contrast to the “hair horns” of
265
Rhinoceros [46]. The keratinous tissue of pronghorns has been construed as homologous to the
266
horn tissue in bovid horn sheaths [67, 70], but the annual replacement of pronghorn sheaths plus
267
a suite of skeletal characters have been seen as homologies linking A. americana to cervids [70].
268
The current molecular evidence indicates a strong connection with giraffids that seemingly
269
invalidates both of these hypotheses of morphological homology [71-74]. Further complicating
270
the homology of antilocaprid headgear are the basal antilocaprids, the paraphyletic
271
“merycodontines” [75-77], which had unshed antler-like headgear of exposed live bone [78-79],
272
suggestive of both unshed antlers of the earliest cervids and the bony tips of okapi ossicones.
273
274
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