Download Analysis of Spatial and Temporal Gene Expression Patterns in

Developmental Biology 245, 187–199 (2002)
doi:10.1006/dbio.2002.0641, available online at http://www.idealibrary.com on
Analysis of Spatial and Temporal Gene Expression
Patterns in Blastula and Gastrula Stage
Chick Embryos
Susan C. Chapman,* ,1 Frank R. Schubert,* Gary C. Schoenwolf,†
and Andrew Lumsden*
*MRC Centre for Developmental Neurobiology, Kings College London, New Hunts House,
Guy’s Hospital, London, SE1 1UL, United Kingdom; and †Department of Neurobiology and
Anatomy, and Children’s Health Research Center, University of Utah School of Medicine,
20 North 1900 East, Salt Lake City, Utah 84132-3401
Studies on the genetic basis of rostral– caudal specification, neural induction, and head development require knowledge of
the relevant gene expression patterns. Gaps in our understanding of gene expression have led us to examine the detailed
spatiotemporal expression patterns of 19 genes implicated in early development, to learn more about their potential role in
specifying and patterning early developmental processes leading to head formation. Here, we report the expression patterns
of these markers in blastula- and gastrula-stage chick embryos, using whole-mount in situ hybridisation. Nodal, Fgf8,
Bmp7, Chordin, Lim1, Hnf3␤, Otx2, Goosecoid, Cerberus, Hex, Dickkopf1, and Crescent are all already expressed by the
time the egg is laid. When the primitive streak has reached its full length, a later group of genes, including Ganf, Six3, Bmp2,
Bmp4, Noggin, Follistatin, and Qin (BF1), begins to be expressed. We reassess current models of early rostral patterning
based on the analysis of these dynamic spatiotemporal expression patterns. © 2002 Elsevier Science (USA)
Key Words: epiblast; forebrain; head; hypoblast; induction; organiser; patterning; trunk; tail; endoderm.
INTRODUCTION
Despite numerous genetic and embryological studies, the
molecular mechanisms underlying the early processes of
rostral– caudal polarisation and neural induction remain
largely unknown. Furthermore, the pattern of expression of
many genes proposed to be involved in these developmental
processes in the chick at blastula and gastrula stages has
been incompletely described. Our aim here is to characterise more fully some of the early gene expression patterns at
Eyal-Giladi and Kochav (EGK) (Eyal-Giladi and Kochav,
1976) stages XI/XII through Hamburger and Hamilton (HH)
(Hamburger and Hamilton, 1951) stages 5/6. A previous
study examined patterns of several genes expressed in the
chick gastrula and primitive streak (Lawson et al., 2000).
We include here genes expressed in various combinations of
tissue layers: primitive streak, the overlying layer (epiblast,
neuroectoderm, and nonneural epidermal ectoderm), the
To whom correspondence should be addressed. Fax: ⫹44 (0)20
7848 6550. E-mail: [email protected].
1
0012-1606/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.
lower layer (hypoblast, rostral, and caudal definitive endoderm), and the later middle layer (ingressing axial mesendoderm, consisting of both the prechordal mesoderm and
rostral notochord).
The lower (ventral) layer initially forms from the polyingression of cells from the overlying epiblast at stage XI/XII
to form islands of cells in the subgerminal cavity (Lawson et
al., 2000). Together with cells moving rostrally from
Koller’s sickle and the posterior marginal zone, this forms a
sheet of cells, the primary hypoblast. The endoblast or
secondary hypoblast begins to form at stages XIII/XIV, with
more cells moving rostrally from Koller’s sickle and the
posterior marginal zone. Together, the primary and secondary hypoblast forms a confluent sheet of cells beneath the
epiblast, which by stages XIV/2, constitutes the primitive
endoderm. The primitive streak appears at stage 2 and
elongates rostrally from its origin at the caudal pole. Definitive endoderm ingresses through the primitive streak at
the time of its formation and continues until stages 4/4⫹,
when the lower layer has fully displaced the hypoblast
sheet rostral to the embryo, forming the germ cell crescent.
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Chapman et al.
The epiblast is the upper (dorsal) tissue layer and forms the
prospective neuroectoderm and epidermis. The neuroectoderm is specified at stage 3 d, at which time it begins to
express neural markers and has a characteristic neuroepithelial morphology (Darnell et al., 1999). Neural induction
(specification) is preceded by an earlier inductive event
leading to the establishment of neural competence (Streit
et al., 2000; Wilson and Edlund, 2001).
We distinguish between prechordal plate endoderm and
prechordal mesoderm. At stages 3a/b, the rostral streak
gives rise to the rostral midline definitive endoderm that
lies beneath the prospective forebrain. This is called the
prechordal plate or prechordal plate endoderm. During
subsequent development, this endoderm buds off mesoderm to form part of the head mesenchyme (Seifert et al.,
1993). The axial mesendoderm that ingresses from stage 4⫹
is the head process, which consists of a mixed cell population of prechordal mesoderm and rostral notochord, which
separate spatially, and is followed by laying down more
caudal notochord as Hensen’s node and the rostral end of
the definitive primitive streak regress caudally. The axial
mesendoderm intercalates between the lower layer definitive endoderm and the overlying neuroectoderm. Prechordal mesoderm lies dorsal to the prechordal plate endoderm. At the very rostral extent of the embryo, no mesoderm intercalates between ectoderm and endoderm that are
directly juxtaposed.
Here, we describe the early expression patterns of genes
encoding the transcription factors Lim1, Hnf3␤, Otx2, Hex,
Ganf, Six3, Qin (BF1), and Goosecoid and the secreted
signalling molecules Nodal, Fgf8, Bmps 2, 4, and 7, Cerberus, Crescent, Dickkopf1, Chordin, Noggin, and Follistatin.
Grouping these dynamically expressed genes in the early
blastula and gastrula stages into any meaningful order is a
difficult task. The spatiotemporal changes, layer of expression, and overall pattern differ enormously. We have, therefore, only loosely grouped genes together where they have
similar expression patterns as seen in midline sagittal
section. Finally, using the currently available models of
head patterning in the chick embryo, we discuss what these
data indicate about when and how head patterning, and in
particular establishment of rostral positional identity, is
specified.
MATERIALS AND METHODS
In Situ Hybridisation of Whole-Mount Embryos
Hens’ eggs (White Leghorn) were incubated at 38°C until the
desired stages were reached. After cracking the egg into a glass petri
dish and removing the albumen over the blastoderm, a square of
filter paper with a hole punched in the middle was placed over the
embryo. After cutting through the vitelline membranes, the filter
paper with attached blastoderm was washed in Tyrode’s solution
(80 g NaCl, 2 g KCl, 2.71 g CaCl 2, 0.5 g NaH 2PO 4䡠H 2O, 2 g
MgCl 2䡠6H 2O, 10 g glucose in 1 liter H 2O, 10⫻ stock) and then fixed
in 4% PFA overnight. After removing the flat, fixed blastoderm
from the vitelline membranes, embryos were stored in 100%
methanol at ⫺20°C.
For the hybridisation step, embryos were rehydrated and permeabilised three times for 20 min in detergent solution (1% IGEPAL,
1% SDS, 0.5% deoxycholate, 50 mM Tris–HCl, pH 8.0, 1 mM
EDTA, pH 8.0, 150 mM NaCl) then fixed for 20 min in 4% PFA.
After several washes in PBT to remove the fixative, the embryos
were incubated for 1–2 h in prehybridisation solution (50% formamide, 5⫻ SSC, pH 4.5, 2% SDS, 2% BBR, 250 ␮g/ml tRNA, 100
␮g/ml Heparin) at 65–70°C before the addition of 10 ␮g/ml of the
appropriate probe, and overnight incubation at 65–70°C. After
incubation, embryos were washed two to four times for 30 min
with solution X (50% formamide, 2⫻ SSC, pH 4.5, 1% SDS) at
65–70°C and then cooled to room temperature in MABT, washing
several times.
For the antibody step, nonspecific binding was blocked by
incubating two times for 30 min in MABT, then 1 h in MABT, 2%
blocking reagent (Roche), 20% normal calf serum. To this solution,
AP-conjugated anti-DIG antibody was added at a concentration of
1:2000 and incubated for 12–72 h at 4°C with rocking. The embryos
were washed six times for 30 min in MABT at room temperature,
followed by two times for 10 min in NTMT (100 mM NaCl, 100
mM Tris–HCl, pH 9.5 or pH 8.0, 50 mM MgCl 2, 1% Tween 20). The
AP-conjugated anti-DIG antibody was detected by a mixture of 200
␮l NBT/BCIP (Roche) in 10 ml NTMT, pH 9.5. The reaction was
stopped by washing in PBT once the required staining intensity was
achieved, then embryos were fixed in 4% PFA overnight.
For double-labelled in situ hybridisation, the following modifications apply. Both the DIG- and FITC-labelled probes were added
during hybridisation. The AP-conjugated anti-DIG antibody was
detected first by using NBT/BCIP, followed by another round of
immunohistochemistry and detection of the AP-conjugated antiFITC antibody separately using Fast Red (Sigma) in NTMT, pH 8.0.
Following overnight fixation, the embryos were cleared in 80%
glycerol/PBS for long-term storage at 4°C. Stained whole mounts
were sectioned at 40 –50 ␮m thickness on a Leica vibratome after
embedding in 20% gelatin and fixation with 4% PFA. Embryos
were examined with a Zeiss Axiophot and imaged with a SPOT
digital camera or Axiocam (Zeiss).
Staging of Early Embryos
Roman numerals are used when applying the criteria of the
staging system of Eyal-Giladi and Kochav (Eyal-Giladi and Kochav,
1976) for prestreak embryos from stages X to XIV. Subsequently,
Hamburger and Hamilton (Hamburger and Hamilton, 1951) is used
to stage embryos from stage 2, primitive streak formation onwards
using Arabic numerals. Refining stages for gastrula embryos use
the criteria as described by Schoenwolf and co-workers (Schoenwolf et al., 1992; Darnell et al., 1999). The first stage in primitive
streak formation, stage 2 has a broad, short triangular streak. Stage
3a is recognised by the appearance of a short and broad linear
streak, followed at stage 3b by a longer and narrower linear streak.
In either case, the defining feature is that the rostral extent of the
streak does not reach the centre of the area pellucida and all such
embryos are labelled as stage 3a/b. The centre of the area pellucida
is the widest zone across the left/right axis of the embryo. At stage
3c, the elongated and grooved streak extends to the centre of the
area pellucida, while stage 3d is characterised by the extension of
the streak rostrally beyond the centre. At stage 4, the maximum
extension of the streak is reached, while a noticeable change in the
morphological character of the epiblast has taken place between
© 2002 Elsevier Science (USA). All rights reserved.
189
Early Chick Gene Expression
stages 3d and 4 as neural induction occurs. A noticeable thickening
at the rostralmost part of the streak indicates the formation of
Hensen’s node. Stage 4⫹ heralds the beginning of ingression of
axial mesendoderm, most often recognised by the triangularshaped ingression rostral to Hensen’s node.
RESULTS
We have examined the expression patterns of 19 genes,
from chick stages XI/XII to stages 5/6, by in situ hybridisation. Expression of many of theses markers is dynamic and
suggests that the 19 genes can be placed into two main
groups. The early onset group is expressed from the earliest
stages studied, and a later group begins to be expressed from
stage 4 onwards, once the primitive streak has reached its
maximum length. The early onset genes included here are
Nodal, Fgf8, Bmp7, Chordin, Lim1, Hnf3␤, Otx2, Goosecoid, Cerberus, Hex, Dickkopf1, and Crescent (Figs. 1 and
2). For the late onset group, consisting of Ganf, Six3, Bmp2,
Bmp4, Noggin, Follistatin, and Qin (BF1), we show their
earliest stages of expression (Fig. 3). We summarise the
expression data in Table 1 and include the type of factor, the
layer of expression at pictured stages, references, and accession numbers where available. Table 2 lists the corresponding mouse knockout, mutant phenotypes, and references.
Early Expressed Genes
We have grouped the results for early expressed genes
into those that are expressed in similar tissues when viewed
in sagittal section: Nodal, Fgf8, Bmp7, Chordin, Lim, and
Hnf3␤ are shown in Fig. 1, and Otx2, Goosecoid, Cerberus,
Hex, Dickkopf1, and Crescent are shown in Fig. 2.
Nodal, Fgf8, and Bmp7. Nodal, Fgf8, and Bmp7 are
expressed mainly in the primitive streak. Nodal and Bmp7
are excluded from the node itself, whereas Fgf8 is asymmetrically expressed within Hensen’s node at stage 4⫹
(Boettger et al., 1998).
Nodal (cNR1) is expressed in a broad caudal domain in
stage XII-XIV embryos at the level of Koller’s sickle. Expression then expands symmetrically in and bilateral to the
caudal two-thirds of the primitive streak from stage 2 to
stage 4/4⫹, excluding the most rostral region (i.e., the
node). At stage 5, Nodal is downregulated, until at stage 6,
only a small patch of cells on the left side of the node
remains. Unlike previous reports, we find continuous expression of Nodal through stages 5–7, although the number
of expressing cells is considerably reduced during these
stages, before being upregulated once more. As can be seen
from the section at stage 4, Nodal is expressed strongly in
all layers of the embryo caudal to the node. Expression
extends in the streak only as far rostral as the node, but
never includes the node itself.
Fgf8 expression begins in a caudal midline region of
Koller’s sickle at stage XIII, and at stage XIV/2 Fgf8 is
expressed at low level in the epiblast cells as they form the
primitive streak. Fgf8 expression gradually moves into the
extending rostral two-thirds of the streak (stage 3c), but it is
excluded from the rostralmost streak until stage 4, where it
is asymmetrically expressed in Hensen’s node (stage 4⫹). At
stage 6, Fgf8 remains confined to the streak and the later
rostral expression domains have yet to appear (Crossley et
al., 1996; Boettger et al., 1998).
Bmp7 is initially expressed in the epiblast of the area
opaca at stages XI-XIII. Transcripts are undetectable by in
situ hybridisation during stage XIV (not shown). At stage
2/3a, expression is reestablished in a small caudal region of
the embryonic epiblast and caudal area opaca. Up to stage
4⫹, Bmp7 is restricted to the caudal epiblast and along the
caudal two-thirds of the streak (see section). The area of
expression expands rostrally and laterally in the caudal
epiblast, forming wings of expression that mark the caudal
limits of the neural plate between stages 4⫹ and 6. At stage
5, a small group of prechordal plate endoderm cells in the
midline expresses Bmp7 in a rostral wedge (see arrow, stage
6). Also at stage 6, transcripts are detected within the rostral
neural/nonneural boundary of the embryo.
FIG. 1. Expression patterns of Nodal, Fgf8, Bmp7, Chordin, Lim1, and Hnf3␤. In Figs. 1 and 2, embryos are orientated with rostral toward
the top of the page and the dorsal side up, unless otherwise indicated. Stages are shown in the bottom right corner, with the earliest stage
on the top of each column and increasingly later stages moving down the page. A midsagittal section is shown for each of the genes, with
rostral to the left and dorsal to the top. A vertical black line on the left side of the whole-mount embryo, from which the section was taken,
indicates the rostrocaudal extent of the section illustrated. The scale bar for whole-mount embryos is 400 ␮m. Nodal is shown from stage
XIV. Levels of transcripts are very high, with a dramatic reduction in expression occurring after stage 4. Stage 6 ventral view. Fgf8 begins
expression at stage XIV in a small caudal patch and levels remain low. Only at stage 4⫹ does asymmetric expression begin in Hensen’s node.
Bmp7 is expressed in the marginal zone (stage XIII) but is downregulated there, only to reappear in the caudal area opaca at stage 2. The
bird-like winged expression at stage 6 marks the caudal boundary of the neural plate. Note the expression in a wedge-like area of the
prechordal plate endoderm (arrow). Chordin expression starts at around stage 2 when the streak begins to form (not shown). Note how
expression moves rostrally, Hensen’s node and axial mesendoderm carrying expressing cells rostrally. Lim1 is expressed throughout the
hypoblast (stage XIII), but once the streak forms, becomes restricted to the rostral third of the streak (stage 3c). At stage 4⫹, expression is
confined to the prechordal plate endoderm and ingressing axial mesendoderm. Hnf3␤ is also expressed in the lower layer at stage XII,
becoming restricted to the rostralmost part of the extending streak (stage 3c). In section, the upper layer expression of Hnf3␤ extends
rostrally beyond Hensen’s node, unlike Lim1. Rostral and bilateral expression surrounds the neural plate.
© 2002 Elsevier Science (USA). All rights reserved.
190
Chapman et al.
© 2002 Elsevier Science (USA). All rights reserved.
191
Early Chick Gene Expression
FIG. 2. Expression patterns of Otx2, Goosecoid, Cerberus, Hex, Dickkopf1, and Crescent. Otx2 expression covers the whole of the embryo
from stage XII. A change in expression occurs at stage 3d, signifying the beginning of the late stage of expression. Expression is at very high
levels in both layers rostral to Hensen’s node (section, stage 4). Arrow indicates a negative zone in lower layer germ cell crescent. Goosecoid
has patchy expression at stage XIII, but by stage XIV, is strongly expressed in the hypoblast. By stage 4⫹, expression is restricted to the
© 2002 Elsevier Science (USA). All rights reserved.
192
Chapman et al.
TABLE 1
Chick Gene Expression
Marker
Type of factor
Nodal (cNR1)
Chordin
transcription
factor; TGF-like
growth factor
secreted signalling;
neural
antagonist
secreted protein
Lim1
transcription factor
Hnf3␤
Otx2
Goosecoid
Cerberus
transcription factor
homeobox gene
homeobox gene
secreted bmp and
wnt antagonist
homeobox gene
Fgf8
Bmp7
Hex
Dickkopf1
Crescent
Ganf
Six3
Bmp2
Bmp4
Noggin
Follistatin
Qin(BF1)
secreted bmp,
nodal and wnt
antagonist
frizzled-like,
secreted signal
homeobox
homeobox
secreted signalling,
neural
antagonist
secreted signalling
secreted bmp
antagonist
secreted bmp
antagonist
transcription factor
Layer of expression
Reference a
Accession No.
Gift of:
E of KS, PS, E
Levin et al., 1995
E, PS, HN
EE, caudal PS, PCP
Crossley et al., 1996
Streit et al., 1998
U41467
AF223970
Gail Martin
Lori Zeltser
rostral PS, HN, ingressing
axial MS and N
H, DE, HN, ingressing
axial MS and N
rostral PS, HN, PE, DE
PE, DE, E, HN, NE
H, PS, PCP
H, PCP, DE
Streit et al., 1998
AF031230
Anthony Graham
Tsuchida et al., 1994
L35569
Sarah Guthrie
Ruiz i Altaba et al., 1995
Bally-Cuif et al., 1995
Izpisua-Belmonte et al., 1993
Zhu et al., 1999
L38904
AAB34353
X70471
AF139721
Marysia Placzek
Massimo Guilisano
Georgy Koentges
Andrew Lassar
S78063
Graham Goodwin
PS, H, DE, HN
Crompton et al., 1992;
Yatskievych et al., 1999
Foley et al., 2000
AY049017
Ed Laufer
H, PCP, DE
Pfeffer et al., 1997
AF006508
Peter Pfeffer
rostral NE
rostral NE, DE
EE, caudal PS, PCP
Zaraisky et al., 1992
Bovolenta et al., 1998
Francis et al., 1994
U65436
O42406
X75914
Andrey Zaraisky
EE, caudal PS
axial MS and N
Francis et al., 1994
Connolly et al., 1997
X75915
BAA75065
Anthony Graham
Jonathan Cooke
DE, MS
Connolly et al., 1997
AAA92005
Anthony Graham
rostral PCP, later NE
Li and Vogt, 1993
A55909
Gord Fishell
H, PCP, DE
Jerome Collignon
Anthony Graham
Note. Abbreviations: DE, definitive endoderm; E, epiblast; EE, epidermal ectoderm; HN, Hensen’s node; H, hypoblast; KS, Koller’s sickle;
MS, mesendoderm; NE, neuroectoderm; N, notochord; PCP, prechordal plate; PE, primitive endoderm endoderm; PS, primitive streak.
a
References are included for the first report of the gene and/or the most relevant reference relating to the stages discussed.
Chordin, Lim1, and Hnf3␤. Chordin, Lim1, and Hnf3␤
are expressed in Hensen’s node and rostral to Hensen’s node
in endoderm and mesoderm from stage 4 onward.
Chordin is expressed in the midline epiblast at stage XII
just rostral to Koller’s sickle in a small, caudally restricted
group of cells (not shown). With the formation of the
prechordal plate endoderm (section). Cerberus is still only patchily expressed at stage XIV. Expression is extremely dynamic and is
downregulated in the midline to the prechordal plate endoderm by stage 4 (section) and further restricted to lateral wings by stage 4⫹
(Chordin in red, in Hensen’s node and ingressing axial mesendoderm). Note arrow points to rostral boundary of ingressing axial
mesendoderm, which is negative for transcripts. Hex is in the hypoblast at stage XIV, but by stage 3c, is restricted to the rostral streak and
a patch of lower layer cells rostral to the streak. A wide zone of expression rostrally becomes restricted to the prechordal plate endoderm
by stage 5 (section, again Chordin in red). Arrow marks boundary between ingressing axial mesendoderm and Hex-positive prechordal plate
endoderm. Note strong expression in future blood islands of the area opaca. Dickkopf1 is shown from stage 2 where expression is found in
the forming streak. Arrow in section marks negative region in lower layer. At stage 5⫹, arrow marks crescent of expression of at rostral
boundary in lower layer. Crescent is found in the primary hypoblast at stage XIV, but is excluded from the endoblast moving rostral from
the caudal marginal zone. This pattern of expression remains. Note that stronger midline expression is not from axial mesendoderm (arrow
in section), but prechordal plate endoderm. Only lower layer is positive, while ingressing axial mesendoderm is negative. Arrow marks
rostral boundary of intercalating axial mesendoderm.
© 2002 Elsevier Science (USA). All rights reserved.
193
Early Chick Gene Expression
TABLE 2
Mouse Knockout Phenotypes
Gene
Knockout phenotype
Reference
Lim1
Development is arrested at early gastrulation; contains little or no
embryonic mesoderm
Fails to express Fgf4 and most cells then fail to move away from
the streak; therefore, no embryonic mesoderm- or endodermderived tissues develop, although extraembryonic tissues form;
patterning of the prospective neurectoderm is greatly perturbed
in the mutant embryos
Postnatal lethal mutation; Bmp7 is an important signaling
molecule for normal development of skeleton, kidney, and eye
Severe prosencephalic defects in combination with noggin null
mutation
Lacks rostral head structures
Hnf3␤
Affects primitive streak, node and notochord formation
Otx2
Early developmental lethal; embryos fail in specification of rostral
neurectoderm and proper gastrulation
Normal and fertile
No phenotype
Variable rostral forebrain truncations.
Rostral head truncation at the level of the midbrain hindbrain
boundary. AVE initially expressed correct markers, but then
fails to develop.
Nodal
Fgf8
Bmp7
Chordin
Goosecoid
Cerberus
Hex
Dickkopf1
Crescent
Hesx1
Six3
Bmp2
Bmp4
Noggin
Follistatin
Qin (BF1)
Variable rostral CNS defects and pituitary dysplasia; reduced
prosencephalon; anopthalmia or micropthalmia; defective
olfactory development; bifurcations in Rathke’s pouch; neonates
have an abnormal corpus callosum, rostral and hippocampal
commissures, and septum pellucidum
Embryonic lethal; fails to close proamniotic canal; malformation
of amnion/chorion; abnormal development of the heart in the
exocoelomic cavity
Embryonic lethal between 6.5 and 9.5 dpc. with a variable
phenotype
Severe prosencephalic defects in combination with chordin null
mutation
Retarded growth; decreased diaphragm and intercostal muscle
mass; shiny taut skin; skeletal defects of hard palate and
thirteenth pair of ribs; abnormal whisker and tooth
development; failed respiration; die perinatally
Die at birth; reduced cerebral hemispheres
primitive streak, expression is observed in its rostral twothirds and slightly bilateral to the streak (stages 3a–3d). By
stage 3d/4, Chordin is expressed only in the rostral third of
the streak and Hensen’s node. With the onset of axial
mesendoderm ingression at stage 4⫹, Chordin transcripts
are detected in Hensen’s node (Garcia-Martinez et al., 1993)
and the ingressing axial mesendoderm (see Fig. 1, chordin
section, stage 4⫹).
Lim1 is strongly expressed in the hypoblast at stages
XI-XIV. As the primitive streak forms, transcripts become
restricted to the rostralmost portion of the streak prior to
Conlon et al., 1991, 1994
Sun et al., 1999; Trumpp et al., 1999
Jena et al., 1997
Bachiller et al., 2000
Shawlot and Behringer, 1995;
Shawlot et al., 1999
Ang and Rossant, 1994; Dufort et
al., 1998
Acampora et al., 1995
Wakamiya et al., 1998
Shawlot et al., 2000
Martinez Barbera et al., 2000
Mukhopadhyay et al., 2001
not yet described
Dattani et al., 1998; MartinezBarbera et al., 2000
not yet described
Zhang and Bradley, 1996
Winnier et al., 1995
Bachiller et al., 2000
Matzuk et al., 1995
Xuan et al., 1995
formation of Hensen’s node. During ingression of the axial
mesendoderm from stage 4⫹, expression is detected in the
node, ingressing axial population, and prechordal plate
endoderm, whilst being excluded from the overlying neuroectoderm (see section).
Hnf3␤ expression is detected in embryos before stage XII,
in the hypoblast and Koller’s sickle (not shown). By stage
XII, expression extends more laterally than Koller’s sickle
to the edge of the area opaca before condensing in Koller’s
sickle by stage XIV. When the primitive streak forms,
Hnf3␤ transcripts are downregulated in the caudal part of
© 2002 Elsevier Science (USA). All rights reserved.
194
Chapman et al.
FIG. 3. Expression pattern of the late onset group of genes, Ganf, Six3, Bmp2, Bmp4, Noggin, Follistatin, and Qin. The whole-mount
embryos are shown with rostral to the top, sections with rostral to the left and dorsal to the top. A vertical line marks the rostral– caudal
level of each sagittal section. Both Ganf and Six3 are double whole-mount in situ hybridisations with Chordin to illustrate the ingressing
axial mesendoderm. Ganf is expressed in the neuroectoderm at stage 4 before the axial mesendoderm has ingressed. The gap is clearly
visible in both the whole-mount and section (arrow). Six3 expression begins at stage 4⫹/5, and the section shows that Six3 is expressed in
the overlying neuroectoderm while the ingressing cells have not yet reached that rostral level yet (arrow). Bmp2 is found outside the
margins of the neural plate and in the caudal third of the primitive streak. The arrow in stage 5⫹ shows a gap between the midline
prechordal plate endoderm and rostral nonneural ectoderm. Bmp4 has a similar expression pattern at stage 4, but by stage 6, Bmp4 has high
levels of transcripts in surrounding the neural plate. Noggin is only expressed in the first axial mesendoderm cells to ingress through
Hensen’s node. By stage 5⫹, the ingressing axial mesendoderm has high levels of transcripts. Follistatin is in the lower layer both in the
rostral streak and caudal neural plate. A caudal to rostral gradient appears to be in place. This dynamic expression changes by stage 6, while
variable levels of transcripts rostrocaudally. Interestingly, Qin (BF1) is initially expressed in only the lower layer and not yet in the
overlying neuroectoderm as previously reported (stage 5/6). This expression is in a very rostral region of the embryo, in the prospective
foregut that will form as the head fold forms.
the embryo, but are still strongly detected in the rostral
third of the primitive streak (stage 3c). From stage 4 onward,
expressing cells are seen in Hensen’s node, prechordal plate
endoderm, ingressing axial mesendoderm, and in the germ
cell crescent. Hnf3␤ signals are also apparent in the neuroectodermal cells of area “a” (Garcia-Martinez et al., 1993),
contrary to previous reports (Ruiz i Altaba, 1993). At stages
5⫹ and 6, the node and axial mesendoderm both retain high
levels of expression, whereas the crescent of expression is
better defined in the stomodeal ectoderm rostrally, germ
cell crescent, and areas bilateral to the neural plate.
Otx2. Expression is detected in epiblast and hypoblast
cells of the unincubated embryo (stages XIII/XIV). As the
primitive streak forms, expression in the epiblast becomes
restricted to the rostral third of the streak. At stage 3a/b,
Otx2 is seen only in the rostral streak and underlying
hypoblast/rostral definitive endoderm. This marks the end
of the early phase of Otx2 expression and is immediately
followed by the second stage of expression. At stages 3d/4,
when the streak is at its maximal extension, and subsequently during streak regression, transcripts are detected in
the neuroectoderm of the prospective fore- and midbrain as
well as the underlying mesoderm and rostral definitive
endoderm (see Fig. 2, Otx2, stage 4). In transverse sections,
a low level of expression in the forming floor plate and
notochord is visible (not shown), whereas sagittal sections
reveal strong expression in the neuroectoderm and definitive endoderm rostral to Hensen’s node. The most rostral
hypoblast, which now forms the germ cell crescent, is
negative for Otx2 expression (arrow).
Goosecoid, Cerberus, and Hex. Goosecoid, Cerberus,
and Hex are expressed early in the rostral streak, but are
later excluded from Hensen’s node and are only expressed
rostrally in the midline lower layer of the prechordal plate
endoderm/rostral definitive endoderm, but never in the
overlying neuroectoderm.
© 2002 Elsevier Science (USA). All rights reserved.
195
Early Chick Gene Expression
Goosecoid expression is detected in the loose cells of the
forming hypoblast at stage XI. Transcripts are also detected
in Koller’s sickle and the posterior marginal zone (stage
XIII). During primitive streak formation, stage 3a/b, transcripts become progressively localised to the rostral third of
the streak. By stage 4, expression is restricted to Hensen’s
node (not shown), and at stage 4⫹, expression is only
observed in the fan-shaped prechordal plate endoderm (see
section). When mesendoderm ingresses at stage 4⫹, expression is downregulated in Hensen’s node, but is detected in
both the prechordal plate endoderm and ingressing axial
mesendoderm. Note that the very rostral hypoblast forming
the germ cell crescent is negative for transcripts (arrow).
Cerberus expression at gastrulation stages has not been
reported previously. From stage XII, the cells of the hypoblast express Cerberus (see stage XIV). From stage 2 onwards, the forming primitive streak expresses strongly (see
stage 3a/b). Hypoblast expression continues with the elongation of the streak, while expression within the streak
itself is downregulated (stage 3c). At stage 4, the streak is
completely negative for Cerberus expression. Expression is
being downregulated from the centre of the lower layer
toward the lateral margins at the level of Hensen’s node. In
sagittal section transcripts in the prechordal plate,
endoderm and rostrally located hypoblast are detected (rostral to arrow). From stage 4⫹ (double stained with Chordin
to show ingressing axial mesendoderm), until by stage 6
(not shown), only two lateral wings remain. Cerberus is
excluded from the ingressing axial mesendoderm and the
overlying neuroectoderm layer.
Hex (Prh) expression is first detected in Koller’s sickle
and the hypoblast at stages XII-XIV. This expression is
restricted to the rostral streak and scattered groups of cells
in the lower layer rostral to the streak at stage 3c. At stages
3d/4, strong expression is detected in the future blood
islands of the caudal and lateral area opaca. Hypoblast cells
are displaced rostral to the node and into the forming germ
cell crescent, which extends bilaterally and caudally. Hex
continues to be strongly expressed in this population, as
well as in a wedge of midline rostral definitive endoderm,
including the prechordal plate endoderm. The definitive
endoderm expression becomes further localised to within
the fan-shaped prechordal plate endoderm at stage 5 (see
section). As can be seen in the embryo double stained for
Hex and Chordin, and accompanying section, the ingressing axial mesendoderm extends rostrally toward the fanshaped wedge of prechordal plate endoderm expressing Hex
transcripts, making contact with the caudal margin. By
stage 6, strong Hex staining in the germ cell crescent is
maintained, but only a small number of cells in the midline
prechordal plate endoderm still express Hex.
Dickkopf1 (Dkk1) and Crescent. Dkk1 encodes a Wnt
inhibitor expressed in the hypoblast at low levels during
stages XI-XIV (not shown). At stage 2, expression is associated with cells of the forming primitive streak, but also in
a few scattered cells within the rostral area opaca. During
stages 3c/3d, transcripts are detected along the whole ex-
tent of the streak. At stage 3d, a broadly scattered domain of
expressing cells is seen in the lower layer, in an area
beneath the presumptive neural plate. Higher levels of
transcripts are detected lateral to the streak, compared with
the area rostral to the primitive streak. By stage 4⫹,
expression has become more defined, with a strong area of
expression detected in a rostral crescent of hypoblast,
which underlies the presumptive stomodeal ectoderm and
marks the future border of the overlying neural and nonneural tissue (rostral to arrow in section (Pera et al., 1999).
The definitive streak and Hensen’s node at stage 4⫹ express
a medium level of transcripts, whereas the ingressing axial
mesendoderm expresses a high level of transcripts, giving
the appearance of very strong staining at the rostral end of
the streak. A broad band of negative tissue is observed in
the area of the prospective fore- and midbrain territory and
directly underlying prechordal plate endoderm (arrow). At
stage 5⫹, low-level scattered expression marks the embryo
out. Only a faint outline of the neural plate-negative zone
remains, bordered rostrally by a narrowed boundary of
expression, marking the position at which the head fold
will form (arrow). The ingressing axial mesendoderm and
Hensen’s node express high levels of transcripts, while in
the streak, only a small caudal region still expresses high
levels of Dkk1.
Crescent (Frzb2), encodes another Wnt inhibitor. Expression begins from stage XI/XII (not shown) and by stage XIV
it is in the primary hypoblast layer, and excluded from the
more caudal secondary hypoblast, also known as the endoblast. As the primitive endoderm is pushed rostrally by the
ingression of the definitive endoderm, Crescent becomes
bilaterally and rostrally restricted, to the germ cell crescent
and anterior definitive endoderm. From stage 4 onward,
dense midline staining is detected. In sections, this staining
appears not to be in the ingressing axial mesendoderm,
rather we consider that it is expressed at high levels in
prechordal plate endoderm (arrow in section).
Late Onset Genes
We have analysed seven genes in this group: Ganf, Six3,
Bmp2, Bmp4, Noggin, Follistatin, and Qin (BF1), by in situ
hybridisation from stages XI/XII, but could find no detectable levels of transcripts until definitive streak stages. All
are pictured in Fig. 3.
We first detect Ganf (blue) at stage 4 in the rostral
neuroectoderm. Hensen’s node and the ingressing axial
mesendoderm are labelled by Chordin (red). A gap is visible
between the rostral domain of Ganf expression and Hensen’s node (arrow). This contradicts earlier findings where
Ganf transcripts were detected only from stage 4⫹, once the
axial mesendoderm comes to underlie the rostral neuroectoderm (Knoetgen et al., 1999). Furthermore, Ganf is expressed only in the upper layer in the stage 4 chick, unlike
in the mouse, where its orthologue Hesx1/Rpx is first
expressed in the anterior visceral endoderm and then in the
overlying neuroectoderm (Hermesz et al., 1996). The region
© 2002 Elsevier Science (USA). All rights reserved.
196
Chapman et al.
of expression narrows as convergence extension and neural
tube formation commence (stage 6). Ganf expression remains in the rostral most region of the embryo, stopping
rostrally at the level of the forming proamnion.
Six3 transcripts are first detected at stage 5 in both the
rostral hypoblast and overlying rostral most neuroectoderm. The comparison with Chordin shows the Six3 expression abutting the rostral extent of the ingressing axial
mesendoderm. At later stages, Six3 transcripts are found in
both very rostral neuroectoderm and the prechordal plate
endoderm (stage 6).
Bmp2 is expressed from stage 4 onward in the nonneural
ectoderm bordering the neural plate. It is also expressed
strongly in the caudal third of the primitive streak, with
very low levels of transcripts in the rostral streak. From
stage 5, a small patch of expressing cells, similar to that
seen with Bmp7, appears in the rostral endoderm. Arrows
highlight the gap between nonneural ectoderm rostrally and
the small area of midline expression in the prechordal plate
endoderm (blue) and the expression in the ingressing axial
mesendoderm (Chordin, red, stage 5⫹ pictured).
Bmp4 has a similar region of expression as Bmp2 in the
early stages, and it is similarly detected only from stage 4
onward, although very low levels of expression have been
reported transiently at stages XI-XIII (Streit et al., 1998). By
stage 6, the nonneural ectoderm around the neural plate
expresses strongly. The caudal two-thirds of the streak also
has high levels of transcripts, whereas the rostral streak has
very low levels.
Noggin has a very small and weak domain of expression
in the midline lower layer, just rostral to Hensen’s node.
This expression is in the leading group of cells that have
just ingressed from the node at stage 4⫹ (section). At stage
5⫹, the ingressing axial mesendoderm has high levels of
transcripts.
Follistatin shows a graded level of expression with transcripts detected from stage 4⫹ onwards. The highest level
of expression is at the caudal extent of its domain, which
seems to mark the caudal most region of the neural plate.
Decreasing levels of transcripts are detected toward the
rostral region of the neural plate. Whereas Follistatin is
excluded from most of the streak, only having a low level of
transcripts in Hensen’s node, it is expressed in the ingressing axial mesendoderm. By stage 6, expression underlies
most of the neural plate. Note the very rostral-negative
region (arrow) and the variable levels of expression throughout the domain (arrow). The caudal boundary of expression
is asymmetrical and the streak is wholly negative for
transcripts (stage 6).
Qin (BF1) is expressed consistently from stage 5 onward.
Occasionally, low levels of transcripts are detected at stage
4/4⫹. This variation probably represents a few hours variation in embryonic development and thus the state of
commitment of cells in the rostral part of the embryo.
Interestingly, we detect expression initially only in the
lower layer and not in the neuroectoderm as expected
(arrow). This early expression is in the rostral region of the
prospective head fold (stage 6). Only at later stages does
expression commence in the prospective telencephalic territory (not shown).
DISCUSSION
Patterning the early embryo is a dynamic process involving several tissues and numerous genes. We have compared
the expression patterns of 19 genes in early chick development with a view to exploring their candidature for roles in
establishing rostral identity, neural induction, and head
formation. Current models for these processes fall broadly
into two camps: the activation/transformation model of
Nieuwkoop (Nieuwkoop et al., 1952; Nieuwkoop and
Nigtevecht, 1954) and the organiser model of Spemann
(Spemann, 1931, 1938), which proposes separate head and
trunk/tail organisers. Both of these models assume a twostep process: neural induction (specification), followed by
signals that pattern the neural tissue to take on specific
rostrocaudal regional identity.
The Timing of Neural Induction in Chick
Using an improved model system, Darnell et al. (1999)
produced transverse rostral blastoderm isolates, obtained
from gastrulation-stage chick embryos, to establish the
timing of neural induction (specification). They show
that the prospective neuroectoderm is unable to selfdifferentiate before stage 3d. Rostral blastoderm isolates fail
to acquire neural morphology or express neural markers
unless area “a” is included in the isolate. Area “a” is a group
of cells rostral to the streak at early gastrula stages that is
capable of acting as a neural inducer (Schoenwolf et al.,
1989). At stage 3c, area “a” becomes incorporated into the
extending rostral streak, which has been defined as the
chick organiser (Waddington, 1933). Neural induction in
chick embryos, however, remains a controversial issue.
Two different groups have suggested separate mechanisms
for neural induction within the epiblast at blastula and
early gastrula stages (Streit et al., 2000; Wilson and Edlund,
2001). Markers of neural competence, L5 and Sox3 (Darnell
and Schoenwolf, 1997; Rex et al., 1997), are expressed from
prestreak stages, whilst markers of neural specification
begin to be expressed at stage 3d, e.g., Sox2 (Rex et al., 1997;
and data not published) and Ganf, the earliest specific
marker of anterior neural plate, at stage 4. Establishment of
competence may involve secreted signalling molecules,
such as FGFs, BMPs, and WNTs, which may later also be
involved in neural induction/specification. However, the
work on neural competence and specification remains incomplete, and clarifying the expression profiles of the
various ligands and receptors as well as in vivo functional
studies are still required.
© 2002 Elsevier Science (USA). All rights reserved.
197
Early Chick Gene Expression
Early Signalling Centres Are Involved in Rostral
Patterning before Neural Induction
Anterior visceral endoderm (AVE) is an early signalling
centre identified in mouse, which appears to be involved in
rostral patterning by signalling to the rostral epiblast before
and during gastrulation. The AVE is an outer cell layer in
the early mouse embryo, fated to form extraembryonic
visceral yolk sac endoderm. It underlies the rostralmost
epiblast that will form the forebrain and midbrain. As the
AVE moves rostrally, it signals to the overlying epiblast and
is responsible for the induction of Hesx1 in the overlying
prospective forebrain epiblast (reviewed in MartinezBarbera and Beddington, 2001). Further genes with differential expression in the AVE include Hnf3␤, Gsc, Hex, Nodal,
Otx2, and Lim1 (Martinez-Barbera and Beddington, 2001
and references therein). Lim1, Hnf3␤, and Gsc are expressed
in AVE, early gastrula organiser (EGO), and later, the node.
The AVE on its own does not have neural-inducing ability,
as grafts of AVE are unable to induce neural character in
nonneural ectoderm (Tam and Steiner, 1999). However,
ablation experiments show that the AVE is required for
neural plate formation (Beddington and Robertson, 1999;
Martinez Barbera et al., 2000; Martinez-Barbera et al., 2000;
Martinez-Barbera and Beddington, 2001), whereas transplant experiments provide evidence that the AVE is not
sufficient to induce a complete head without the EGO (Tam
and Steiner, 1999; Martinez-Barbera and Beddington, 2001).
The EGO is a group of cells rostral to the primitive streak,
similar to area “a” in the chick, capable of neural induction
and can even produce a rudimentary axis (Schoenwolf et al.,
1989; Tam et al., 1997). Only a combination of AVE, rostral
epiblast, and EGO together is able produce a complete
neural axis (Tam et al., 1997; Tam and Steiner, 1999). Thus,
two tissues outside the organiser, the AVE and EGO, have
now been identified as important in establishing positional
and neural identity, respectively, in the ectoderm.
Gene Expression Patterns Indicate That Positional
Identity Is Established in the Chick before Neural
Induction
The gene expression data reported here indicate that
regionalised gene expression occurs before the onset of
neural induction at stage 3d. Nodal, Fgf8, Bmp7, Chordin,
Lim1, and Hnf3␤ are expressed in the primitive streak,
before stage 3d, when neural induction begins. Otx2, Cerberus, Hex, and Crescent are expressed in the prechordal
plate endoderm both before and after neural induction.
Bmp7, Lim1, Hnf3␤, and Gsc are expressed in prechordal
plate endoderm only after neural induction. Dickkopf1 is
conspicuously absent from the hypoblast underlying the
future rostral neural plate until after neural induction.
Crescent is never expressed in the streak, only the hypoblast and definitive endoderm below the prospective rostral
neural plate. Moreover, we show that Ganf is expressed at
stage 4, before the ingression of Chordin-positive axial
mesendoderm. These characteristic expression patterns in-
dicate that rostral– caudal patterning is underway before
neural induction.
None of the proposed chick models takes into account
the possibility that rostral identity is established before and
distinct from neural induction. Recent results in mouse,
Xenopus (Jones et al., 1999), and zebrafish (Houart et al.,
1998; Koshida et al., 1998; Jones et al., 1999; Martinez
Barbera et al., 2000; Martinez-Barbera et al., 2000), however, do lend support to this idea. In chick, the debate is
ongoing as to which tissue is equivalent to the mammalian
AVE, and whether the lower layer— hypoblast and rostral
definitive endoderm— has any patterning role. Based on
similarity of gene expression patterns, Foley et al. (2000)
have suggested that the mouse AVE and chick hypoblast are
equivalent tissues. Interestingly, they show that at stage
XII, hypoblast transiently induces expression of Sox3 and
Otx2.
Based on our expression data, we suggest that candidate
lower layer tissues capable of inducing rostral– caudal patterning in the epiblast before neural induction include: at
stage XII/XIII, the rostral hypoblast underlying the future
rostral neural plate; the hypoblast underlying the rostral
neural plate at primitive streak stages 2–3a/b; and rostral
definitive endoderm (i.e., the prechordal endoderm) which
begins ingressing from stage 3a/b and underlies the epiblast
at stages 3c, 3d, and 4; but not the ingressing head process,
comprising the mixed prechordal plate mesoderm and rostral notochord population, as these cells only ingress at
stage 4⫹, following neural induction. A number of studies
support a role for the prechordal mesoderm/axial mesendoderm in patterning the rostral neural plate (Dale et al., 1997;
Foley et al., 1997; Pera and Kessel, 1997; Rowan et al.,
1999), but this tissue arises too late to establish positional
identity in epiblast prior to neural induction. Further investigation of the lower layer tissues is required, focusing on a
possible role in establishing positional identity in the
overlying epiblast.
In summary, we have described the expression patterns of
19 genes in early blastula- and gastrula-stage chick embryos. These expression patterns and recent experimental
data challenge the current models of rostral identity, neural
induction, and head patterning. The establishment of rostral positional identity before neural induction is a topic
under investigation that must be taken into account in any
model that hopes to explain how the embryo specifies and
patterns the head.
ACKNOWLEDGMENTS
This work was supported by the MRC, a Wellcome Prize
Studentship, and travel grants from The Physiological Society and
the Guarantors of Brain (to S.C.C.) and NIH Grant NS18112 (to
G.C.S.).
© 2002 Elsevier Science (USA). All rights reserved.
198
Chapman et al.
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© 2002 Elsevier Science (USA). All rights reserved.
Received for publication January 9,
Revised February 26,
Accepted February 26,
Published online April 14,
2002
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