Vol 454 | 17 July 2008 | doi:10.1038/nature07020LETTERS
Control of segment number in vertebrate embryos
Céline Gomez1, Ertuğrul M. Özbudak1, Joshua Wunderlich1, Diana Baumann1, Julian Lewis2 & Olivier Pourquié1,3
The vertebrate body axis is subdivided into repeated segments,
best exemplified by the vertebrae that derive from embryonic
somites. The number of somites is precisely defined for any given
species but varies widely from one species to another. To determine the mechanism controlling somite number, we have compared somitogenesis in zebrafish, chicken, mouse and corn snake
embryos. Here we present evidence that in all of these species a
similar ‘clock-and-wavefront’1–3 mechanism operates to control
somitogenesis; in all of them, somitogenesis is brought to an end
through a process in which the presomitic mesoderm, having first
increased in size, gradually shrinks until it is exhausted, terminating somite formation. In snake embryos, however, the segmentation clock rate is much faster relative to developmental rate than in
other amniotes, leading to a greatly increased number of smallersized somites.
Vertebrate segments are formed during early embryogenesis, when
vertebrae precursors, called somites, bud off in a rhythmic fashion
from the anterior part of the presomitic mesoderm (PSM). The periodic formation of somites is proposed to be controlled by a molecular
oscillator—the segmentation clock—which drives the periodic
activation of the Notch, Wnt and fibroblast growth factor (FGF)
pathways in the PSM1,2,4. The periodic signal of the segmentation
clock is converted into a repetitive series of somites by a travelling
front of maturation—the wavefront or determination front—
formed by a Wnt/FGF signalling gradient that regresses caudally in
the PSM in concert with axis elongation5–8.
The number of somites, and hence of vertebrae, is highly variable
among vertebrate species9. For instance, frogs have ,10 vertebrae,
whereas humans have 33 and snakes can have more than 300. To
investigate the mechanisms controlling somite numbers in vertebrates, we compared somitogenesis in the corn snake (Pantherophis
guttatus; Fig. 1a, b), which makes a large number of somites (,315),
with that in three other vertebrate species that make far fewer: zebrafish (Danio rerio, 31), chicken (Gallus gallus, 55) and mouse (Mus
musculus, 65).
We examined the expression of the corn snake homologues of
genes involved in PSM patterning and somitogenesis (Fig. 2a–l).
The genes coding for fibroblast growth factor 8 (FGF8) (Fig. 2b)
and its targets sprouty 2 (SPRY2; Fig. 2c) and dual specificity phosphatase 6 (DUSP6)4,10 (Fig. 2d), as well as WNT3A (Fig. 2e) and its
targets AXIN2 (ref. 7; Fig. 2f) and mesogenin 1 (MSGN1) (ref. 11;
Fig. 2g), ephrin receptor A4 (EPHA4; Fig. 2h), the retinoic acid
biosynthetic enzyme (RALDH2; Fig. 2i), paraxis (TCF15) (Fig. 2j),
UNCX4.1 and MYOD (Supplementary Fig. 1) were cloned and their
expression analysed by in situ hybridization. All of these genes (except
SPRY2) were expressed in domains comparable to those observed in
their fish or amniote counterparts4–7,12–19, supporting the existence of
a Wnt/FGF posterior gradient opposing an anterior retinoic acid
gradient in corn snake, as in other species.
We compared the dynamics of this gradient in snake with that in
the other species. As a readout for the posterior gradients, we used
MSGN1 expression, which is controlled by the Wnt/FGF gradient
3 vertebrae
a
226
vertebrae
222
vertebrae
296
vertebrae
3
cervical
219
thoracic
4
cloacal
70
caudal
b
118 somites
165 somites
235 somites
255 somites
Figure 1 | Vertebral formula and somitogenesis in the corn snake.
a, Alizarin staining of a corn snake showing 296 vertebrae, including 3
cervical, 219 thoracic, 4 cloacal (distinguishable by their forked
265 somites
280 somites
290 somites
315 somites
lymphapophyses) and 70 caudal. b, Time course of corn snake development
after egg laying (118-somite embryo on the far left) until the end of
somitogenesis (,315 somites).
1
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA. 2Vertebrate Development Laboratory, Cancer Research UK, London Research Institute, London WC2A
3PX, UK. 3Howard Hughes Medical Institute, Kansas City, Missouri 64110, USA.
335
©2008 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 454 | 17 July 2008
a
Somites
s1
Determination
front
Axis
elongation
b FGF8
c
SPRY2
d
DUSP6
e WNT3A
f
AXIN2
g
MSGN1
h EPHA4
i
RALDH2
j
TCF15
k
m
LFNG
LFNG
n
o
l
LFNG
p
Figure 2 | The corn snake determination front and segmentation clock.
a, Schematic drawing of a corn snake tail showing the position of the
determination front (dashed line) in the PSM relative to the newly formed
somite (s1). b–l, Whole-mount in situ hybridizations of 230-somite
(b–h, k, l) and 260-somite (i, j) corn snake embryo tails: FGF8 (b), SPRY2
(c), DUSP6 (d), WNT3A (e), AXIN2 (f), MSGN1 (g), EPHA4 (h), RALDH2
(i) and TCF15 (j). Two different phases of LFNG expression in 230-somite
corn snake embryos (k, l), two-day-old chicken embryos (m, n) and E9.5
mouse embryos (o, p). Lateral views in a–l, o and p. Dorsal views in m and
n. Anterior to the top.
and for which the sharp anterior boundary marks the position of the
mesoderm posterior 2 (MESP2) stripe20 at the determination front3
(Fig. 2a–g). We measured the regression speed of the MSGN1
anterior boundary during somitogenesis in all four species
(Fig. 3a–x) and found that it moves by one somite length during
one period of somite formation, independent of the somitogenesis
stage and the species (Fig. 4a and Supplementary Fig. 2). This validates an important prediction of the clock-and-wavefront model—
that somite size corresponds to the distance travelled by the wavefront during one oscillation period. We plotted the ratio of MSGN1
expression domain to PSM size as a function of stage for each species
(Fig. 4b). Notably, a similar ratio was observed throughout somitogenesis in all four species, suggesting that similar processes (scaled
proportionately) are occurring. Thus, the characteristics of the gradient system involved in PSM patterning seem to be conserved
between corn snake and the other species examined.
We examined the cyclic gene expression associated with the
amniote segmentation clock. No dynamic expression of SPRY2,
DUSP6 (ref. 4) or AXIN2 (ref. 7) was evident in the snake PSM
(Fig. 2c, d, f). However, Lunatic fringe (LFNG) exhibited an unexpected expression pattern that consisted of up to nine stripes of
variable size and spacing in the PSM (Fig. 2k, l)21,22. Thirty-nine snake
embryos were hybridized with LFNG and all showed a different
expression pattern (Fig. 2k, l and data not shown). These data support the existence of an oscillator driving cyclic gene expression in
snake embryos. The number of stripes of LFNG expression in corn
snake is, however, several times larger than in other vertebrate species
(Fig. 2k–p), suggesting that the segmentation clock might be regulated in a different way.
Assuming that one somite is formed during each clock oscillation
cycle, we can deduce the period of the clock oscillations by counting
somite numbers in embryos from the same clutch at various incubation times (Supplementary Methods). In the corn snake, the average
somite formation rate is one pair every 100 min, compared to rates of
one pair every 30, 90 and 120 min in zebrafish, chicken and mouse,
respectively. The somite formation rate was found to be one pair
every ,60 min in the house snake (Lamprophis fuliginosus) embryos,
which has very similar developmental characteristics and LFNG
expression pattern to the corn snake (Supplementary Methods and
Supplementary Fig. 3).
To appreciate the significance of these periods, we need to compare them to the general rate of development, which differs between
species. Comparison of the time required to reach conserved morphological landmarks (Supplementary Fig. 4) suggests that the
development rate is at least three times slower in corn snake than
in chicken. We also examined the lizard Aspidoscelis uniparens, which
has the same slow general development rate as the snake23
(Supplementary Methods). This species makes only ,90 somites
and has a much longer somite formation time (,4 h). Therefore,
relative to the development rate, the clock ticks much faster in snake
than in chicken or lizard embryos.
Analysing the relationship between somitogenesis rate and growth
rate of the PSM tissue confirms this hypothesis. According to the
clock-and-wavefront model, each somite consists of the cells emerging from the PSM in one clock cycle. This must equal the quantity of
new cells generated in the PSM by growth, at least when the PSM
maintains a steady size. Thus, the somite size as a fraction of the PSM
size directly reflects the duration of the clock cycle as a fraction of the
average PSM cell generation time (that is, average cell-cycle time;
Supplementary Box 1). In snake, this fraction is approximately
one-quarter of the value observed in other species (Fig. 4c). Thus,
the snake segmentation clock runs approximately four times faster
relative to the average PSM cell generation time than in the other
species.
We estimated the average cell generation time and the total number of PSM cell generations required to produce a complete set of
somites. For this, we took into account the way in which the PSM size
336
©2008 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 454 | 17 July 2008
changes over the course of somitogenesis (Figs 3a–x and 4d). In
zebrafish, the PSM length decreases from the beginning of somitogenesis; in amniote embryos, the PSM first increases and then
decreases in size until somitogenesis ends24 (Fig. 4d). From the
detailed measurements of PSM length and the size of the most
recently formed somite s1 as a function of developmental stage
(Fig. 4d, e), combined with a knowledge of the period of the segmentation clock and the total number of somites formed, we can
estimate the average cell-generation time and the total number of
PSM cell generations required to generate the full set of somites
(Supplementary Box 1). This calculation provides an average cell
generation time in the PSM that is much longer in corn snake
(,24.4 h) than in mouse (,8 h), chicken (,6.3 h) or zebrafish
(,5.5 h). Direct cell-cycle measurements, using 5-bromodeoxyuridine (BrdU) incorporation followed by flow cytometry, gave a cycle
time of ,34 h in corn snake embryonic tail and ,30 h in lizard,
compared to ,9 h in chicken (as measured using tritiated thymidine25; Supplementary Table 4, Fig. 4f and Supplementary Fig. 5),
confirming that the cell generation time is almost fourfold slower in
snake and lizard than in chicken. Remarkably, the calculated number
of cell generations required to generate the 315 somites in the snake
(,21 generations) is only slightly greater than for the 65 somites in
the mouse (,17 generations) or 55 somites in the chicken (,13
generations), although much larger than for the 31 somites in the
zebrafish (,2.8 generations). Therefore, the exceptionally large
number of somites in the snake, compared with that in other
amniotes, is not primarily the result of a large number of generations
Zebrafish
Corn snake
b
Zebrafish
Mouse
Chicken
c
d
u
Size (µm)
a
of PSM growth, but reflects a clock rate that is rapid in relation to the
cell-cycle rate in the elongating axis.
Finally, we investigated the basis for the unusually large number of
LFNG stripes observed in snake embryos. The number of stripes of
expression reflects the number of clock cycles by which the cells at the
anterior end of the PSM lag behind those in the posterior PSM. By
measuring the stripe spacing, one can deduce how the clock rate
changes with position in the PSM26. Using measurements of LFNG
stripe spacing in corn snake embryos, we obtained a graph depicting
the slowing down of gene oscillations in the PSM. The graphs for
snake and zebrafish are almost identical (Fig. 4g), suggesting that the
mechanism controlling the slowing down of cyclic gene oscillations is
similar in the two species. The same manner of slowing down of gene
oscillations in snake and zebrafish entails very different numbers of
PSM stripes simply because of the different ratios of oscillator rate to
growth rate (Supplementary Box 2). The faster the oscillator runs
relative to PSM growth rate, the more stripes of cyclic gene expression
are observed in the PSM.
Thus, our data show that the basic clock-and-wavefront mechanism operates according to similar principles in snake, chicken,
mouse and zebrafish. In all four species, somitogenesis ends with a
progressive shrinking of the PSM. This shrinking presumably reflects
a gradual extinction of the signals that maintain the PSM character of
cells at the tail end of the embryo27. In chicken and mouse embryos,
termination of somitogenesis is an active process associated with
extensive cell death in the tail bud28. Apoptosis of tail bud cells leading to axis truncation can be induced by retinoic acid treatment29;
e
f
g
h
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
1 4
8 12 15 19 23 26 30
Somite
Anterior PSM
MSGN1
domain
Somite number
Corn snake
i
j
k
l
Size (µm)
v
n
o
p
Size (µm)
w
m
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
x
r
s
t
Size (µm)
q
Figure 3 | Dynamics of the PSM size in zebrafish, corn snake, chicken and
mouse. a–t, Developmental series of four vertebrate species hybridized with
MSGN1 in whole mount. Zebrafish embryos are shown at 1 (a), 8 (e), 12
(i), 19 (m) and 30 (q) somites; corn snake embryos at 165 (b), 202 (f), 251
(j), 291 (n) and 310 (r) somites; Chicken embryos at 6 (c), 17 (g), 22 (k), 30
(o) and 44 (s) somites; and mouse embryos at 6 (d), 20 (h), 35 (l), 45 (p) and
111 143 170205 237 256 295
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
0
200
400
600
800
1,000
1,200
1,400
1,600
1,800
Somite number
Chicken
3
6 12 17 22 30 38 44 50
Somite number
Mouse
6
20
25
35
45
55
Somite number
55 (t) somites. Dorsal views (a, e, i, m, q, c, g, k, o, s, d) and lateral views
(b, f, j, n, r, h, l, p, t). Anterior to the top. Scale bars correspond to 100 mm
(zebrafish) and 200 mm (corn snake, chicken and mouse). u–x, Graphs
showing the time evolution (in somite numbers) of the PSM (blue), of the
somite s1 (green) and the MSGN1 domain (dark blue) sizes in chicken, corn
snake, mouse and zebrafish embryos.
337
©2008 Macmillan Publishers Limited. All rights reserved
LETTERS
NATURE | Vol 454 | 17 July 2008
thus, it is possible that the proximity of the anterior retinoic acid
domain to the tail bud caused by PSM shrinking triggers the death of
paraxial mesoderm precursors. Therefore, PSM shrinking could
explain the arrest of axis elongation and termination of somitogenesis. In zebrafish, the switch to PSM shrinkage occurs very early in
comparison with that in amniotes, and this correlates with a small
somite number. In amniotes, the switch to PSM shrinkage occurs at
the transition between the trunk and the tail region, suggesting that it
might be under the control of regional regulators such as HOX genes.
Our modelling suggests that the number of PSM generations for
which somitogenesis continues changes relatively little among
amniotes. In contrast, over the course of evolution there have been
large changes in the ratio of the segmentation clock rate to the developmental growth rate. Variation in this ratio accounts for much of
the evolutionary divergence in amniote segment number.
METHODS SUMMARY
In total, 192 corn snake eggs, 13 house snake eggs and 34 whiptail lizard eggs were
used in this study. Corn snake genes were cloned by PCR using standard protocols
(Supplementary Table 1). Orthology was established by calculating the percentage
of similarity with orthologous genes in other vertebrate species using Vector NTI
(Supplementary Table 2). Whole-mount in situ hybridization was performed as
described30 with a hybridization temperature of 58 uC (Supplementary Table 3).
For measurements, embryos were hybridized with a MSGN1 probe in whole
mount and were photographed. The size in micrometres of the PSM, the somite
s1 and the MSGN1 domain were measured using Zeiss LSM image browser software. Embryos were pooled in groups of five on the basis of their somite number.
Measurements corresponding to each pool were averaged, and the standard deviation calculated. For the calculation of the slowing down of the period along the
PSM in corn snake, interstripe distance was measured using the Zeiss LSM software in two-day post-oviposition embryos (n523) stained with LFNG, and calculations were performed as described26. Cell cycle length was measured using
BrdU incorporation followed by flow cytometry analysis. Alizarin staining was
performed according to standard procedures.
Received 4 October 2007; accepted 21 April 2008.
Published online 18 June 2008.
a
3.0
b
MSGN1 front regression speed
1.
2.
0.6
2.0
Ratio
Ratio (Vd/Vs)
2.5
1.5
0.4
1.0
0
0
c
0
20
40
60
80
100
Percentage of total somite number
Size (µm)
30
20
5.
PSM size
6.
1,200
7.
800
8.
400
0
20
100
40
60
80
Percentage of total somite number
9.
0
20
40
60
80
100
Percentage of total somite number
10.
f
Somite s1 size
200
11.
Size (µm)
Corn
snake
150
45
Whiptail
lizard
100
House
snake
12.
40
50
g
13.
35
20
40
60
80
100
Percentage of total somite number
Oscillations slow down along the PSM
6
5
4
30
14.
25
15.
20
Chicken
15
16.
3
10
2
0
Cell cycle duration (h)
0
0
Period (1/To)
4.
1,600
10
250
20
40
60
80
100
Percentage of total somite number
2,000
40
e
0
d
PSM/S1 ratio
0
0
3.
0.2
0.5
Ratio
MSGN1/PSM ratio
0.8
17.
5
0.2
0.4
0.6
0.8
Length (1/PSM size)
1.0
Chicken
Mouse
Snake
Zebrafish
0
18.
Species
19.
20.
Figure 4 | Comparison of somitogenesis parameters. a, Ratio of the speed
of the MSGN1 front regression (in mm per period, Vd) to the speed of
somitogenesis (in mm per period, Vs). b, Variation of the ratio of the MSGN1
domain size to the PSM size during somitogenesis. c, Variation of the ratio of
PSM size to s1 size during somitogenesis. d, Dynamics of the PSM size during
somitogenesis. e, Dynamics of s1 size during somitogenesis. f, Graph
comparing the cell-cycle time in four amniote species. Error bars represent
standard deviation. Sample sizes are provided in Supplementary Methods
(a–e) and in Supplementary Table 4 (f). g, Slowing down of the oscillation
period along the PSM (To, clock period at the tip of the tail).
21.
22.
23.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements The authors thank M. Gibson, B. Rubinstein, P. Francois and
members of the Pourquié laboratory for critical reading and discussions, M. Wahl
for the mouse LFNG pictures, members of the Reptile and Aquatics Department,
J. Chatfield for editorial assistance, and S. Esteban for artwork. Research was
supported by Stowers Institute for Medical Research, and in part by a Defense
Advanced Research Projects Agency (DARPA) grant (O.P.). J.L. is supported by
Cancer Research UK. Zebrafish were obtained from the Zebrafish International
Resource Center (ZIRC) at the University of Oregon, which is supported by a grant
from the NIH-NCRR. O.P. is a Howard Hughes Medical Institute Investigator.
Author Contributions C.G. and O.P. designed the experiments, C.G. cloned the
snake genes and performed the mouse, chicken and snake in situ hybridizations,
E.M.O. performed the fish in situs, C.G. and E.M.O. performed the measurements
and analysed the data with O.P. D.B. established the corn snake and zebrafish
colony and produced the embryos. C.G. and J.W. performed the cell cycle analysis.
J.L. performed the mathematical modelling. C.G., E.M.O., J.L. and O.P. wrote the
manuscript. All authors discussed the results and commented on the manuscript.
Author Information Sequences of genes described in this paper have been
deposited into GenBank under accession numbers EU196456, EU196465,
EU232010, EU196457, EU196458, EU196459, EU196460, EU196466, EU196461,
EU196464, EU196462 and EU196463. Reprints and permissions information is
available at www.nature.com/reprints. Correspondence and requests for
materials should be addressed to O.P. (olp@stowers-institute.org).
339
©2008 Macmillan Publishers Limited. All rights reserved
Paper Review Guidelines
You should include all of the following components in your reviews, but not
necessarily in the order indicated.
Do your reviews in 12 point font, single spaced, with standard (0.5 inch) margins.
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1. Background 1pt: Discuss the scientific context in which the experiments
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about our work and the work of colleagues. It’s what peer review is all
about.
6. Future Experiments 2pts: Design an additional experiment (or two) that
addresses deficiencies in the paper, or provides corroborative evidence
for the conclusions it draws. Be creative, but do attempt to stay within the
bounds of what is plausible. Typically, one point in this component is
reserved for the appropriateness of the experiment, the other for the
feasibility of the experiment.
REVIEWS MUST BE NO LONGER THAN ONE PAGE!
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