Vertebral Development and Ossificationin the Siberian Sturgeon Acipenser Baerii, with New Insights on Bone Histology and Ultrastructure of Vertebral Elements and Scutes AMANDINE LEPR EVO
Trang 1Vertebral Development and Ossification
in the Siberian Sturgeon (Acipenser Baerii), with New Insights on Bone Histology and Ultrastructure
of Vertebral Elements and Scutes
AMANDINE LEPR EVOST,1THIERRY AZA€IS,2 MICHAEL TRICHET,3ANDJEAN-YVES SIRE1*
1Sorbonne Universites, UPMC Univ Paris 06, CNRS, Institut de Biologie Paris-Seine,
Department Evolution Paris Seine, Equipe ‘Evolution et Developpement du Squelette,
Paris, France
2
Sorbonne Universites, UPMC Univ Paris 06, CNRS, Colle`ge de France, Laboratoire de
Chimie de la Matie`re Condensee de Paris (LCMCP), Paris, France
3Sorbonne Universites, UPMC Univ Paris 06, Institut de Biologie Paris-Seine, CNRS,
Service de Microscopie Electronique, Paris, France
ABSTRACT
In order to improve our knowledge on the vertebral development, struc-ture and mineralization in Acipenseriformes, we undertook a study in a growth series of reared Siberian sturgeons (Acipenser baerii) using in toto clear and stain specimens, histological and ultrastructural observations, X-ray micro-tomography, and solid state NMR analyses Scutes were also studied to compare the tissue structure and mineralization of endoskeletal and dermal skeletal elements This study completes and clarifies previous investigations on vertebral development and architecture in sturgeons, and brings original data on the structure of (i) the perichondral bone that is pro-gressively deposited around the vertebral elements during ontogeny, (ii) the typical cartilage composing these elements, and (iii) the scutes In addition
we provide data on the mineralization process, on the nature of the bone mineral phase, and on the growth dynamics of the vertebral elements Anat Rec, 00:000–000, 2016 V C2016 Wiley Periodicals, Inc
Key words: Acipenseriformes; Siberian sturgeon; vertebral
skeleton; development; mineralization; ossifica-tion; 3D microtomography
INTRODUCTION While reviewing current knowledge on the vertebral
column in Acipenseriformes prior to study vertebral
defor-mities affecting reared sturgeons in France, it appeared
that knowledge on the vertebral architecture,
develop-ment and mineralization in Acipenseriformes was limited
and incomplete (Leprevost and Sire, 2014) It was
howev-er for long known that (i) in sturgeons the endoskeleton is
mostly cartilaginous and combined with the presence of
an extensive dermal skeleton (five scute rows along the
body), (ii) the vertebrae are composed of several elements
organized around a persistent and unconstricted noto-chord, and (iii) mineralization only concerns a few of these elements and occurs late in ontogeny (e.g., Kolliker, 1860;
*Correspondence to: Jean-Yves Sire; E-mail: jean-yves.sire@ upmc.fr
Received 17 May 2016; Accepted 14 October 2016.
DOI 10.1002/ar.23515 Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
THE ANATOMICAL RECORD 00:00–00 (2016)
Trang 2Goette, 1878; Hasse, 1893; Klaatsch, 1893; Gadow and
Abbott, 1895; Goodrich, 1909)
In sturgeons, the axial skeleton is characterized by
the lack of vertebral centra, which means that the
noto-chord is the only support for the cartilaginous elements
composing the vertebrae (Arratia et al., 2001; Hilton
et al., 2011; Zhang et al., 2012) In Acipenseriformes,
each vertebra is composed of four elements organized
around the notochord, the basidorsal and interdorsal,
and the basiventral and interventral, which form the
dorsal and the ventral arcualia, respectively The
basi-dorsals carry short, fork-shaped neural spines that
enclose the longitudinal ligament running along the
ver-tebral axis (Gadow and Abbott, 1895; Arratia et al.,
2001) In addition to paired ribs, the abdominal
verte-brae also support a median supraneural In
Acipenseri-dae, the supraneural series is interrupted beneath the
dorsal fin (Findeis, 1997; Hilton et al., 2011)
Studying Acipenser brevirostrum Lesueur, 1818,
Hil-ton et al (2011) divided the vertebral column into two
regions, the abdominal region composed of vertebrae
supporting ribs and the caudal region, in which ribs are
absent Within the caudal region, these authors
distin-guished the preural region, located between the anterior
base of the dorsal fin and the base of the caudal fin, and
the ural region that includes the caudal fin and its
sup-port The border between the preural and the ural
regions consists of the vertebra supporting the
parhypu-ral The boundary between the abdominal and caudal
regions is not well defined, especially in young
speci-mens, in which ribs have not developed yet
In adult Acipenseriformes, all elements composing the
vertebrae, with the exception of interdorsals and
inter-ventrals, are supposed to be able to mineralize Several
authors named this mineralization “perichondral
ossification” (Grande and Bemis, 1991; Arratia et al.,
2001; Hilton et al., 2011) However, to our best
knowl-edge, the presence of perichondral bone around
cartilagi-nous elements or of calcified cartilage was not supported
in the literature by accurate histological data A single
study by Meunier and Herbin (2014) displays a
trans-verse section of a “neurapophysis” of Acipenser sp (from
a material dated 1876), showing the presence of primary
bone More precisely, this section shows “a crown of
peri-osteal bone tissue surrounding a large, circular surface
resembling amorphous cartilage” This bony tissue
tains osteocytes and presents Sharpey’s fibres and
con-centric growth ridges
The few descriptions available on the axial skeleton
often led to confusions between neural spines and
supra-neurals, and to controversy concerning their identification
For example, while Arratia et al (2001) make a distinction
between these two elements, Hilton et al (2011) do not
mention the presence of neural spines and include them in
the term ‘basidorsals’, as neural spines form by dorsal
growth of the basidorsals Also, Zhang et al (2012) wrongly
termed the supraneural “neural spine”
These few examples illustrate how limited are the
data available on the axial skeleton mineralization in
Acipenseriformes and support the need of new
descrip-tions Therefore, we undertook a series of experiments
using growth series of the Siberian sturgeon (Acipenser
baerii Linnaeus, 1758), the most frequently reared
stur-geon in France, chosen as a model species to fill these
gaps
We used (i) cleared and stained specimens and histolog-ical sections in a growth series to document the develop-ment of the vertebral axis in sturgeon, (ii) X-ray micro-tomography and mineralization rate measurements to localize and quantify the mineralization, (iii) histological and ultrastructural observations in adult specimens to describe the morphology of vertebral elements, and (iv) solid state nuclear magnetic resonance (NMR)
spectrosco-py to identify the nature of the mineral phase To complete these observations, the scutes were also studied in order
to compare the mineralized tissue of these dermal skele-ton elements to the endoskeletal elements composing the vertebrae
MATERIALS AND METHODS Biological Material
Sturgeons were randomly sampled in several fish farms of the company Sturgeon SCEA (Saint-Seurin-sur-l’Isle, Soumeras, Colombiers, Saint-Fort-sur-Gironde, France) between 2013 and 2015 Particular attention was paid to retain normally shaped specimens only, i.e., that did not show external deformities
Five specimens aged 1, 2, 3, 5, and 24 years (57, 70,
80, 88, and 148 cm total length, TL, respectively) and six specimens aged 7 years (108 cm TL in average) were used for X-ray micro-tomography The specimens aged 2,
3, 5, 7, and 24 years were also used for mineral content (MC or ash fraction) measurements, in addition to one specimen aged 20 years (144 cm TL)
A growth series from 9 to 43 days posthatching (dph) was sampled every 2 days (two specimens for each sam-ple, from 1.7 to 9.5 cm TL) for in toto clear and stain and for histological observations on paraffin sections Two specimens aged 7 years (105 and 109 cm TL) were used for histological and ultrastructural observa-tions of neural spines, supraneurals, and lateral scutes
on Epon sections (1 to 2 lm-thick and ultrathin sections) and the 105 cm individual was also used for solid state NMR spectroscopy analysis of the mineral phases The specimens aged 7 years and more were collected after being sacrificed for caviar production in the
laborato-ry of the company Sturgeon SCEA (Saint-Genis-de-Sain-tonge, France), stored at 218 8C and sent to the laboratory where they were dissected for X-ray microtomography and MC measurements Younger specimens were lethally anesthetized in MS222, then either immediately dissected and immersed in fixative (histological analyses), or immersed in PBS (solid state NMR spectroscopy) The experiments conformed to the directives of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes (Direc-tive 2010/63/EU) and the French Rural Code (Article R214-87 to R214-137, Decree no 2013-118 of 1st February 2013) As a food producer the company received the agree-ment n8FR 17.331.003 CE
Dissections of 7-year-old specimens were performed according to the following protocol: once defrozen, the vertebral column of each specimen was roughly
dissect-ed, the head was cut between the first and second dorsal scutes, and the caudal fin was cut at its basis, anterior
to the dorsal fulcrum (Fig 1) Vertebral columns were then immersed in a water bath at 65–70 8C for 7–10 min Remaining soft tissues were removed carefully and
2 LEPR EVOST ET AL.
Trang 3the notochord was separated from surrounding vertebral
elements
Vertebrae were counted from the border between the
first and second dorsal scutes, at the point where the
head was cut (Fig 1)
In Toto Cleared and Stained Specimens
Entire specimens were fixed immediately after death
in a mixture of 4% paraformaldehyde (PFA) in PBS (1X)
for 24 h at room temperature They were then rinsed in
PBS and stored in ethanol 70% We used the cleared and
double-stained method described by Hanken and
Was-sersug (1981) Briefly, after fixation, specimens were
eviscerated and the eyes removed They were washed in
distilled water several times for at least 2 days, and
car-tilage stained with alcian blue Then the specimens were
dehydrated in absolute ethanol, rehydrated through a
graded series of ethanol, and macerated in a mixture of
a saturated solution of sodium borate and a solution of
0.5% potassium hydroxide (used as a clearing agent
instead of trypsin) This step can last from a few days to
a few months according to specimen size Bone was then
stained with alizarin red and specimens were finally
transferred into glycerol via a graded series of glycerol
in a 0.5% potassium hydroxide solution A few crystals
of thymol were added to glycerol for storage
Photo-graphs were taken with a SZ X12 Olympus binocular
magnifier equipped with a Spotflex camera and Spot
software (Media Cybernetics, Bethesda, MD)
Histological Analyses on Paraffin Sections
Until 40 dph the specimens were not dissected before
fixation; in older specimens, the head and the caudal fin
were removed These biological materials were fixed
using the same protocol as described for the cleared and
double stained method They were gently decalcified in
acetic acid (10%) for one month, the solution being
changed every week Samples were then dehydrated
through a graded series of ethanol, shortly immersed in
toluene and embedded in Paraplast (Sigma-Aldrich,
France) The 10 mm-thick sections were obtained in the
abdominal and caudal regions with a Leica RM2245
microtome and deposited on Superfrost slides (Fisher
Scientific, France) The sections were dewaxed in
tolu-ene, rehydrated through a decreasing series of ethanol,
stained with toluidine blue and coverslipped with EukittV R mounting medium (Sigma-Aldrich, France) Photographs were taken with an Olympus BX61 micro-scope equipped with a QImaging camera and Image Pro Plus software (Media Cybernetics, Bethesda, MD)
X-Ray Microtomography and 3D Modelization Three segments including two vertebrae were collected (i) in the abdominal region around vertebra 15, (ii) in the caudal region around vertebra 30, and (iii) between the end of the dorsal fin and the caudal fin The samples were dehydrated in a graded series of ethanol and stored in butanol for at least 2 days Butanol improves the contrast
of cartilaginous elements and of the notochord on X-ray microtomography images The vertebrae were placed in plastic tubes and wedged with polystyrene pieces, and the atmosphere inside the tubes was saturated with butanol
in order to avoid desiccation Ribs could not be kept as they were often lost during skeletal preparation
X-ray microtomography images were obtained in the AST-RX platform of the Museum National d’Histoire Naturelle (Paris) with a microtomograph reference v|tome|x 240 L (GE Sensing and Inspection Technolo-gies Phoenix X|ray), equipped with a RX microfocus 240 kV/320 W tube 3D modelizations were performed using ImageJ and Avizo softwares Cartilage diameter and bone thickness (mm) were measured on supraneurals from 1 year onwards The virtual section orientation was re-adjusted with ImageJ in order to obtain frontal sections, and measurements were performed with Adobe Photoshop CS6
Mineralization Rate Measurements The vertebral axis located between the head and the posteriormost vertebra supporting a supraneural was divided into three parts and three vertebrae were col-lected in the central region of each part (Fig 1) Neural spines and supraneurals located above the neural canal were cut and separated from the rest of the vertebra The samples were dehydrated in a graded series of ethanol (70, 95, and 100%, 24 h each), and the lipids were removed in acetone (48 h) and in trichloroethylene (24 h), according to Deschamps et al (2009) Then, the samples were dried for 48 h at room temperature under
a laboratory hood, for 24 h at 37 8C and then weighed
Fig 1 Drawing of a sturgeon in lateral view including a photograph of the vertebral axis, modified after Lepr evost et al (subm.) A and B indicate the level where the head and the caudal fin were cut,
respec-tively The dashed line delimits the abdominal and caudal regions The arrow points to the end of the
supraneural series The asterisks point to the regions sampled for X-ray micro-tomography; the numbers
1, 2, 3 correspond to the regions sampled for mineralization rate measurements.
VERTEBRAL OSSIFICATION IN SIBERIAN STURGEON 3
Trang 4(Wdry) at the nearest mg They were incinerated for 8 h
at 800 8C in a muffle furnace (reference LE 14/11,
Naber-therm), the ashes weighed (Wash) at the nearest mg, and
mineral content (ash fraction) (MC, %) was calculated
according to the formula: MC 5 (Wash/Wdry) 3100
Control was made on muscle samples in order to
eval-uate MC in an unmineralized tissue The MC value of
muscles being 1.80% in average, samples having MC
1.80% were considered unmineralized
Histological and Ultrastructural Observations
Samples of neural spines, supraneurals, and scutes
were fixed in a mixture of PFA 1.5% and glutaraldehyde
1.5% in PBS 13, overnight, at room temperature
Tis-sues were then decalcified for one month in the same
fix-ative, to which EDTA 4% was added The samples were
postfixed in 1% osmium tetroxide for 2 h, rinsed in PBS,
dehydrated through a graded series of ethanol then
immersed in propylene oxide prior embedding in Epon
812 (EMS); 2 mm thick, transverse sections were
ob-tained using a Reichert OMU-3 ultramicrotome,
de-posited on a glass slide, stained with toluidine blue,
mounted, and photographed
These sections also enabled to select the region to be
studied at the ultrastructural level using transmission
electron microscopy (TEM) Ultrathin, 80 to 90 nm-thick
sections were cut with a Leica Ultracut ultramicrotome
and collected on 200 Mesh copper grids (Agar) Sections
were stained with 2.5% aqueous uranyl acetate (Prolabo)
and 1% lead citrate (Agar) TEM observations were
car-ried out at 80 kV in a Zeiss 912 Omega, equipped with
side-mounted 2k 3 2k Veleta (Olympus) CCD camera
controlled with iTEM (Olympus) software
Solid State NMR Spectroscopy
Samples of neural spines and scutes were dissected
(about 5 mm long 3 2 mm wide) after slaughter, then
stored in PBS 13 Solid state NMR analyses were
rapid-ly performed to avoid tissue degradation and
dehydra-tion 1H and 31P solid state NMR experiments were
performed in an Avance 300 Bruker spectrometer
oper-ating at mL(1H) 5 300.13 MHz and mL(31P) 5 121.5 MHz
Intact samples were placed in-between two Teflon
spacers into a 4 mm zirconia rotor and analyzed under
magic angle spinning (MAS) at mMAS5 8 kHz 1H and
31
P chemical shift was referenced (d 5 0 ppm) to TMS
and 85 wt.% aqueous H3PO4, respectively Direct
acqui-sition31P MAS experiments were performed with recycle
delay RD 5 30 s Two dimensional 1H–31P CP MAS
experiments were recorded through the HetCor
(Hetero-nuclear Correlation) sequence 1H–31P HetCor
para-meters were as follow: RD 5 3.5 s, CT 5 1 ms, 400
transients for each 128 t1 increments were acquired
High power proton decoupling was applied during
acqui-sition (SPINAL-64, mRF(1H) 5 60 kHz) for each 31P
experiments
RESULTS Development of the Vertebral Elements
During development, cartilage anlages initiate above
and below the notochord They will give rise to the
basi-dorsals and basiventrals, respectively (Fig 2A) Then,
the two series extend in opposite directions: the basidor-sals develop anteroposteriorly and the basiventrals develop postero-anteriorly (Fig 2A,B) At 25 dph each vertebra possesses paired basidorsals and paired basi-ventrals, while neural spines start to develop anteropos-teriorly through dorsal extension of the basidorsals (Fig 2C) At 31 dph, the interdorsals, interventrals, and ribs have initiated and they develop in the same directions
as basidorsals and basiventrals (Fig 2D) Supraneurals have also started to develop anteroposteriorly above the neural spines They form independently from the other vertebral elements (Fig 2D) then extend dorsally in an oblique direction (Fig 2E,F)
Vertebrae display different morphologies along the vertebral column (Fig 2D–F) Abdominal vertebrae are tall and easily recognizable as they support ribs, neural spines, and supraneurals The supraneural series is interrupted beneath the dorsal fin In the caudal region, ribs are absent, and neural spines become shorter, so that the caudal vertebrae look rounder
In toto alizarin red staining did not enable to visualize accurately the onset of mineralization of the vertebral elements, because from 10 months onward the muscles could not be cleared enough to see the skeleton It is the reason why we followed the mineralization using X-ray microtomography
Localization of the Mineralization
in the Vertebral Axis Mineralization of the vertebral axis in A baerii is lim-ited to a few vertebral elements that become perichon-drally mineralized at a relatively late ontogenetic stage Supraneurals are the first elements being mineralized at the age of 1 year (Fig 3A), then neural spines start to mineralize around 2 years (Fig 3B) The mineralized matrix completely surrounds the neural spines in the abdominal region of the specimen aged 3 years (Fig 3C) Mineralization of neural spines and supraneurals follows the growth of these elements, then extends to basidor-sals and parapophyses in older specimens (Fig 3D–F) The mineralization is regionalized along the vertebral axis and progresses anteroposteriorly during develop-ment In our growth series, mineralization of caudal ver-tebrae, both anterior and posterior to the dorsal fin, was only detected in the specimen aged 24 years (Fig 3F–H) The anteroposterior progression of the mineralization along the vertebral column was confirmed when measur-ing mineral content (MC) of neural spines and supra-neurals (Fig 4) In all specimen studied, MC decreased anteroposteriorly and in a given region of the vertebral column, MC increased with age For example, in the sampling zone 1, between years 2 and 24 MC increased from 14.2 to 23.4% in these two elements, while in sam-pling zone 3, it increased from 3 to 17.5%
In order to determine the growth process of these ver-tebral elements, we measured the cartilage diameter and bone thickness in different regions of the supraneu-ral on virtual sections obtained with X-ray microtomog-raphy (Table 1) In our growth series, the diameter of the cartilage in the medial region of this element was almost constant during ontogeny (except in the oldest specimen), whereas it increased in distal and proximal regions Bone thickness was always higher in the medial region, and increased in all regions with ageing It
4 LEPR EVOST ET AL.
Trang 5Fig 2 Development of the vertebral elements in a growth series of
Acipenser baerii Left columns: In toto cleared and stained specimens;
cartilage in blue, mineralized tissues in red Only the scutes are
miner-alized in this series (dorsal row first, then lateral and ventral rows).
Right columns: Transverse, paraffin sections through the abdominal
(A2, B2, C2, D3, E3, F3) and caudal regions (C3, D4, E4, F4) The
lev-els of section are shown on A1 Specimens of same age as on the left
both 2.0 cm TL; C: 25 dph, 2.6 and 2.8 cm TL; D: 31 dph, 5.6 and 5.0 cm TL; E: 37 dph, 7.4 and 6.5 cm TL; F: 43 dph, 8.1 and 9.5 cm
TL bd: basidorsal, bv: basiventral, dsc: dorsal scute, id: interdorsal, iv: interventral, lsc: lateral scute, nc: neural canal, no: notochord, ns: neural spine, p: parapophysis, r: rib, sn: supraneural, vsc: ventral scute Asterisks: posteriormost vertebra supporting a supraneural Scale bars: A1, B1, C1, D1–D2, E1–E2, F1–F2 5 1 mm; A2–A3, B2–B3, VERTEBRAL OSSIFICATION IN SIBERIAN STURGEON 5
Trang 6increases of about 22 mm per year in the proximal region, of 51 mm per year in the medial region, and of 45
mm per year in the distal region, between years 1 and
24 These findings indicate that the supraneurals extend
in length by means of cartilage deposition at both extremities, allowing also extension of the diameter of the cartilage matrix Once bone is deposited around the cartilage, the latter can no longer extend in diameter and supraneural growth in diameter is then ensured by bone apposition as shown in the medial region (Fig 3I)
Histological Analyses of Vertebral Elements and Comparison with Scutes
In order to compare the microstructure and organiza-tion of the endoskeleton (neural spines and supraneu-rals) with those of an element of the dermal skeleton, the same histological analyses were performed on bone tissues of vertebral elements and of scutes
Histological observations of the vertebral elements using light microscopy and TEM revealed that the bone matrix directly lines the cartilage, which typologically corresponds to perichondral bone This matrix is entirely composed of randomly distributed bundles of collagen fibers, a feature that characterizes fibrous
(woven-Fig 3 A–H: 3D modelization (lateral view) of two vertebrae in a
growth series of Acipenser baerii The mineralized vertebral elements (in
red) were revealed using X-ray microtomography Anterior to the left.
Vertebra drawings are modified from Gurtovoy (1976) I: Growth
dynam-ic scheme in a vertdynam-ical section of a supraneural (red: bone; blue:
carti-lage) The numbers 1, 2, 3 indicate the region where the measurements
of table I were taken A: 1-year-old (y.o.), 57 cm total length (TL); B: 2
y.o., 70 cm; C: 3 y.o., 80 cm TL; D: 5 y.o., 88 cm TL; E: 7 y.o., 108 cm TL; F, G, H: 24 y.o., 148 cm TL A–F: abdominal vertebrae; G: caudal ver-tebrae of the region where the supraneural series is interrupted (around vertebra n830); H: caudal vertebrae between the posterior base of the dorsal fin and the caudal fin bd: basidorsal, bv: basiventral, l l: longitudi-nal ligament, n c: neural calongitudi-nal, no: notochord, n s: neural spine, p: para-pophysis, r: rib, sn: supraneural Scale bars 5 2 cm.
Fig 4 Anteroposterior distribution of neural spine and supraneural
mineral content (MC %) in a growth series of Acipenser baerii The
numbers 1, 2, 3 correspond to the sampled regions of the vertebral
axis defined in Fig 1.
6 LEPR EVOST ET AL.
Trang 7fibered) bone (Fig 5A–D) A few osteocytes are located
within the bone matrix, but no vascular canals were
iden-tified Taken altogether these findings allow to define the
mineralized matrix of supraneurals as a perichondral,
avascular, cellular woven-fibered bone Supraneurals and
neural spines (not shown) display the same features The
organic matrix of the scutes is also cellular and similarly
organized as in the vertebral elements (Fig 5E–H)
How-ever, the bone matrix of the scutes is formed in absence of
a cartilage anlage and is vascularized
In both, vertebral elements and scutes, an irregular
layer of scattered osteoblasts lines the bone surface,
roughly delimiting the interface between the
unmineral-ized bone matrix, osteoid, and the surrounding
mesen-chyme, which is largely composed of layers of oriented
bundles of collagen fibrils (Fig 5B,F) The osteoblasts
are elongated and the nucleus occupies a large part of
their volume The cytoplasm does not house a large
number of rough endoplasmic reticulum cisternae and of
Golgi apparatus that are generally associated to an
active protein synthesis In contrast, the osteoblasts
exhibit a large number of cytoplasmic extensions facing
the forming bone matrix and surrounding patches or
col-lagen bundles (Fig 5B,F,G) These features indicate that
these bone cells are rather modeling the pre-existing
bundles of collagen fibrils of the mesenchyme than
syn-thesizing new collagen matrix The modeled collagen
bundles progressively organize into a woven-fibered
bone matrix, a process which characterizes metaplastic
ossification Some cells are entrapped into this forming
bone matrix and become osteocytes (Fig 5C,D,H) The
osteoid tissue resulting from this process is first
unmin-eralized, then is mineralized As samples were
decalci-fied, the mineral phase is not visible on these sections,
but the region where the collagen matrix was
mineral-ized is charactermineral-ized by the electron dense, thin
extrafi-brilar matrix, probably deposited by the osteoblasts prior
to mineral deposition (Fig 5C) The only structural
dif-ference between the vertebral elements and scutes is
that the latter are anchored to the dermis by large
colla-gen bundles (Fig 5E,F)
In supraneurals and neural spines the interface
between the perichondral bone and the cartilage matrix
is devoid of cells and no remnants of pre-existing cells
were present The boundary between the two tissues is
rather sharp (Fig 5D) These features indicate that (i)
the cartilage was deposited first by chondroblasts located
in the perichondrium, (ii) these cells stopped depositing
cartilage, (iv) either the chondroblasts disappeared from
the cartilage surface or they differentiate into osteo-blasts (perichondrium to periosteum transition), and (v) the latter start to form bone tissue at the cartilage sur-face using pre-existing bundles of collagen fibers of the surrounding mesenchyme In addition to the measure-ments presented in Table 1, these observations lead to our interpretation of dynamic growth schematically pre-sented in Fig 3I
In the two vertebral elements studied the cartilage matrix has an unexpected appearance: the matrix is dense compared to various cartilage types and the em-bedded chondrocytes are scarce and not hypertrophied, looking like rounded osteocytes (Fig 5A,D) In addition, the number of chondrocytes appears higher in young that in adult specimens (Fig 2)
Nature of the Mineral Phase in Vertebral Elements and Scutes
To complete our comparison between endoskeletal and dermal elements of the sturgeon skeleton, we analyzed the mineral phase from neural spines and scutes using
31
P solid state NMR The samples were processed as rapidly as possible after being dissected from the animal
in order to preserve the native hydration of the samples Indeed, dehydration could lead to the precipitation of unwanted calcium phosphate phases from ionic precur-sors present in the biological medium 31P quantitative MAS (direct acquisition) spectra revealed that the min-eral phase in these samples is similar to bone minmin-eral from mature ewe bone (2-year-old) used as control, which is representative of mammalian bone hydroxyapa-tite (Fig 6A) Indeed, for neural spines and scutes, the 1D 31P MAS spectra exhibit one single broad resonance centered around 2.9 ppm that is similar in terms of posi-tion and line width to the31P resonance of bone apatite from ewe bone
The 2D {1H}31P HetCor spectrum of scute (Fig 6B) is similar to ewe bone and displays two different spectral regions that correspond to two different mineral domains
as already demonstrated for bone mineral (Wang et al., 2013): (i) the apatitic core of the crystals where ortho-phosphate ions correlate with hydroxyl ions (d(1H) 5 0 ppm), and (ii) the hydrated disordered surface, for which HPO224 ions correlate with water molecules (d(1H) 5 4.8 ppm) Despite our efforts, a partial dehydration of the sample occurred in the rotor and the 2D {1H}31P HetCor spectrum of scute reveals the spectral signature of pro-tons from HPO224 surface ions (d(1H) 5 6–16 ppm) 31P resonances corresponding to phosphate ions from the
TABLE 1 Measurements (mm) of the cartilage diameter and bone thickness in different regions of the
supra-neural in a growth series of Acipenser baerii
Location of measurements (1, 2, and 3) are shown in Fig 3
VERTEBRAL OSSIFICATION IN SIBERIAN STURGEON 7
Trang 88 LEPR EVOST ET AL.
Trang 9two domains possess the same position (d(31P) 5 2.9
ppm), but differ from their line width: 31P resonance
from the surface domain is twice larger than the one
from the apatitic core The similarity of the spectral
fin-gerprints between scute and ewe bone of similar
hydra-tion degree (Fig 6C) emphasizes that the mineral phase
in scute is similar to bone mineral and possesses the
same core/layer organization However, the proportion of
the apatitic domain seems to be lower for scute Indeed,
the correlation resonance corresponding to the apatitic
phase is less intense for scute compared to ewe bone
This result might be related to a lower degree of
maturi-ty/crystalinity of the apatite crystals in scute (Wang
et al 2014)
DISCUSSION Previous Descriptions are Confirmed, but Largely Completed and Clarified
Our study largely completes and clarifies previous fragmented observations available on the vertebral col-umn of sturgeons and paddlefishes (Gadow and Abbott, 1895; Grande and Bemis, 1991; Bemis et al., 1997; Fin-deis, 1997; Arratia et al., 2001; Hilton et al., 2011; Zhang
et al., 2012) In the past, most data were obtained in various species and in a few individuals, which did not allow a clear understanding of the growth and minerali-zation of the axial skeleton By using growth series in a single species, the Siberian sturgeon Acipenser baerii,
Fig 5 Transverse, epon sections of a supraneural (A–D) and of a
lat-eral scute (E–H) in Acipenser baerii (7 y.o., 105 cm TL) A, E: 1 to 2
lm-thick sections, toluidine blue staining B–H: TEM micrographs of 80 to
90 lm-thick sections B: Interface between the mesenchyme and the
osteoid tissue; C: Transition area between osteoid and maturing bone;
dermis and the scute; G: Detail of osteoblasts at the scute surface Cytoplasmic extensions encircling collagen fibers; H: Detail of the maturing bone tissue of a scute ab: anchoring bundle, b: bone, c: carti-lage, ch: chordocyte, ep: epidermis, ld: loose dermis, m: mesenchyme, osb: osteoblast, osc: osteocyte, ost: osteoid, vc: vascular canal Scale
Fig 6 Solid state NMR analyses of mineralized elements of a
speci-men of Acipenser baerii aged 7 years (108 cm TL) A: Direct
acquisi-tion 31 P quantitative MAS spectra of neural spines and scutes
compared to mature ewe bone (control) Asterisk: phosphate ions
from the phosphate buffer solution, in which samples were stored B, C: 2D {1H}31P HetCor spectra of scute and ewe bone highlighting the two different domains present in the apatite crystals: (i) the apatitic region and (ii) the hydrated, amorphous calcium phosphate surface VERTEBRAL OSSIFICATION IN SIBERIAN STURGEON 9
Trang 10our study brings new and accurate information to our
knowledge
As previously observed by Hilton et al (2011) in A
brevirostrum, our observations in A baerii confirm that
the elements of the vertebral axis develop in two
oppo-site directions The basidorsals develop anteroposteriorly
and the basiventrals develop postero-anteriorly, which is
an original process among vertebrates The paired
neu-ral spines develop by dorsal extension of basidorsals as
in all actinopterygians and enclose the longitudinal
liga-ment, as noticed by Arratia et al (2001) The
supraneu-rals develop independently from the other cartilaginous
elements and articulate with the neural spines Neural
spines and supraneurals are therefore distinct elements
and their identification should not be controversial, even
though some fusions between adjacent cartilaginous
ele-ments can occur, especially in the caudal region (Hilton
et al., 2011)
Our study brings new information concerning the
mineralization of the vertebral axis in A baerii In
Aci-penseriformes, the mineralization of the vertebral
col-umn was known to be restricted to a few vertebral
elements (Gadow and Abbott, 1895; Grande and Bemis,
1991; Bemis et al., 1997; Findeis, 1997; Arratia et al.,
2001; Hilton et al., 2011) Here, for the first time we
described the timing of mineralization in a sturgeon
The supraneurals are the first elements to mineralize,
then neural spines start to mineralize on their outer
lat-eral sides, before minlat-eralization extends to their inner
sides besides the longitudinal ligament Basidorsals and
parapophyses are the last elements to mineralize but
late in ontogeny, as seen in a 24-year-old specimen
Basi-ventral mineralization was not detected in our oldest
specimen, but according to Hilton et al (2011), it can
occur late in ontogeny in A brevirostrum These findings
confirm that only a few elements mineralize in this
Aci-penseriformes, and also indicate that the mineralization
process starts late then progresses slowly during
ontogeny
Mineralization of the vertebral column starts in the
abdominal region and extends anteroposteriorly during
ontogeny, a finding that is in accordance with the
previ-ously described timing of mineralization of each
verte-bral element This process results in an anteroposterior
regionalization of mineralization in all specimens In
addition to morphological differences that exist
bet-ween abdominal and caudal vertebrae, this feature
reinforces the differentiation between the abdominal
and the caudal region of the vertebral column in
Acipenseriformes
Previous authors (Grande and Bemis, 1991; Arratia
et al., 2001; Hilton et al., 2011) used the term
“ossification” to describe the mineralization observed in
the vertebral column of various sturgeons However,
in the literature there was no histological
demons-tration of the nature of the mineralized tissue
sur-rounding the vertebral elements, except one image of a
“neurapophysis” of Acipenser sp dated 1876 (Meunier
and Herbin, 2014) Here, we confirmed that the
peri-chondral, mineralized tissue deposited around the
supra-neurals and neural spines consists of bone, and for the
first time, we provided TEM ultrastructural data of this
tissue, with comparison to the bone tissue of a scute of
the dermal skeleton
Vertebral Elements and Scutes Possess
a Similar, but Unexpected Bone Tissue
In all sturgeon species, the reduced mineralization of the endoskeleton, mostly in the form of “perichondral” bone, is combined with the presence of an extensive der-mal skeleton consisting of five rows of scutes extending along the trunk (Bemis et al., 1997; Findeis, 1997) Vari-ous types of bone tissues often occur in a same species, particularly in osteichthyans, which are characterized
by the possession of a wide range of skeletal tissues (Meunier and Huysseune, 1991) Our study in A baerii
of the morphology and ultrastructure of the perichondral bone of vertebral elements and the dermal bone compos-ing the scutes revealed that in both bone tissue is
entire-ly woven-fibered bone
According to Meunier and Huysseune (1991), all liv-ing, non-teleost fishes, including Acipenseriformes, pos-sess cellular bone, a condition that was confirmed by our study Scutes have been the subject of a few studies, most of them describing their development and morphol-ogy, rather than their histological features (Sewertzoff, 1926; Jollie, 1980) Nevertheless, Meunier et al (1978) carried out a histological and micro-radiographic study
of various “scales” in several non-teleost extant actino-pterygians In A sturio they found that the scutes have
a homogenous structure, composed of cellular bone with parallel fibers, not much vascularized On their deep surface, large resorption cavities were sometimes observed, on the surface of which secondary bone with woven fibers had deposited Periodic differences of min-eralization were visible on X-ray images, and the orna-mentations appeared more mineralized Here, in A baerii our observations revealed that the structure of the bone matrix was not parallel-fibered, but entirely woven-fibered (fibrous)
The bone tissues of neural spines, supraneurals, and scutes have not only the same morphological aspect but also the same mineral phase, i.e., mainly composed of bone-like apatite Ultrastructurally, the formation of this bone tissue resembles that of a primordium of fibrous dermal bone, characterized by mesenchymal metaplasia First, osteoblasts appear to remodel pre-existing collagen fibers of the mesenchyme, then secrete non collagenous extracellular components that fill the interfibrillar spaces, then initiate mineralization Some osteoblasts can be trapped within the matrix and differentiate into osteocytes, while new osteoblasts can be recruited from undifferentiated pre-osteoblasts located in the surround-ing mesenchyme Dursurround-ing the formation of a typical der-mal bone in an actinopterygian, the initial woven-fibered osseous metaplasia is followed by the deposition of newly synthesized collagen fibrils from osteoblasts organized around the elements This results in the formation of parallel-fibred bone then of lamellar bone later in ontog-eny (Sire and Huysseune, 2003) In A baerii, the bone matrix either around the vertebral elements or in the scutes was woven-fibered type in its entire thickness, which strongly suggests that the same metaplastic pro-cess is conserved throughout ontogeny, a propro-cess that was never reported elsewhere to our knowledge
In all vertebrates, bone is composed of three basic components: cells [osteocytes, osteoblasts, bone-lining cells (inactive osteoblasts at the bone surface) and osteo-clasts], an organic matrix (predominant network of type
10 LEPR EVOST ET AL.