Chapter 4

Invertebrate Cartilages, Notochordal

Cartilage and Cartilage Origins

The Cell Theory, which states that all organisms are organisms consist of one or more cells, that the cell
composed of cells, is usually traced to observations is the basic unit of structure indeed of life for
made in the seventeenth century by English polymath all organisms and that every organism, whether plant or
Robert Hooke (1635 1703). Using a microscope he had animal, comprises combinations of cells arranged in
made, Hooke (1665) described the honeycomb-like predictable patterns.
network in thin slices of cork as a network of cellulae,
Latin for small storage rooms. Although what Hooke
saw represented only the thickened walls of previously
CHONDROID AND CARTILAGE
living plant tissue cork is dead and dried bark derived Schaffer (1930) exhaustively summarised and reviewed
from the cork oak Quercus suber Hookes cellulae are the nineteenth and early twentieth century studies on the
what we now know as cells. tissues identified as chondroid, chordoid and mucoid by
As introduced in Chapter 2, the Dutch microscopist Schwann. Schaffer did not regard theses tissues as real
Antony van Leeuwenhoek published a description of cartilages. Their immature histology, combined with a
the microstructure of bone in 1693. A decade earlier, scant extracellular matrix and inability to detect glycosa-
Leeuwenhoek was the first to publish on the presence in minoglycans or collagen in their matrices features diag-
pond water of single-celled organisms (now known to be nostic of vertebrate cartilage and their failure to
protozoans). Leeuwenhoek described red blood cells and mineralise, led Schaffer to regard these invertebrate tissues
sperm as single cells. It was another 150 years, however, as chondroid tissues. By chondroid, Schaffer meant carti-
before cells were recognised as the basic units of all life. lage-like, implying a less fully evolved skeletal tissue than
That recognition came within the span of 12 months in cartilage. Schaffer classified chondroid tissues into grades
1838 1839. It resulted from the research of two professors of greater or lesser resemblance to cartilage without,
at Jena University in Germany, a physiologist named however, implying any phylogenetic trends or affiliations1.
Theodor Schwann (1810 1882) and a botanist named Schaffers view prevailed, and skeletal biologists
Matthias Jakob Schleiden (1804 1881). turned to other problems. Indeed, no research publications
Based on his extensive microscopical observations on on invertebrate cartilages appeared until 1959. Then in
plant tissues and plant development, a Cell Theory was the 1960s and 1970s, primarily through the work of one
articulated by Schleiden (1838), who asserted that all man, Philip Person, several structural studies appeared.
plant tissues are composed of cells, that each plant begins Person was thorough. He used light and electron micros-
its life as a single cell and that the cell is the basic build- copy, biochemical characterisation of extracellular matri-
ing block of all plant matter. Cartilages played a central cial products, histochemistry and mineralisation, and he
role for Schwann (1839), who described cellular struc- provided some results on the regenerative ability of inver-
tures in cartilages from animals. tebrate cartilage2, With the exception of one study by
One of the generalities that led to the formulation of Person, no further information on the development of
the Cell Theory was the recognition by Schwann that invertebrate cartilages was available until Alison Cole
many invertebrates have a cartilage-like chondroid, began studies in my laboratory this century (Cole and
chordoid, mucoid connective tissue, structurally simi- Hall, 2002, 2004a,b, *2009, and see Chapter 5).
lar to the parenchymal tissues of plants; both plant cells By 1969, sufficient data had accumulated that Person
and cartilage cells were surrounded by a rigid extra- and Philpott could review the work of the previous
cellular matrix or wall. Schwann (1839) concluded that all decade to present evidence in favour of regarding these

Bones and Cartilage. DOI: http://dx.doi.org/10.1016/B978-0-12-416678-3.00004-5

2015 Elsevier Inc. All rights reserved. 63
64 PART | II Origins and Types of Skeletal Tissues

invertebrate tissues as cartilages. Evidence included cells G tentacular cartilages of two polychaete annelids, the
in lacunae embedded in a matrix containing collagen feather duster worms Eudistylia polymorpha and
and chondroitin sulphate-A or sulphate-C (chondroitin- Sabella melanostigma; and
4-sulphate and chondroitin-6-sulphate, respectively), G lophophore cartilage in the lampshell, Terebratalia
producing a tissue providing mechanical support and a transversa, an articulate brachiopod7.
site for muscle and ligament attachment (Person and
Philpott, 1969a)3.
A deep homology of extracellular matrices, and so a ODONTOPHORE CARTILAGE
monophyletic origin of the animal kingdom, has been IN CAENOGASTROPODS
proposed on the basis of numerous lines of evidence. In
Caenogastropods (sea snails, whelks), the most diverse
an analysis placing fibrillar collagen in a key position in
and dominant gastropods of marine habitats, comprise
vertebrate evolution fibrillar collagen arose before the
some 60% of all gastropod species. Odontophoral carti-
genome duplication at the base of vertebrate evolution
lages are present in the buccal mass dorsal to the radula
Boot-Handford and Tuckwell (2003) discuss invertebrate
where they support the chitinous and toothed radula
collagens only briefly. Older studies saw collagen as
during feeding. In one of the few functional morpho-
first present in sponges, in part because sponges were
logical studies on invertebrate cartilage, Guralnick and
considered the most basal metazoans4. Although that
Smith (1999) examined the phylogenetic distribution of
phylogenetic position has been challenged, the presence
cartilage in gastropods including criteria to classify
in basement membranes at the divergence of sponges
cartilages and looked at correlations between the evolu-
from cnidarians of type IV collagen cross-linked by
tion of gastropod cartilages and the radula, the cartilage
sulphilimine bonds, the extracellular matrix associated
performing a supporting role.
peroxidase peroxidasin (which catalyses formation of
The channelled whelk, Busycon canaliculatum, has
sulphilimine bonds), and hypohalous acids (which inter-
a large odontophore cartilage consisting of hypertrophic
act with unsaturated phosphatidylcholines) lies at the
cells in capsules separated by scant amounts of extra-
basis of the evolution of tissues such as cartilage (Fidler
cellular matrix (ECM) (Figure 4.1). The scant ECM of
et al., 2013).
these invertebrate cartilages has been a stumbling block
Although type I is the most widespread collagen,
to accepting them as cartilages. However, a number of
invertebrates contain several collagen genes. Type II
vertebrate cartilages are characterised by scant amounts
collagen in cartilage arose with the vertebrates, which
of ECM, including xiphisternal, secondary, and callus car-
Cole and Hall (2004a,b) interpreted as indicating the
tilages in jawed vertebrates and lampreys, and cell-rich
primitiveness of collagen with three identical chains dur-
cartilage in teleosts.
ing evolution. Indeed, a phylogenetic analysis of fibrillar
In general, the glycosaminoglycans of invertebrate
collagen evolution by H. Wada et al. (2006) provided evi-
cartilages are oversulphated in comparison to those from
dence that the common ancestor of all deuterostomes had
vertebrate cartilages. Fitting this pattern, odontophore
three fibrillar collagen genes that were co-opted into for-
cartilage lacks chondroitin sulphate, having instead a
mation of a notochordal sheath with the origin of the
polyglucose sulphate akin to chitin8. Echinoderm connec-
chordates, a topic taken up again in Chapters 41 and 425.
tive tissues have a similar polyfucose sulphate, but also
The distribution of elastin in elastic cartilages and in
have chondroitin sulphate. Odontophore cartilages have
lamprey mucocartilage was discussed in Chapter 3. periodicity and an X-ray dif-
collagen fibres with a 640 A
Previously regarded as absent from all invertebrates
fraction pattern similar to that from vertebrate collagen.
and present in all craniates other than hagfishes and lam-
Busycon cartilage contains myoglobin within its cells.
preys, elastin arose phylogenetically with the evolution of
This is unusual because the blood pigment in molluscs is
closed, high-pressure circulatory systems, although elastin
haemocyanin and cartilages are normally not pigmented9.
is arranged differently from vertebrate to vertebrate6.
Busycon cartilage was the first cartilage from any
The nature of invertebrate cartilages can, perhaps, best
taxon vertebrate or invertebrate found to have cyto-
be reviewed by summarising our knowledge of the carti-
chrome oxidase. Indeed, the low utilisation of oxygen
lages in different taxa. To that end I discuss
by vertebrate cartilage prompted Person et al. (1959) to
G odontophore cartilages in caenogastropods; examine invertebrate cartilages in the first place. They
G branchial (gill book) cartilage in an arthropod, the were prescient. Paradoxically, low oxygen utilisation was
horseshoe crab Limulus polyphemus; coupled with high levels of dehydrogenases requiring
G cranial cartilages of several cephalopods the long- cytochrome oxidase and considerable synthesis of sugars
fin squid Loligo pealii, the common cuttlefish Sepia requiring oxygen, an unexpected mix of metabolic
officinalis, and the common octopus Octopus vulgaris; parameters.
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 65

A B C

FIGURE 4.1 The histology of invertebrate cartilages. (A) Odontophoral cartilage of the marine snail, Busycon canaliculatum, with large cells and
scant extracellular matrix. (B) Cranial cartilage of the longfin squid, Loligo pealii, with abundant extracellular matrix. (C) Branchial cartilage of the
horseshoe crab, Limulus polyphemus, again with scant extracellular matrix. All stained with haematoxylin and eosin. Adapted from Person (1960).

Taking advantage of micro-computed tomography Hydroxyproline and hydroxylysine are present in

(micro-CT) imaging and computer-based methods of amounts typical of vertebrate cartilage, and with X-ray
three-dimensional (3D) reconstruction, Golding et al. diffraction patterns typical of collagen. Lipid and glyco-
(2009) reconstructed odontophoral cartilages from 18 gen are found as storage products, whereas cytochrome
species of caenogastropods10. The images obtained oxidase indicates potential aerobic metabolism. As with
illustrate the diversity and complexity of odontophoral all invertebrate cartilages, gill book cartilages are unmi-
cartilages across the gastropods. Basal species have neralised. The presence of lipids, which in vertebrate skel-
a previously unknown cartilage beneath the radula etal tissues play a role in mineralisation, is of interest
(a subradular cartilage), which may be a homologue of (see the section on mineralisation below).
the dorsal odontophoral cartilage of more derived taxa. As gill book chondrocytes divide, a phragmosome-like
Little else is known about mollusc cartilages outside gas- structure12 reminiscent of the membrane formed during
tropods (above) and cephalopods (below) aside from an the division of supporting tissue in plants is deposited
intriguing study on regeneration of the proboscis. After between the daughter cells and provides the basis for
the proboscis is amputated, members of a number of new ECM. As the cells mature, the entire chondrocyte is
genera of boring prosobranch molluscs can regenerate a chondrified; its cytoplasm fills in with matrix products
new proboscis. A single aggregation of cells gives rise to and the cell dies, a process reminiscent of lignification in
cartilage and muscle of the new proboscis (Carriker plants. This unusual pattern of chondrification may
et al., 1972). A single aggregation is consistent with explain, in part, the entirely appositional growth of gill
muscle and cartilage arising from a single population of book cartilage, which contrasts with interstitial growth of
cells as occurs in amphibian limb regeneration, which is cartilages in other cephalopods (below)13.
discussed further in Chapter 14.
CRANIAL CARTILAGES IN SQUID,
CUTTLEFISH AND OCTOPUSES
BRANCHIAL (GILL BOOK) CARTILAGE
IN THE HORSESHOE CRAB, LIMULUS The longfin squid Loligo pealii, the common cuttlefish
Sepia officinalis, and the common octopus Octopus
POLYPHEMUS
vulgaris all contain extensive cranial cartilage that pro-
The gill books of the horseshoe crab, Limulus polyphe- tects the brain and a scleral cartilage that protects the
mus, contain a cartilaginous endoskeleton (Figure 4.1). eye (Figure 4.1). Unlike cartilages in the whelk and horse-
The scant extracellular matrix contains chondroitin-4- shoe crab, these cranial cartilages have extensive matrix,
sulphate which the whelk does not and reacts as sparsely scattered cells and cell densities more typical
does vertebrate cartilage to histochemical tests for glyco- of vertebrate bone than cartilages. The matrix may be
saminoglycans, although a novel sulphated oligosaccha- fibrous, often with a canalicular system coursing through
ride containing 3-o-sulphated glucuronic acid was isolated it. Cell processes lie within the canals, connecting cells
from chondroitin sulphate K from Limulus cartilage11. one to another and giving them the appearance of
66 PART | II Origins and Types of Skeletal Tissues

osteocytes. Some squid have been reported to have carti- aggrecan or aggrecan link protein in cultured fibroblasts or
laginous epidermal scales, which would be well worth chondrocytes disrupts cell substrate interaction17.
further investigation14.

Collagens
Composition of the Extracellular Matrix Cranial cartilage collagen in the Japanese flying or com-
mon squid, Todarodes pacificus, resembles vertebrate
Great diversity of matrix products is seen in cephalopods. type I collagen (Table 4.1).
It is unclear if such diversity exists in other invertebrate A combined light, polarising and ultrastructural micro-
groups. Although a number of cephalopod species have scopical study of the head cartilages of Sepia officinalis
been examined, only one or two species have been stud- and Octopus vulgaris revealed 10 25-nm-diameter colla-
ied in most other groups. Histochemical evidence shows gen fibres similar to vertebrate type I (albeit with some-
fewer glycosaminoglycans and a different spectrum of what different banding patterns), polymeric aggregates
matrix products in cephalopod cartilage than in Limulus, and a defined perichondrium features typical of verte-
emphasising the need for more biochemical characterisa- brate cartilages but with chondrocytes connected to
tion of matricial products. one another by cell processes and blood vessels within
the cartilage, which are features of vertebrate bone and
not cartilage (Figure 4.1). Bairati et al. (1987) proposed
Glycosaminoglycans that vascularisation of these cartilages provides a high
The predominant glycosaminoglycan is chondroitin-4- level of nutrition, eliminating the need for mineralisa-
sulphate, as in Limulus. Squid cartilage lacks hyaluronan, tion. This and subsequent studies by Bairati et al. (1989,
and the core protein differs from vertebrate cartilage *1999) provide the most detailed information on any
glycosaminoglycan core protein. Squid chondroitin sul- squid or octopus cartilage. They undertook
phate contains novel tetrasaccharide sequences; the G a transmission electron microscopic (TEM) study of
common squid, Ommastrephes sloani pacificus, has a
the perichondrium, demonstrating a fibrous layer that
glucuronic acid containing glycopeptide distinct from
was discontinuous at connective tissue or muscle
any other glycosaminoglycans15.
attachments, and remarkably vertebrate-like;
Cartilages of the brown or short-finned squid, Ilex ille- G a TEM study of chondrocytes from the cranial cartilages
cebrosus coidentii, contain hyaluronan (Box 4.1), a previ-
of both species, demonstrating many long processes
ously undescribed heavily oversulphated chondroitin
connecting cells (considered reminiscent of vertebrate
sulphate, and a previously undescribed 39,200 molecular
osteocyte processes), absence of secretory granules,
weight polysaccharide composed mainly of glucuronic
presence of rough endoplasmic reticulum cisternae and
acid, galactose, and mannose in the ratio of 1:2:1.
of vesicles opening to the cell surface, and haemocyanin
Extraction and characterisation of proteoglycans from the
in many chondrocytes and gap junctions; and
Japanese flying squid, Todarodes pacificus, show that one- G an immunological study of collagens from Sepia offi-
fourth of the proteoglycans from cranial cartilages do not
cinalis using vertebrate and cuttlefish collagen antibo-
aggregate with hyaluronan. Cranial cartilage is low in pro-
dies. Response to antibodies against cuttlefish type I
teoglycan content, the proteoglycans do not interact with
and rat type V was intense. Reactions to chick anti-
hyaluronan, and keratan sulphate is absent (Box 4.1)16.
type I and to calf anti-type II were weaker. Indeed,
Cranial cartilage from the arrow squid, Nototodarus gouldi,
and surprisingly, suggesting that Sepia collagen is not
contains highly sulphated chondroitin sulphate E. The
highly derived, except for antibodies against mamma-
epidermis contains unsulphated, but highly glycosylated,
lian type I collagen, all antibodies tested cross-react
glycosaminoglycans (Falshaw et al., 2000).
with Sepia cartilage, implicating type I collagen at an
Tsilemov et al. (1998) showed that a 35-kDa protein
early step in skeletal evolution (Chapter 2).
from squid cartilage species not specified, but the carti-
lage lacks hyaluronan reacts to antiserum against sheep Sepia cranial cartilage contains a novel collagen with
link protein and binds to the same G1 domain of aggrecan, three distinct chains named C1 C3 consisting of
which is the globular domain at the N-terminus that 105, 115 and 130 kDa, respectively. This collagen is more
functions as the hyaluronan-binding region. This finding cross-linked than shark cartilage-type I collagen. Of inter-
suggests a role for this protein similar to that carried out est, an antibody against shark cartilage cross-reacts with
by aggrecan in vertebrates, which is to bind to hyaluronan Sepia cartilage but a Sepia cartilage antibody does not
to modulate the osmotic properties of cartilage (Box 4.2). cross-react with shark cartilage. Based on rotary shadow-
In vertebrates, synthesis of aggregan is enhanced in mes- ing electron microscopy, the matrix collagen fibres have
enchyme subjected to compression, whereas expressing been compared with those of chick sternal cartilage18.
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 67

BOX 4.1 Hyaluronan

Hyaluronan, a high-molecular-weight polyanion with a highly
negative charge, occurs in low concentrations in many animal 40
tissues. A single molecule may have a molecular weight of
10 million. Consisting of an alternating polymer of glucuronic
acid and N-acetylglucosamine joined by a 1 3 linkage, hya- 30

Hexosamine
luronan expands enormously in solution to form a net of
extended, random-coiled molecules. Hydrated, it typically
occupies the volume of a sphere 400 nm in diameter a thou- 20
sand times greater than the volume of the nonhydrated chain.
Hyaluronan then known as hyaluronic acid was iso- Y = 3.7415x 0.9505
lated in 1934. Hyaluronan is degraded by hyaluronidase. 10 r = 0.996
Much of the research over the 40 years after the discovery of
hyaluronan was biochemical, with few suggestions of associa-
tions with cell proliferation, migration, or aggregation, or of 0
the presence of hyaluronidase and therefore hyaluronan 0 2 4 6 8 10
removal with subsequent cell differentiation. Perhaps the Hydroxyproline
first to demonstrate these links with cell behaviour were Mauer
and Hudack (1952), who showed that a large amount of hya- FIGURE 4.2 Concentrations of hexosamine and hydroxyproline as
luronan is synthesised in the early stages of callus formation mg/pair of tibiae from chick embryos incubated for 12 days are sig-
during repair of fractured long bones. Hyaluronan plays impor- nificantly correlated (r 5 0.996) in this study comparing results from
tant roles in vertebral and limb chondrogenesis, migration of control embryos and from embryos treated with vitamin C or with
neural crest cells, regeneration of amphibian limbs, ectopic the metal thallium. See the text and Hall (1973b) for details of
treatments.
chondrogenesis and osteogenesis (for which, see Chapter 14),
and corneal development, to name only a few examples.
Although some hyaluronan is intracellular, most lies within
extracellular matrices, where it forms the backbone to which We have some indications of how levels of hyaluronan
proteochondroitin sulphate is attached. This complex forms and chondroitin sulphate (CS) are correlated (Figure 4.2).
large aggregates with aggrecan within cartilaginous ECM Synthesis of hexosamine is enhanced when tibial cartilage is
(for which see Box 4.2). Both hyaluronan and the aggregate treated for 2 days with hyaluronidase and then organ-cultured
impede transport and help to maintain the greatly hydrated for 4 days in the absence of hyaluronidase. Less CS is synthe-
nature of cartilaginous ECM. Prechondrogenic cells produce sised than in untreated cartilage; the CS has a shorter chain
only monomeric proteoglycan. With progressive differentia- length than normal; and CS is uncoupled from the hyaluronan
tion, larger and more aggregated forms of proteoglycan are backbone of the hyaluronan proteochondroitin sulphate aggre-
deposited. Of interest, as they age in vivo or in vitro, chondro- gate. Synthesis of CS is depressed when hyaluronan is added to
cytes revert to synthesising small monomer proteoglycans, a cultures of chondrocytes from embryonic or adult mammals or
change that may be a form of dedifferentiation, as occurs, to birds, which suggests that synthesis of CS is controlled at
some extent, in some tumours. With chondrocyte differentia- least in part by the level of hyaluronan. The effect of hyaluro-
tion, hyaluronan is localised preferentially in the hypertrophic nan is specific. Other glycosaminoglycans chondroitin and
zone of the growth plate, concentration correlating with keratan or heparin sulphates do not stimulate synthesis of
lacuna size, reflecting water-binding properties; lacunae fail CS. Oligosaccharides derived from hyaluronan dob.
to enlarge if chondroblasts are cultured in hyaluronidasea. Because hyaluronan is primarily extracellular, little atten-
Hyaluronan is removed from regions at which joints tion has been paid to how hyaluronan might be internalised
between adjacent skeletal elements will form. Spicer and Tien by cells. Mammalian articular chondrocytes use the hyaluro-
(2004) review the role played by hyaluronan in morpho- nan receptor CD-44 . Using rat cranial base synchondroses as
genesis, especially condensation and joint cavitation during a model, Gakunga et al. (2000) localised hyaluronan to the
skeletogenesis. As summarised in Table 1 in their paper, hypertrophic but not other chondrocyte zones; autoradio-
Spicer and Tien identify 30 genes associated with the synthe- graphic and histological analyses of growth of the cartilages in
sis, function and/or degradation of hyaluronan. The enzyme the midline cranial base of the Norwegian rat Rattus norvegi-
hyaluronic acid synthase-2, coded by the gene Has2, plays an cus to 16 months after birth show that cell proliferation is spe-
important role in removing hyaluronan at presumptive joint cific to individual cartilages. Hyaluronidase and CD-44
sites through a pathway involving Shh regulation of Gli3, colocalise with hyaluronan. H. Jiang et al. (2001) maintain that
which binds to a promoter in the Has2 gene (J. Liu et al., hyaluronan is internalised by hypertrophic chondrocytes and
2013). This study using mouse limb buds sheds light on the degraded via a CD-44 based mechanism. Synchondroses cul-
known reduction in Gli3 in limbs buds of talpid2 chick tured in Streptomyces hyaluronidase show reduced lacunar
embryos, discussed in Chapter 39. expansion, a finding consistent with hyaluronan-mediated

(Continued )
68 PART | II Origins and Types of Skeletal Tissues

BOX 4.1 (Continued)

hydrostatic pressure playing a role in lacunar expansion during deals especially well with the role of hyaluronan in the pericellular matrix
that surrounds chondrocytes.
chondrocyte hypertrophyc. b. See Hardingham et al. (1972) for synthesis of hexosamine in tibial carti-
lage, and Wiebkin and Muir (1973) and Solursh et al. (1974) for addition of
a. See Larsson and Kuettner (1974) for intracellular hyaluronan; Wiebkin hyaluronan to cartilage cultures.
and Muir (1973), Goldberg and Toole (1984), W. Knudson and Knudson c. See H. Jiang et al. (2001) for the CD-44 receptor and see Roberts and
(1991), Pavasant et al. (1996) and Toole (*1973, *2001) for aggregate for- Blackwood (1984) for the study with the Norwegian rat. CD-44 is expressed
mation; and Ovadia et al. (1980) and Pacifici et al. (1981) for synthesis of in mouse development (9.5 12.5 days) in high levels in the somites, heart,
monomers by prechondroblasts and ageing chondrocytes. See Pavasant limb condensations, apical epithelial ridge of limb buds, tooth primordia
et al. (1996) for lacunar enlargement, and Toole (*2001) for a review of and upper and lower jaw mesenchyme (Wheatley et al., 1993).
hyaluronan and its binding proteins, the hyaladherins. The 2001 paper

BOX 4.2 Aggrecan

Aggrecan (cartilage-specific proteoglycan core protein, chon- an important structural property to cartilage, namely to main-
droitin sulphate proteoglycan 1, aggregating chondroitin sul- tain an osmotic swelling pressure and the highly hydrated
phate proteoglycan) is the product of the Acan gene and a nature of cartilaginous extracellular matrix. Compressive
member of the aggrecan/versican/proteoglycan family of extra- forces, such as those acting on articular cartilages, stimulate
cellular matrix proteins. A much as 10% of the dry weight of synthesis of aggrecan. The core protein of aggrecan is broken
some cartilages is aggrecan. As many as 100 aggrecan mono- down by proteases, a breakdown that facilitates vascular inva-
mers bind to a single hyaluronan chain via a link protein to sion of cartilage.
form a high-molecular-weight aggregate, with a globular Acan mRNA is expressed in periosteal cells of membrane
domain (G1, the hyaluronan-binding region) at the N-terminus bones immediately before they begin to differentiate as chon-
where the keratan sulphate chains are preferentially located. droblasts (see the section on evidence for bipotentiality in
Aggrecan shares numerous sequence similarities with versican Chapter 12). Different mutations in Acan result in dwarfism
(discussed in Box 21.3) and with CD-44 (Ayad et al., 1994). in different lineages, including Cartilage matrix deficiency
Aggrecan is a structural proteoglycan, by which I mean (Cmd) in mice and Nanomelia (nm) in domestic fowl
that the aggregation to which aggrecan contributes provides (Chapter 27).

TABLE 4.1 Amino Acid Composition of Collagen and Alpha Collagen Chains from Cranial Cartilage of the Japanese
Flying Squid, Todarides pacificus, Compared with Type I Collagen from Rat Tail Tendon and Rat Skina

Squid Rat Tail Tendon Rat Skin

Amino Acid Collagen 1(I) 1(II) Collagen Collagen

3-Hydroxyproline 1 1 1 4.2 1
4-Hydroxyproline 90 92 88 90 92
Alanine 94 102 76 107 106
Arginine 59 58 60 50 51
Aspartic acid 58 53 65 45 46
Glutamic acid 91 92 90 71 71
Glycine 338 341 339 331 331
Histidine 6 6 8 4 5
Hydroxylysine 14 11 24 7 6
Isoleucine 15 11 22 10 11
Leucine 27 25 31 24 24
Lysine 10 12 9 27 28
Methionine 11 11 8 8 8
Phenylalanine 9 10 8 12 11
Proline 90 88 90 122 121
Serine 42 44 36 43 43
Threonine 23 24 20 20 20
Tyrosine 2 3 1 4 2
Valine 20 16 24 23 25
a
From data in Kimura and Karasawa (1985) for squid and in Serafini-Fracassini and Smith (1974) for rat tail tendon and skin. Based on limited pepsin proteolysis
and expressed as residues/1,000 amino acid residues.
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 69

TENTACULAR CARTILAGE IN POLYCHAETE crown) supported by large vacuolated cartilage cells,

ANNELIDS which are in turn surrounded by a supporting sheath of
extracellular matrix and a vascular supply. This generali-
The feather duster worms Eudistylia polymorpha and sation was reinforced by an extensive histological analysis
Sabella melanostigma have extensive, branched and sub- of the tentacle cartilages in 30 species (18 genera) of
divided (segmented?) cartilages at the base of the tentacle sabellids by Capa et al. (2011). In all species, the base of
complex, within the tentacles, and in the pinnae that the tentacles (dorsal lips) is supported by cartilage, as
branch from the tentacles (Figure 4.3). These cartilages described earlier. With elongation, tentacles are supported
(presumably) give considerable support and flexibility to either by cartilage with irregularly arranged chondrocytes
the tentacles as a food-gathering organ. The cartilaginous or by an acellular extracellular matrix. Capa and collea-
matrix is sparse and vascularised, causing it superficially gues review past studies on these tissues, including issues
to resemble osteoid. The glycosaminoglycan is a highly of terminology, recommending supporting cellular axis
sulphated chondroitin-6-sulphate. Ultrastructural studies for the basal tissue, supporting acellular axis for the
of Sabella cartilage show heterogeneity of matrix acellular tissue and hyaline cartilage for the cartilage.
structure19. Members of the subfamily Fabriciinae, such as
All members of the subfamily Sabellinae to which Fabricia stellaris, on the other hand, support their tenta-
these two species belong have tentacles (a branchial cles with an acellular skeleton, and their tentacles lack
blood vessels. Members of the Serpulidae lack skeletal
support in their tentacles (Randel and Bick, 2012 and
references therein). Such divergence of skeletal structures
in three subfamilies that form a monophyletic clade of
sabellid polychaetes demonstrates the advances that could
be made by future comparative analyses of polychaete
branchial crowns. Indeed, Randel and Bick (2012) con-
sider that the radioles and pinnules that make up the tenta-
cles in these three subfamilies may not be homologous, a
conclusion consistent with the variable nature of the
supporting tissues.

LOPHOPHORE CARTILAGE IN AN
ARTICULATE BRACHIOPOD,
A 10 m TEREBRATALIA TRANSVERSA
Cartilage in the pedicle of an articulate (two-valve) bra-
chiopod, Terebratalia transversa, was discovered seren-
dipitously during an ultrastructural study of the
development of the pedicle, which is the fleshy stalk that
attaches the animal to the substrate. The cartilage consists
of a weakly metachromatic connective tissue that devel-
ops in the pedicle after metamorphosis. The connective
tissues of the tentacles are acellular and not metachro-
matic after toluidine blue. At the ultrastructural level they
resemble (indeed, appear no different from) vertebrate
fibrous cartilage. The connective tissue in the remainder
B 200 m of the lophophore is metachromatic and clearly
cartilaginous20.
FIGURE 4.3 Polychaete and gastropod cartilages. (A) A longitudinal
section of a tentacle of the marine polychaete, the feather duster worm,
Eudistylia polymorpha, showing the central cartilage and the cartilages
of the pinnae. (B) Regenerating odontophoral cartilage of the gastropod, MINERALISATION OF INVERTEBRATE
the marine oyster drill, Urosalpinx cinerea follyensis, 11 days after CARTILAGES
amputation of the proboscis. Note the proliferating, blastema-like carti-
lage on the right. Adapted from Person and Mathews (1967) and Box 4.3 contains an overview of the mineralisation of
Carriker et al. (1972). vertebrate cartilage. Chapter 22 contains a discussion
70 PART | II Origins and Types of Skeletal Tissues

BOX 4.3 Cartilage Mineralisation

Interest in mineralisation of cartilage and bone goes back to
Honor Fells pioneering studies 80 years ago. Because minera- (Figure 4.4), where its distribution coincides with mineralisa-
lisation is so regular, dynamic and precisely controlled tion. The interface between nonmineralised and mineralised
and can be approached from so many different levels and cartilage, which is known as the tidemark, is illustrated in
backgrounds crystallography, mineralogy, endocrinology, Figure 3.11. Tidemarks occur in hyaline and fibrocartilage if a
cell biology, pharmacology, pathology the literature is junction exists between mineralised and nonmineralised carti-
extensive. Throughout the book I touch on mineralisation as a lage (Figure 3.11, and see Havelka et al., 1984, for a review).b
developmental process. Here, I survey mineralisation using Osteonectin (SPARC secreted protein acidic and rich in
the mineralisation of cartilage to highlight some important cysteine) is a 32,000 MW protein that makes up around one-
featuresa. tenth of bone protein. Linking collagen to hydroxyapatite and
acting as a nucleus for mineralisation, osteonectin is present
The Players
in osteoblasts, osteoprogenitor cells, young but not old osteo-
Robin Poole from Montreal contributed greatly to our basic
cytes, chondrocytes of mineralising cartilage, fracture callus
understanding of cartilage mineralisation and its relationship
and ectopic ossifications such as myositis ossificans (illustrated
to growth of long bones at growth plates. Poole et al. (1989)
in Figure 1.6). Osteonectin is expressed in hypertrophic chon-
summarised and reviewed the roles of alkaline phosphatase,
droblasts, in mineralised ECM, and in the perichondrium of
collagen, chondrocalcin and proteoglycans in cartilage miner-
rat mandibular condylar cartilage. Osteonectin is present in
alisation. Here, I outline calmodulin, chondrocalcin and
the cells of all zones of chicken tibial growth cartilage, but
osteonectin.
only in the matrix in the mineralising zoneb.
Calmodulin is so named because this abundant cyto-
The role of proteoglycans in the mineralisation of cartilage
plasmic constituent is a calcium-modulated protein. It func-
is complex, little understood, and cartilage- or taxon-specific.
tions by sensing Ca21 levels and then signalling calcium-
Acridine orange preserves proteoglycans, allowing their visuali-
sensitive enzymes or calcium-sensitive ion channels.
sation in rosettes in the longitudinal septa of the rat growth plate
Calmodulin is present in chondrocytes (detected in mature
at sites where mineralisation occurs. Proteoglycans, monomers
and mineralising chondrocytes of rodent primary and condy-
and link protein also are retained during the mineralisation of
lar cartilages), but not in perichondrial or periosteal cells
mammalian and avian epiphyseal growth plates (Figure 4.7)c.
(Lewinson and Boskey, 1984).
Chondrocalcin (the c-propeptide of type II procollagen) is
a calcium-binding extracellular protein. The c-propeptide
therefore has two distinct roles assembling the triple helix
of type II collagen, and binding calcium in mineralising car-
tilage (A. R. Poole et al., 1984). Although not present in
the proliferative layers of bovine fetal epiphyseal or growth
plate cartilages, chondrocalcin is present in the hypertrophic
zone and in the longitudinal septa of the extracellular matrix

100 m 100 m
A B

A B C D
FIGURE 4.5 The organisation of growth plates. Proximal tibial
FIGURE 4.4 The relative amounts of longitudinal and transverse growth plates from 16- (A) and 25- (B) day-old mice to show organi-
septa (trabeculae) depicted by the thickness of the lines in sation of the cell columns. The articular surface is at the top. P, pro-
normal bone (A), under conditions of rapid growth in length (B), in liferative, H, hypertrophic and R, resting cartilage cell zones. M,
arrested growth (C), and with atrophy (D). The lengths of the bone mineralisation front. Modified from Wikstrom et al. (1984).
segments in A D are proportional to each physiological situation.
From H. A. Harris (1933). (Continued )
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 71

BOX 4.3 (Continued)

100 m
A B

FIGURE 4.7 Link protein. This longitudinal section of a portion of

the hypertrophic zone of a bovine fetal growth plate shows link pro-
tein (green in original) localised to the longitudinal septa (L) and to
spicules of mineralised cartilage matrix (S). Little link protein can be
visualised in the transverse septa. Cytoplasmic staining (orange in
original) reflects a counterstain, not link protein. Modified from A. R.
Poole et al. (1982a).

Cartilage Specificity
C D Mineralisation of cartilage varies in a cartilage-specific
manner. Mammalian small-celled cartilage normally does not
FIGURE 4.6 Reduced development of the proximal tibial growth mineralise. Why? Because mineralisation is the prerogative
plates in Brachymorphic (Bm/Bm) mice at two ages after birth: 16- of hypertrophic chondrocytes. Secondary cartilages on avian
day-old normal (A) and bm/bm (B) mice; 25-day-old normal (C) membrane bones mineralise, but Meckels cartilage (a small-
and Bm/Bm (D) mice. Cartilage and mineralisation (black) is disor- celled cartilage) does not. Mineralisation and alkaline phos-
ganised in the growth plates from the Bm/Bm embryos. The entire phatase are virtual synonyms, but mineralisation of rat
growth plate is reduced in height in mutant embryos of both ages tracheal cartilages is not associated with deposition of
(all at same scale). P, proliferative, H, hypertrophic and R, resting alkaline phosphatase. Guinea pig mandibular condylar, tibial
cartilage cell zones. M, mineralisation front. Modified from epiphyseal and articular cartilages exhibit different patterns
Wikstrom et al. (1984).
of mineralisation in response to scurvy. The jaw cartilages of
(Continued )
72 PART | II Origins and Types of Skeletal Tissues

BOX 4.3 (Continued)

A B C

FIGURE 4.8 Mineralisation of cartilage in vitro. Mandibular mesenchyme removed from chick embryos of H.H. stage 21 (3.5 days of incuba-
tion), dissociated and established in micromass culture (10 L drops of 2 3 107 cells/mL) undergoes chondrogenesis and matrix mineralisation
(as seen with Kossa staining) when cultured in Hams F12:BGJb [3:1] 1 10% fetal calf serum 1 ascorbic acid (150 g/mL). (A) Cartilage nodules
(c) with initial signs of mineralisation (m) after 12 days of culture. (B) Increasing mineralisation (m) after 14 days in vitro; see also Figure 4.9.
(C) After 20 days in vitro, only the central cartilage (c) is unmineralised. Adapted from Ekanayake and Hall (*1994b).

A 40 m B 40 m

C 80 m D 80 m

FIGURE 4.9 Mineralisation of cartilage in vitro. Mandibular mesenchyme, removed from chick embryos of H.H. stage 21 (3.5 days of incuba-
tion), dissociated, and established in micromass culture (10 L drops of 2 3 107 cells/mL) undergoes chondrogenesis with matrix mineralisation.
Culture media were Hams F12:BGJb [3:1] 1 10% fetal calf serum (A); Hams F12:BGJb [3:1] 1 10% fetal calf serum 1 150 g/mL ascorbic
acid, and 1027 M dexamethasone (B D). Culture period was 12 or 14 days; see also Figure 4.8. Mineralisation visualised with the Kossa reac-
tion. (A and B) Histological sections after 12 days of culture. Cartilage (c) is present in both, mineralisation in neither (cf. Figure 4.8). Cultures in
test medium: 12-day (C) and 14-day (D). Onset of mineralisation can be seen in one region of the cartilage (c) after 12 days of culture with ascor-
bic acid and dexamethasone (C), and more extensive and heavier mineralisation after 14 days (D). Adapted from Ekanayake and Hall (*1994b).
(Continued )
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 73

BOX 4.3 (Continued)

Vitamin D deficiency can result in an inability to mine-
clam-crushing myliobatid stingrays are stiffened internally ralise cartilage because hypertrophic chondrocytes fail to
with mineralised struts and rows of tesserae in a manner seen mature, resulting in stunted growth that can lead to rickets in
only in bone in other vertebratesd. Clement (1992) analysed children and osteomalacia in adults. Rickets and osteomalacia
the fine structure of mineralisation of elasmobranch skeletons are two terms for the same condition of softening of bones,
in 53 species; Summers et al. (2004) correlated CT-scanning although the consequences differ the earlier the onset.
images with measures of stiffness in mineralised cartilages of Collagen synthesis and calcium nucleation are both enhanced
the California horn shark, Heterodontus francisci. when rachitic bone is exposed to vitamin D. Some of the
action of vitamin D on bone growth and mineralisation may
Mineralisation In Vitro
be indirect through enhancement of plasma Ca21 and PO432 .
Many instances in which cartilage mineralises in vitro are
Demineralised bone matrix implanted into vitamin
discussed throughout the text (Figures 4.8 and 4.9). For exam-
D deficient rats induces normal amounts of cartilage. Less
ple, in what B. Zimmermann et al. (1990) termed organoid
bone is produced than in controls, and the bone is minera-
cultures, explanted mouse limb cells had become almost
lised abnormally unless vitamin D is addedf.
completely mineralised after 21 days of culture in medium
containing 10-nM -glycerophosphate. Examination at the a. For reviews of the microstructure and mineralisation of skeletal tissues,
ultrastructural level revealed that almost all chondrocytes in see J. G. Carter (1990), Francillon-Vieillot et al. (1990) and de Ricqle`s et al.
the mineralised areas had died. In a later study, addition of (1991).
b. See Termine et al. (1981), Stenner et al. (1986), Jundt et al. (1987) and
osteoblastic calvarial cells enhanced mineralisation, whereas Nomura et al. (1988) for osteonectin; Copray et al. (1989) and Pacifici et al.
addition of fibroblasts decreased mineralisation, presumably (1990) for expression in condylar cartilage and tibial growth cartilage. See
by the release of soluble molecules or the disruption of inter- Figures 4.5 and 4.6 for proximal tibial growth plates in 16- and 25-day-old
mice.
actions between mineralising cells; addition of dexametha-
c. See Shepard and Mitchell (1985) for acridine orange, and Davis et al.
sone induces these cells to chondrify (B. Zimmermann and (1982) and A. R. Poole et al. (1982c) for retention of proteoglycans during
Cristea, 1993). mineralisation.
d. See Hall (1971b) and Sasano et al. (1993a) for mineralisation of avian
Vitamins and tracheal cartilages; Irving and Durkin (1965) and Durkin et al. (1969a,
Vitamin D plays a critical role in the mineralisation of carti- b) for guinea pig; and Summers (2000) and Summers et al. (1998) for mylio-
batid stingrays, which include such species as the cow nose ray,
lage and bone, both of which are commented on here. Rhinoptera bonasus; eagle ray, Aetobatus narinari, and bat ray, Myliobatis
Vitamin D elevates plasma Ca21 and PO432 to levels high californica. Rhioptera bonasus also develops a fibrocartilage where the ven-
enough for bone to mineralise. Of the two major hormones tral intermandibular tendon inserts onto the mineralised jaw cartilage
(Summers et al., 2003).
that mediate calcium metabolism, parathyroid hormone is
e. See G. L. Wong (*1984) for the skeletal actions of PTH, and see Pacifici
vitamin D dependent and calcitonin is not. The first direct evi- et al. (1980), Ornoy and Zusman (1983), Narbaitz (1992) and Boskey et al.
dence that vitamin D3 stimulates movement of calcium into (2001) for vitamins and cartilage.
bone came from studies on calvariae from Japanese quail f. See Cousins and DeLuca (1972) and P. H. Stern (1980) for vitamin D
and bone, Underwood and DeLuca (1984) for plasma regulation, and
maintained in vitro by Gunasekaran et al. (1986), who para- Van der Steenhoven et al. (1988) for bone implanted into vitamin
doxically found that Ca21 uptake into mouse bone was D deficient rats.
reduced by vitamin D3e.

of mineralisation in relation to chondrocyte hypertrophy of invertebrate skeletal tissues relates to the evolution of
and the synthesis of type X collagen. vertebrate mineralising skeletal tissues22. Preparations
Although many invertebrates have cartilaginous skele- of cartilage from Loligo, Limulus and Busycon can
tal matrices, no instance of in vivo mineralisation is mineralise in vitro if incubated in a solution
known, and no osseous tissue or bone has been found in metastable to hydroxyapatite. Mineralisation occurs only
any invertebrate. Of interest, many invertebrate taxa at a temperature (37 C) well above normal environmental
with unmineralised cartilages do have mineralised non- temperatures of 20 C. Mineralisation was as hydroxyapa-
cartilaginous endoskeletons, raising the question of why tite, which is the normal form of calcium phosphate in
the cartilages do not mineralise. Do they lack the ability vertebrate cartilages. The initial abstract reports accumu-
to mineralise in vivo, or are they inhibited from minera- lation of 18 g Ca21/g cartilage in Loligo. Ca21 accumula-
lising? Would they mineralise in vitro under conditions tion follows formation of perichondrial and matrix
discussed in Chapter 3 that permit lamprey cartilage to granules and progressive intracellular mineralisation of
mineralise?21 the chondrocytes (Figure 4.10). Such a pattern of intracel-
Mineralisation of cartilage in invertebrates is blocked lular mineralisation, normally associated with cell death
by temperature, perhaps coupled with the absence of in vertebrate chondrocytes (Hall, 1972a), is seen when
sufficient concentrations of calcium and phosphorus in lamprey cartilage mineralises in vitro23. The rate of
seawater. Hall (*1975a) discusses how the mineralisation mineralisation in vitro is correlated positively with the
74 PART | II Origins and Types of Skeletal Tissues

Hemichordates
Often taken as a surrogate vertebrate ancestor, amphioxus
(14 species of hemichordates) such as Branchiostoma lan-
ceolatum, Saccoglossus bromophenolosus, S. kowwalevskii
and Asymmetron spp. have a pharyngeal set of gill bars
that contain skeletal rods. These gill bars contain a fibril-
lar collagen and proteoglycans. Based on a phylogenetic
approach using the triple helical domain, Rychel et al.
(2006) demonstrated that the fibrillar collagen is similar
A B to both type I and type II vertebrate collagens. Presence
of collagen and proteoglycans in an extracellular matrix is
FIGURE 4.10 Squid cartilage can mineralise. Head cartilage of consistent with this being a cartilaginous tissue but the
the longfin squid, Loligo pealii, after incubation for 32 (A) or 40 (B) gill bar skeleton is acellular the cells remain outside
hours in a hydroxyapatite mineral solution at 37 C. (A) Phase-contrast the extracellular matrix they secrete and is derived
microscopy reveals accumulation of mineral (arrows) beneath the peri-
chondrium. (B) von Kossa staining with toluidine blue counterstain
from the endoderm, two pivotal features that remove
shows accumulation of mineral granules (black) over the ECM. these tissues from the vertebrate and invertebrate cartilage
Modified from Libbin et al. (1976), which contains details of the incuba- families (Gillis et al., 2012b; Hall and Gillis, *2013).
tion solution. However, Rychel et al. (2006) hypothesised that the
earliest gill cartilages were acellular, and these were grad-
concentration of phosphatidyl-serine within cartilage, ually replaced by neural crest cells in vertebrates (p. 543,
consistent with lipid involvement in mineralisation of and see Chapter 17 for the neural crest origin of pharyn-
vertebrate cartilage and bone24. geal arch cartilages).
Similar key transcription factors (Pax1/9, Hox1, Eya,
Six1 and FoxC) are expressed in the pharyngeal gill arches
CARTILAGE ORIGINS of Saccoglossus kowwalevskii and the pharyngeal arches
of vertebrates. A major difference is that expression that
Theories of the evolutionary origin(s) of cartilage abound.
is in derivatives of all three germ layers in vertebrates is
Most start from the premise that cartilage is exclusively a
restricted to the pharyngeal endoderm in S. kowwalevskii,
vertebrate tissue.
which lacks Tbx1-expressing mesodermally derived mes-
Berrill (1955) supported Garstangs earlier conclusion
enchyme in the pharyngeal arches (Gillis et al., 2012b).
that vertebrates arose from free-swimming larvae of
The genetic basis for the patterning of the visceral arches
Precambrian tunicates. He proposed that cartilage evolved
of vertebrates, in which derivatives of all three germ
as an extracellular secretion by tunicate connective tissue
layers are involved, arose as signalling from pharyngeal
cells to serve a mechanical function, and that bone formed
endoderm, the homology of which is well substantiated.
an outer, impermeable layer because of the tendency for
The endodermal pharyngeal pouch supporting skeletal tis-
calcium phosphates to be excreted from the skin. In tele-
sue in hemichordates cannot readily be homologised as
ost fish, in fact, calcium is excreted via the gills, and
cartilage with vertebrate or nonchordate invertebrate
phosphorus is transported in the intestine with little if any
cartilages (Gillis et al., 2012b; Hall and Gillis, *2013).
skeletal involvement25.
In addition, it has been proposed that cartilage or a
Such a premise is either untenable or, if tenable, relates
cartilage-like tissue is present in the tentacle-like oral cirri
to the origin of vertebrate cartilage, not to the origin of car-
around the oral hood of species of hemichordates. The pre-
tilage per se. Why? Because as we have just seen, cartilage
cise nature of this tissue is unclear from early studies: the
is present in many invertebrates. Consequently, several
presence of an ECM suggests cartilage. Although retention
important questions remain:
of vacuoles in the cells is shared with notochord (see
G When and how many times did cartilage evolve as a below), the proposal that the tissue is endodermal in origin
tissue? (like the pharyngeal tissue) is not consistent with a rela-
G What is the precursor of cartilage among the earliest tionship to notochord or to its classification as a cartilage.
Metazoans? Examination of the regeneration of these oral cirri in a
G Does the evolution of vertebrate cartilage(s) relate in Japanese species Branchiostoma belcheri sheds consider-
any way to the evolution of invertebrate cartilage(s)? able light on the nature of the skeletal supports. Kaneto and
G Can we separate the evolution of cartilage as a tissue Wada (2011) used expression of fibrillar collagen and SoxE
from the evolution of type II (cartilage type) collagen? genes in the regenerating tissues to classify it as cartilage
Cole and Hall (2004a,b) think we can; see below. and propose the following: an evolutionarily conserved
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 75

Chondroid connective tissue/ FIGURE 4.11 A summary proposal of

Mesenchyme the evolutionary relationships among chon-
droid, notochord, cartilage and bone in
relation to invertebrate and vertebrate carti-
lages, presence of type II collagen in noto-
Bone chord and vertebrate cartilages but not in
invertebrate cartilages, and the presence of
Type I/V collagen + several lineages of chondrocyte differentia-
Mucopolysaccharides tion among invertebrates. (Reproduced
from Cole, *2011, with permission of the
Cartilage author.)
Vertebrate

Type II
? collagen
Notochord

Independent
chondrocyte
specialisation? Invertebrate cartilages

genetic regulatory system is involved in amphioxus mRNA (Ringuette et al., 1991), a finding that raises fasci-
cirrus and vertebrate skeletogenesis (p. 409). Even more nating issues: Do invertebrates do with cartilage all that
intriguing is the demonstration of known osteogenic mar- vertebrates do with cartilage and bone? Cartilaginous
kers, Runx and osteonectin, in the regenerated cartilage, fishes certainly do with cartilage what teleosts and tetra-
aligning this tissue with the intermediate tissues discussed in pods do with bone. Whatever properties of invertebrate
Chapter 5. Fibrillar collagen and osteonectin genes also are cartilages brings them into the cartilage family perhaps
coexpressed in ascidian notochord and in mineralised bone. superfamily would be a more appropriate term, depending
An important generalisation is that molecular and genetic on issues of homology we must project the origin(s)
data provide insights into mechanisms that phenotype that of cartilage much further back than the invertebrate
morphology often cannot. The scenario proposed is that the chordates such as hemichordates or tunicates.
common chordate ancestor had a genetic regulatory system We have seen the enormous differences among
from which both chondrogenesis and osteogenesis emerged lamprey, hagfish, gnathostome and invertebrate cartilages.
as separate differentiated cell types and tissues. Indeed, because extant vertebrates are subdivided into the
Living species of amphioxus (hemichordates) clearly three groups (lampreys, hagfishes and gnathostomes),
are not vertebrate ancestors and may not even be good can- invertebrates should be subdivided into groups (cephalo-
didates as surrogate vertebrate ancestors. Fossils, as illus- pods, brachiopods and annelids) to reflect the diversity of
trated by amazing fossil finds over the past two decades invertebrate cartilaginous tissues. Figure 4.11 shows sev-
and by enhanced methods of palaeohistology, will increas- eral independent lines of chondrocyte origins in inverte-
ingly come into play in our discussions of the origin(s) of brates. Extant agnathans and invertebrates have cartilage
vertebrate skeletal tissues. Cathaymyrus diadexus and as a tissue but not type II collagen as a matrix product.
Cathaymyrus haikoensis have been identified as cephalo- Type II collagen, a sine qua non for the identification of
chordates on the basis of the presence of a notochord and cartilage in jawed vertebrates, was not associated with ini-
segmented body muscles. Equally old, however, are two tial evolution of cartilage as an invertebrate tissue, shown
species of hemichordates, Haikouella lanceolata (over 300 in Figure 4.11. Consequently, we must widen our defini-
individuals) and Haikouella jianshanensis, from the Early tion and concepts of what constitutes cartilage. One set of
Cambrian Chengjiang formation in China (530 mya) that data causing us to reinvestigate the definition of cartilage
had gills, a tail, six visceral arches presumed to be carti- outlined below is the differentiation of cartilage by
laginous and pharyngeal teeth, which may represent the notochordal cells. A second set of data, outlined in
earliest mineralised tissues26. Chapter 16, relates to the first steps in formation of verte-
No invertebrate has bone, although some invertebrate brae in teleost fishes in which mineralisation of the noto-
cartilages contain such bone-cell markers as osteonectin chord sheath initiates vertebral development through
76 PART | II Origins and Types of Skeletal Tissues

osteogenesis, not chondrogenesis. A third set of data on study of notochordal cartilage (chordoid and chondroid)
similarities between notochord and cartilage, discussed in in the notochords of geckos by Jonasson et al. (2012) is
Box 42.2, leads us to ask whether cartilage is a form of discussed in Box 42.2 in the context of whether notochord
notochord or notochord a form of cartilage, or whether should be considered a type of cartilage29.
both are forms of a larger set of connective tissues. During regeneration after amputation of the tail of the
glass knifefish, Eigenmannia virescens, cartilage develops
at the end of the severed notochord. The chondrocytes do
NOTOCHORDAL CARTILAGE not hypertrophy, and the matrix does not mineralise.
Notochord is an unusual tissue with large vacuolated Cartilage on the ventral surface is removed by multinucle-
cells connected by desmosomes and surrounded by an ated cells and replaced by perichondral bone (Kirschbaum
expanded basement membrane as a perinotochordal and Meunier, *1988).
sheath. Notochordal cells synthesise and deposit type II A further fascinating example, which comes to us
collagen, which we think of as cartilage-type collagen. from the fossil record, illustrates the advantages to be had
We may have this backward, however. Type II collagen when an informed palaeontologist incorporates a develop-
is notochordal collagen, a notochordal molecule that was mental approach in his/her analysis. The example is a
taken over by cartilage and elaborated into an ECM when group of extinct, limbless, enormously elongate reptiles,
vertebrate cartilage originated27. the aystopods, with many hundreds of vertebrae. The
Even more intriguing issues are whether notochord challenges in laying down so many vertebrae without
should be included in the cartilage superfamily; how greatly extending ontogeny are considerable. R. L. Carroll
notochord as a tissue relates to connective tissues; and how (1989) identified two patterns of vertebral development
it relates to tissues such as chondroid (Figure 4.11) or myx- in aystopods and in Palaeozoic tetrapods, both of
oid, typically regarded as intermediates between two other which reveal unexpected involvement of the notochord.
tissue types (Chapters 1, 5 and 42). The most parsimonious Vertebrae developed either from a perichondral tube
relationship between these tissues is outlined in around the notochord or from a medial notochordal
Figure 4.11, in which notochord and vertebrate cartilage fibrous sheath, a mechanism that allows more rapid ossifi-
share expression of type II collagen. As discussed in this cation than does perichondral ossification, and that is the
chapter, the origin of type I collagen was much older, pre- mode of origin of vertebral bodies in teleosts (Bensimon-
dating any of these skeletal tissues (Figure 4.11). Britto et al., 2012b, and see Chapter 16).
It is well known that the notochord induces sclerotomal A final example of extremes of vertebral development
mesenchyme around it to chondrify as vertebral cartilage comes from the prolacertiform reptile Tanystropheus
(Chapter 42). Metachromatic, glycosaminoglycan-rich longobardicus from the Middle Triassic (Wild, 1973).
matrix accumulates around the notochord before chondro- At least three-fourths of the total body length of these
genesis of the adjacent mesenchyme. This sequence was 6-m-long ancient reptiles was neck and tail (Figure 4.12).
interpreted as indicating that these notochordal matrix The enormously elongate neck has been described as
products progressively become the matrix of the vertebral the longest neck possible within the laws of physics.
cartilage28.
More intriguing, cartilage can arise from the noto-
A
chord itself. Although not common, notochordal chon-
drogenesis is part of normal vertebral development in
some taxa and is a regenerative response in at least one
taxon of teleost fishes. 1 cm
D. B. Wake and Lawson (1973) and Lawson (1966)
described the formation of notochordal (intravertebral)
cartilage in the centra of a salamander, Eurycea bislinea-
ta, and in the Frigate Island caecilian, Hypogeophis ros-
tratus. In both species, the cartilage developed from cells
of the notochordal epithelial sheath and not from invad-
ing mesenchymal cells derived from the sclerotome.
Absence of a distinct sclerotome but plentiful notochordal
B 50 cm
cartilage also characterises vertebral development in the
northern two-lined lungless salamander, Eurycea bislineata
FIGURE 4.12 The longest cervical vertebrae (A) may have belonged
(D. B. Wake and Lawson, 1973). As Lawson (1966) to the middle Triassic reptile, Tanystropheus longobardicus (B). A cervi-
emphasised, urodele centra ossify from connective tissue, cal vertebra is shown to scale beside the neck in (B). Based on informa-
whereas anuran centra ossify in cartilage. A more recent tion in Wild (1973) and Tschanz (1988).
 Chapter | 4 Invertebrate Cartilages, Notochordal Cartilage and Cartilage Origins 77

Amazingly, it consists of no more than 12 (often only 10) 10. See Metscher (2009a,b) and Wise et al. (2013) for an analysis of
enormously elongated cervical vertebrae (Figure 4.12). As micro-CT methodology, 3D reconstruction, volume rendering and
determined by Tschanz (1988) from an analysis of eight colour enhancement, illustrated with superb images of unmineralised
embryos and fetal rabbit and rat skeletons.
individuals from Switzerland that comprise an ontogenetic
11. T. Sugahara et al. (1996), who did this study, use the common name
series, the largest individuals had cervical vertebrae as
king crab, an older name for the horseshoe crab, but it is clear
long as 26 cm. Average length of cervical vertebrae 3 11 that they were examining Limulus. The common name king crab
was 17.5 cm in the largest individual and 3.5 cm in the (red, blue or golden king crab) is more commonly used for the three
smallest, a fivefold increase in size. species of Alaska king crab, the red king crab Paralithodes
The enormous diversity of cartilages in invertebrates camtschaticus, golden king crab Lithodes aequispinus and the blue
and vertebrates demonstrates the lability of skeletogenic king crab Paralithodes platypus.
cells, a lability that is further reinforced when we con- 12. Descriptions of phragmosomes differ, in part reflecting differences
sider, as we do in the next chapter, the range of intermedi- in different plant taxa. The phragmosome (partition body) is vari-
ate tissues in vertebrates: tissues with features of two or ously described as the following: (i) a structure within plant cells
more of the four skeletogenic and odontogenic tissues a flat membranous vesicle composed of cell-wall components,
microbodies of the cell plate that form after nuclear division, a
bone, cartilage, dentine and enamel.
raft-like structure composed of actin filaments (see Box 5.1) and
microtubules formed immediately before mitosis at the preprophase
stage; (ii) a region within plant cells the cytoplasmic region con-
NOTES
taining the nucleus during mitosis; or (iii) by function cytoplasmic
1. See W. A. Beresford (*1981) and Dhem et al. (1989) for analyses strands that aid the nucleus in its migrate to a central position before
of chondroid, its contribution to skeletal growth, and various uses of mitosis. All these descriptions fit the membrane formed in dividing
the term. gill-book chondrocytes.
2. See Person et al. (1959), Person (1960, *1983a), Person and Philpott 13. See Person (1960), Person and Philpott (1963) and Cowden (1967)
(1963, 1969a,b), Philpott and Person (1970) and Person in Slavkin for light and transmission microscopic cytology/histology; Mathews
(1972, pp. 140 151, the evolution of cartilage section of a work- et al. (1962), Cowden (1967) and Sugahara et al. (1996) for the gly-
shop held on Santa Catalina Island, CA). cosaminoglycans; Person and Philpott (1969b) for collagen; Philpott
3. See Jeanloz (1960) for a discussion of the nomenclature of the gly- and Person (1970) for cytochrome oxidase; and Person and Philpott
cosaminoglycans or acid mucopolysaccharides, and Anderson (1963, 1969b) for the pattern of chondrification and its resemblance
(1976b), who surveyed the distribution and composition of glycosa- to lignification.
minoglycans and glycoproteins. 14. See Cowden (1967) and Person (1969, *1983a) for cranial, scleral
4. See P. J. Morris (1993) for the origin of extracellular matrix, and and epidermal cartilages; Person (1960), Person and Philpott (1963)
Exposito and Garrone (1990), Har-El and Tanzer (1993) and Garrone and Philpott and Person (1970) for glycosaminoglycan composition;
(*1998) for collagen appearing with sponges. See Hall (*2003a), and the chapters in Wight and Mecham (1987) for the biology of
Stone and Hall (2004, 2006) and Hall and Kerney (2012) for analyses proteoglycans.
of such issues as deep homology and the complex relationships 15. See Kinoshita et al. (1997) for the novel sequences, and see Habuchi
between homology at various levels of the biological hierarchy. et al. (1983) for the distinct glycopeptide.
5. M. B. Mathews (1968, 1975) and E. Adams (1978) review the compo- 16. See Hjerpe et al. (1983), Vynios and Tsiganos (*1990) for these
sition of invertebrate collagens, Kobayashi (1971) and Finerty (1981) studies. Two mannose-6-phosphate receptors are expressed in carti-
the homology of collagens, and Garrone (*1998) the evolution of lage during mouse development (Matzner et al., 1992).
metazoan collagens, whereas Cole and Hall (2004a,b) discuss the col- 17. See Ayad et al. (1994) and Knudson and Knudson (2001) for
lagens of invertebrate cartilages and the evolution of type II collagen. detailed reviews of aggrecan and its functions, and see Yang et al.
6. See Sage and Gray (*1980) and the analysis by G. M. Wright et al. (1998) for expression in cultured cells.
(1988). 18. See Sivakumar and Chandrakasan (1998) for C1 C3 collagens, and
7. For the latest overviews of invertebrate cartilages see Hall (*1978a), Rigo and Bairati (1998) for the rotary shadowing.
Cole and Hall (2004a,b, *2009) and Cole (*2011). 19. See Person and Mathews (1967) for structure and biochemistry, and
8. See Persons (1960) emphasis that invertebrate cartilages possess Cowden and Fitzharris (1975) for ultrastructure.
chitin but lack chondroitin sulphate. 20. See Stricker and Reed (1985, and references therein) and Reed and
9. See Person (1960, *1983a) and Person and Philpott (1963) for Cloney (1977) for these tissues and the proposals for and against
light and transmission microscope cytology/histology, Lash and them as cartilaginous.
Whitehouse (1960) and Katzman and Jeanloz (1969) for polyglucose 21. For descriptions of mineralised tissues in invertebrates, see Watabe
and polyfucose sulphates, and Lash (1959), Person et al. (1959) and (1965), Travis et al. (1967) and J. G. Carter (1990).
Person and Philpott (1963) for myoglobin. See Cole and Hall 22. See Libbin et al. (1976) and Rabinowitz et al. (1976) for lipid
(2004a,b, *2009) for overviews of gastropod cartilages, Ponder and and in vitro mineralisation of invertebrate cartilages; Irving
Lindberg (1997) for a phylogeny of gastropod molluscs (117 charac- (1973), Schuster et al. (1975) and Rogers (2000) for lipids and
ters, 40 taxa, mostly prosobranch), including the use of cartilage as mineralisation of bone and vertebrate cartilages; and Roy et al.
character, and see Guralnick and Smith (1999) and references (2004) for the influence of dietary phosphorus on bone
therein, for gastropod cartilages. mineralisation.
78 PART | II Origins and Types of Skeletal Tissues

23. See Eilberg et al. (1975), Libbin et al. (1976) and Hall (1972a) for (2003a) for Haikouella jianshanensis; and Mallatt and Chen (2003)
intracellular mineralisation of invertebrate chondrocytes, and Langille for these species and craniate origins. Haikouichthys ercaicunensis
and Hall (1988a, 1993a) and Janvier and Arsenault (2002) for minera- from the same Lower Cambrian formation in China, with a noto-
lisation of lamprey cartilage. chord, segmented muscles, a dorsal fin and cartilaginous hill bars
24. Genes that regulate lipid biosynthesis or transport can act dif- and olfactory and optic cartilages, was interpreted as a stem group
ferentially on cartilage and on lipogenesis. The Gonzo (Goz) mutant craniate (an early jawless vertebrate) by Shu et al. (2003b). See
zebrafish, which results from a mutation in the gene Site1 protease J. A. Long et al. (2010) for the proposal that the evolution of carti-
that regulates key enzymes for lipid synthesis and transport, acts laginous optic capsules preceded the origin of jaws.
independently to produce cartilage and lipid defects (Scholmbs 27. See Ekanayake and Hall (1991) for the perinotochordal sheath, and
et al., 2003). For genes and mutations in zebrafish see the website see Hall (*1998a) and Cole and Hall (2004a,b, *2009) for noto-
http://www.Zfin.org. chord cartilage affinities. My thanks to Eckhard Witten for com-
25. See Romer (1942), Hall (*1975a, 1999a), Gans and Northcutt ments on this portion of the text.
(1983), Northcutt and Gans (1983), Hall and Horstadius (1988), 28. See Frederickson and Low (1971), Strudel (1971) and Corsin (1974)
Smith and Hall (*1990, *1993), Janvier (1996), Northcutt (1996), for discussions of studies on the notochordal extracellular matrix.
Cole and Hall (2004a,b, *2009), Cole (2011) and Hall and Gillis 29. See R. L. Carroll et al. (1999) and Hall (2003b) for overviews of
(*2013) for theories of the origin of cartilage. evolution and development, respectively, and see R. L. Carroll
26. See Rahr (1981) and de Beer (1937) for gill and oral hood carti- (1997), Hall (*1998a) and Hall and Olson (2003) for books that inte-
lages; J.-Y. Chen et al. (1999) for Haikouella lanceolata, Shu et al. grate both approaches.