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Patterns of paired-related homeobox genes PRX1 and PRX2 suggest involvement in matrix modulation in the developing chick vascular system

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Graded Retinoid Responses in the Developing Hindbrain
Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
The purpose of this study was to
make an explicit test of the idea that a retinoid
could act as a morphogen, differentially activating genes and specifying anteroposterior (a-p)
level in the developing vertebrate central nervous system (CNS). Our approach was to characterize the concentration-dependent effects of retinoic acid (RA) on the neural expression of a set of
a-p patterning genes, both in vivo and in an in
vitro system for neural patterning. Our results
indicate that a retinoid is unlikely to specify a-p
level along the entire CNS. Instead, our data
support the idea that the developing hindbrain
may be patterned by a retinoid gradient. Sequentially more posterior hindbrain patterning genes
were induced effectively by sequentially higher
RA concentration windows. The most posterior
CNS level induced under our RA treatment conditions corresponded to the most posterior part of
the hindbrain. Dev. Dyn. 1998;213:39–49.
r 1998 Wiley-Liss, Inc.
Key words: retinoids; Hoxb genes; Xenopus; anteroposterior patterning
There is evidence that anteroposterior (a-p) axis
formation in the vertebrate central nervous system
(CNS) occurs in two steps (for review, see Durston et al.,
1998). A neural induction or activation signal in the
gastrula first induces competent ectoderm to develop
into anterior neural tissue. A transformation signal
then converts the posterior part of the newly induced
anterior neuroectoderm into presumptive hindbrain
and spinal cord. Neural transformation activity is
present as a gradient in the gastrula and in early
neurula-stage amphibian embryo. High levels of transforming activity induce spinal cord, and lower levels
induce hindbrain (Nieuwkoop et al., 1952).
Retinoids have been proposed as candidates for the
neural transformation signal in early embryos (for
review, see Durston et al., 1998). Retinoic acid (RA) and
several other acidic retinoids are potent teratogens that
can suppress development of the forebrain and other
anterior structures and can cause enlargement of the
posterior CNS (Durston et al., 1989; Sive et al., 1990;
Papalopulu et al., 1991; Ruiz i Altaba and Jessell, 1991;
Pijnappel et al., 1993; Simeone et al., 1995; Blumberg et
al., 1996). Active retinoids have been identified in
Xenopus gastrulae while neural induction and a-p
patterning of the CNS are in progress (Durston et al.,
1989; Pijnappel et al., 1993; Chen et al., 1994; Creech
Kraft et al., 1994; Blumberg et al., 1996). They are
reported to be concentrated in the late gastrula organizer (source of transformation signals) and to be
present as a posterior-to-anterior concentration gradient in the neurula (Chen et al., 1994). A molecular basis
for retinoid posteriorization is indicated by the finding
that some posterior neural patterning genes, including
some of the Hox genes, are direct retinoid targets
through enhancers that contain RA response elements
(Langston and Gudas, 1992; Pöpperl and Featherstone,
1993; Marshall et al., 1994; Studer et al., 1994; Frasch
et al., 1995; Ogura and Evans, 1995a,b; Doerksen et.
al., 1996; Morrison et al., 1996; Dupé et al., 1997;
Langston et al., 1997).
The purpose of the present study was to test whether
a retinoid gradient could mediate neural transformation, such that sequentially higher retinoid concentrations specify sequentially more posterior zones in the
developing neural plate through differential regulation
of a-p patterning genes. Surprisingly, this idea has
never been tested explicitly before, despite a large
number of publications about retinoid effects on development. We therefore made a systematic investigation
of the effects of the active retinoid, RA, on early neural
patterning. Our results indicated that RA does not
modulate the whole of neural transformation, but that
an RA-like retinoid may be involved in two separable
aspects of the transformation process. RA can transform presumptive forebrain and midbrain into hindbrain and can also modulate hindbrain patterning. We
detected an anterior-to-posterior gradient of RA sensitivity within the developing hindbrain, suggesting that
a retinoid gradient may pattern this part of the CNS in
The idea that a retinoid gradient could mediate CNS
patterning was tested by characterizing the effects of a
range of RA concentrations on the neural expression of
a set of a-p patterning genes (for details, see Experimental Procedures). If a retinoid gradient does convey
Grant sponsor: Netherlands Science Organization Life Sciences;
Grant number: 805–33–021; Grant number: 417442; Grant sponsor:
EU Biotech Programme; Grant number: ERBBIO4CT960378.
*Correspondence to: A.J. Durston, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Received 24 February 1998; Accepted 20 May 1998
Fig. 1. Effect of retinoic acid (RA) treatments on expression of Otx-2,
En-2, and Krox-20 in whole tailbud stage embryos as shown by in situ
hybridization. A: Expression of otx-2 in the forebrain and midbrain of
control embryos. There is also some expression in other anterior structures and associated with the otic vesicles (o). B,C: Expression of Otx-2 in
stage 29–31 tailbud embryos following RA treatment beginning at the
early gastrula stage: 1027 M RA (B) and 1026 M RA (C). Note the smaller
expression domain in B compared with the control (A). However, very
anterior structures, including nasal pits, still developed in these embryos
(data not shown). D: Expression of en-2 at the midbrain-hindbrain border
in untreated embryos. E,F: Expression of en-2 in stage 29–31 embryos
following addition of 1027 M RA (E) and 5 3 1027 M RA (F) to the culture
medium at stage 10. In E, note the extended domain of en-2 expression
along the anteroposterior (a-p) axis and the lack of transcripts dorsally in
the brain. There is no localized expression of otx-2 (C) or en-2 (F)
following treatment with 2.5 3 1027 M (not shown) or higher RA
concentrations. G–I: Krox-20 expression in tailbud stage embryos. Control embryo is shown in G. Expression is visible in rhombomeres 3 and 5
(r3 and r5) and in r5-associated neural crest. H shows an embryo that was
treated with 1028 M RA: The two Krox-20 stripes are broader and closer
together. In I, an embryo that was treated with 1027 M RA shows only one
clear stripe of Krox-20 expression. This is presumably the r5 stripe,
because it is associated with Krox-20 positive neural crest cells. Krox-20
expression is undetectable in embryos treated with 2.5 3 1027 M RA or
higher (not shown).
positional information, then we would expect increasing concentration windows of RA to activate the expression of increasingly more posterior markers. These
experiments employed whole embryos as well as an in
vitro system for CNS patterning, as described below.
Anterior development was inhibited, and the expression of the forebrain and midbrain markers, Otx-2 and
En-2, as well as hindbrain markers back to Hoxb-3
(rhombomeres 5 and 6; r5 and r6) could be downregulated by RA (for details, see Figs 1, 2, Table 1). The
posteriorization caused by RA was mediated at least
partly by posterior transformation of the developing
CNS rather than just anterior deletion. We note, for
example, that repression of anterior markers by increasing concentrations of RA (up to 2.5 3 1027 M) was
accompanied by sequential extension of the expression
zones of Hoxb-3 and Hoxb-4 until these reached the
front end of the embryo. This indicates that the presumptive anterior CNS is transformed entirely to a
state resembling posterior hindbrain by high concentrations of RA.
In contrast to its action on presumptive forebrain,
midbrain, and hindbrain domains, continuous RA treat-
Neural Transformation Is Not Simply Mediated
by a Retinoid Gradient
RA treatment of whole embryos posteriorized the
developing CNS, as reported previously (for review, see
Durston et al., 1998). This RA-induced posteriorization
shows some resemblances to the natural neural transformation process, but it also shows striking differences.
In situ hybridization to markers for different levels
along the developing CNS (shown and described in
detail in Figs. 1 and 2) revealed that markers for the
forebrain, midbrain, and hindbrain are RA sensitive.
Figure 2.
ment had relatively little effect on the expression
domains of 58 Hoxb genes in the spinal cord. High RA
concentrations (2.5 3 1027 M to 1026 M) caused posterior truncations and a corresponding shortening of the
58 Hoxb gene expression domains, but lower concentrations had no detectable effect, and the effects of RA were
generally much less than on hindbrain development
(Fig. 2). These data match previous findings in Xenopus
and mouse embryos that RA has little effect on neural
Hoxb-9 expression (Conlon and Rossant, 1992; Dekker
et al., 1992b). They are also consistent with findings
from loss-of-function experiments using vitamin A deficiency (Maden et al., 1996) or dominant-negative retinoid receptors (Kolm et al., 1997; van der Wees et al.,
1998; but see also Blumberg et al., 1997) in emphasizing that retinoids are important for development of
hindbrain rather than spinal cord. This characteristic
contrasts with the nature of the natural neural transformation signal, which induces spinal cord at the highest
intensities (Nieuwkoop et al., 1952; Sala, 1955). We
conclude that neural transformation is likely to be
mediated by a complex of different signals and that
retinoids could mediate formation and patterning of the
hindbrain part of the developing CNS. It seems likely,
however, that the retinoid responses of neural markers
are somewhat condition dependent, because RA responses have been reported in other studies for the
expression of relatively 58 posterior Hox genes (IzpisuaBelmonte et al., 1991; Sharpe, 1991; Conlon and Rossant, 1992; Dekker et al., 1992b; López and Carrasco,
We observed sharp RA thresholds for inhibition of the
development of particular a-p zones. For example, the
forebrain and midbrain marker, Otx-2, was expressed
in an almost normal pattern following treatment with
1027 M RA or less, but was totally undetectable following treatment with 2.5 3 1027 M RA or more. Similarly
sharp thresholds were observed for repression of other
markers (Figs. 1, 2, Table 1). The pattern of the
thresholds that was observed, as described below, sug-
Fig. 2. (Previous page.) A–U: Effect of RA treatments on Hoxb gene
expression in whole embryos, as shown by wholemount in situ hybridization. Expression patterns are shown of Hoxb-1 (A), Hoxb-3 (E), Hoxb-4 (I),
Hoxb-5 (M), Hoxb-7 (P), and Hoxb-9 (S) in untreated, tailbud stage 29–31
embryos. B–D, F–H, J–L, N, O, Q, R, T, and U show embryos that were
treated with RA from stage 10 and fixed at stage 29–31. B–D: Hoxb-1
expression in embryos treated with 1027 M RA (B), 5 3 1027 M RA (C),
and 1026 M RA (D). Note that the expression of Hoxb-1 occurs adjacent to
the otic vesicle (o) in both control (A) and 1027 M RA-treated embryos (B).
Higher levels of RA (2.5 3 1027 M or more) caused a loss of localized
Hoxb-1 expression. This was also the case when embryos were given
30-min pulse treatments with high concentrations of RA (not shown). F–H:
Hoxb-3 expression following treatment with 1027 M RA (F), 5 3 1027 M RA
(G), and 1026 M RA (H). J–L: Hoxb-4 transcripts in embryos treated with
1027 M RA (J), 5 3 1027 M RA (K), and 1026 M RA (L). RA causes Hoxb-3
and Hoxb- 4 to be expressed in extended domains along the a-p axis. This
is already evident in embryos treated with 1028 M RA (not shown) and
occurs to increasingly greater extents with increasing RA concentrations.
This effect is correlated with a loss of rhombomeric segmentation in the
hindbrain. In F, the strongest Hoxb-3 expression is anteriorized by 1027 M
gested that retinoid effects on the hindbrain and on the
forebrain and midbrain occur through separable mechanisms.
A Gradient of RA Sensitivity in the Developing
We detected anterior-to-posterior sequences of repression and activation in the developing hindbrain. The
most striking feature in vivo was an anterior-toposterior sequence of RA repression thresholds. Embryos treated with less than 1027 M RA expressed all
hindbrain markers. At 1027 M RA, the r3 stripe of
Krox-20 was absent, but all other hindbrain markers
remained. At 2.5 or 5 3 1027 M RA, Hoxb-1 (r4) and the
posterior stripe of Krox-20 (r5) were also repressed. At
1026 M RA, Hoxb-3 (r5 and r6) was also repressed. No
RA inhibition was ever seen for Hoxb-4 (r7 and r8) or for
more posteriorly expressed genes. It is notable that this
sequence of hindbrain inhibition thresholds begins at
an RA concentration (1027 M) that is lower than the
repression threshold for forebrain and midbrain development, suggesting that these aspects are separable
(Figs. 1, 2, Table 1).
Along with repressing gene expression, RA induced
expression of hindbrain markers (but not of forebrain or
midbrain markers) in vivo. This was especially obvious
for Krox-20, Hoxb-3, and Hoxb-4 in whole embryos
(Figs. 1, 2; see also Conlon and Rossant, 1992). Low RA
concentrations have also been shown to induce anterior
expansion of Hoxb-1 expression in murine and chicken
embryos (Morriss-Kay et al., 1991; Conlon and Rossant,
1992; Marshall et al., 1992; Sundin and Eichele, 1992;
Wood et al., 1994; Simeone et al., 1995). We did not see
this in our experiments, possibly because induced
Hoxb-1 expression was too diffuse to be detected by in
situ hybridization, although this was detectable by
RNAse protection (see Fig. 3). RA-mediated induction
was also obvious for a range of markers in explants in
vitro (see below).
RA, so that it overlaps with Hoxb-1 expression adjacent to the otic vesicle.
There are also dispersed spots of expressing cells visible anterior to the
otic vesicle and weak staining in the post-otic hindbrain. In embryos that
were treated with 2.5 3 1027 M RA (not shown) or with 5 3 1027 RA (G),
Hoxb-3 expression extends up to the anterior end of the brain. No
localized Hoxb-3 expression is visible in H. In J, strong Hoxb-4 expression
is anteriorized by 1027 M RA so that it extends up to the otic vesicle, and
weaker expression continues more anteriorly. In K and L and in embryos
that were treated with 2.5 3 1027 M RA (not shown), expression extends
up to the anterior end of the central nervous system (CNS). Note that
somites continued to express Hoxb-4 in RA-treated embryos. N,O: Effect
of 1027 M RA (N) and 1026 M RA (O) on Hoxb-5 expression. Q,R: Effect of
1027 M RA (Q) and 1026 M RA (R) on Hoxb-7 expression in embryos. T,U:
Effect of 1027 M RA (T) and 1026 M RA (U) on Hoxb-9 expression. High
concentrations of RA caused tail truncations and a corresponding shortening of the 58 Hoxb gene expression domains. The spinal cord also appears
somewhat thickened (O,R,U). There was no clear anteriorization of the 58
Hoxb genes, Hoxb-5 through Hoxb-9, within the hindbrain in these
TABLE 1. Concentration-Dependent Effects
of Retinoic Acid on Anteroposterior Marker
Expression in Whole Embryos: Summary
of In Situ Hybridization Dataa
aThe left hand column is a schematic representation of the
central nervous system (CNS). The zones where each marker
gene is expressed most strongly in untreated embryos are
shown in the second column headed ‘‘Control.’’ For simplicity,
Hoxb-3 (strongest expression in rhombomeres 5 and 6; r5 and
r6) is shown adjacent to r6, and Hoxb-5 and Hoxb-7 (anterior
expression border in the posterior hindbrain) are shown
adjacent to the spinal cord. The effects of different concentrations of retinoic acid (RA) on marker gene expression in whole
embryos (see Figs. 1 and 2) are shown in the right hand set of
columns as follows: 1, expression detectable in the CNS; 11
and 111, expression in an extended zone along the CNS; 2,
no expression detectable in the CNS. fb, Forebrain; mb,
midbrain; hb, hindbrain; r1–r8, rhombomeres 1–8; SC, spinal
RA induction obviously occurred at concentrations
below those required for repression of the same genes,
indicating RA concentration windows for inducing gene
expression. We note further that extended gene expression zones, as observed here, are a prediction of gradient models in which anterior and posterior gene expression boundaries are set by activation and inhibition
threshold concentrations of a morphogen and in which
ectopic addition of a signaling molecule causes flattening of the gradient.
The phenotypes described above were observed at
tailbud stage 29–31, when morphological markers facilitated identification of a-p level. Because RA has teratogenic effects at much earlier stages, we examined the
timing of RA effects on gene expression. RA-induced
changes in gene expression occurred early and were
stable; they were very similar in embryos that were
harvested at the early neurula stage (Fig. 4, stage 14)
and in larvae harvested at stage 29–31 (described
Fig. 3. RNase protection analysis of Otx-2 and 38 Hoxb gene expression in fragments of control and RA-treated embryos. Our in situ
hybridization data showed that high concentrations of RA abolished
hindbrain-localized expression of Hoxb-1 and Hoxb-3. Similar treatments
were previously shown by RNase protection analysis to cause increased
expression of these genes (Dekker et al., 1992a,b; Leroy and De
Robertis, 1992). Here, we investigated the localization of RA-induced
transcripts. RNA from untreated (CON) tailbud (stage 28) embryos and
from embryos that were treated with 1026 M RA from stage 10 onward
(RA) was analyzed for expression of Otx-2 (otx), Hoxb-1 (b1), and Hoxb-3
(b3). Xom 62/9 (xhom) was used as an internal control for the loading. The
RNA samples analyzed were from whole embryos (WE) and from
embryos that were cut into three fragments: an anterior piece (Ant) that
included all tissues up to the level of the hindbrain/spinal cord border, a
posterior dorsal piece (PD) that was cut just ventral to the somites, and the
remaining posterior ventral structures (PV). Otx-2 expression was restricted to the anterior fragment in control embryos and was absent in
RA-treated embryos. In control embryos, Hoxb-1 and Hoxb-3 were
expressed most strongly in the fragments that included dorsal tissue both
in the fragment containing hindbrain (Ant) and in the more posterior dorsal
fragment (PD). There was also very weak expression in the posterior
ventral fragment, in which bands were clearly visible only after longer
exposure of the autoradiograph. In RA-treated embryos, there was a clear
up-regulation of both Hoxb-1 and Hoxb-3 in all fragments, suggesting that
there is widespread, RA-inducible expression that is not detectable by
using in situ hybridization.
The effects of RA on whole embryos are complex, and
RA induction is difficult to detect against the background of endogenous gene expression. We therefore
used an in vitro system to examine RA posteriorization
of developing anterior neural tissue directly: animal
caps from embryos injected with mRNA for the anterior
neural inducer noggin (Lamb et al., 1993). The results
were assayed by using RNAse protection assays (Fig. 5)
and with wholemount in situ hybridization (Fig. 6).
We found that noggin injection induced expression of
the general neural marker N-CAM. These neutralized
explants also expressed the forebrain marker Otx-2 but
not six (hindbrain and/or spinal cord expressed) Hox
genes that were examined. This supports previous
conclusions that noggin-induced neurectoderm has anterior specification (Lamb et al., 1993). RA treatment of
these explants down-regulated Otx-2 but not N-CAM,
Fig. 4. Effect of RA treatment at the early gastrula stage on Hoxb-3
expression in early neurula stage embryos, as shown by wholemount in
situ hybridization. A: Hoxb-3 transcripts in a control stage 14 embryo.
B,C: Hoxb-3 expression in stage 14 early neurulae following treatment at
the early gastrula stage (stage 10) with 1027 M RA (B) and with 1026 M RA
(C). Anterior is to the left in all cases. The expression patterns are very
similar to those seen at stage 29–31 (Fig. 2). RA-induced effects on the
Hoxb-1, Hoxb-4, and Hoxb-9 expression patterns also showed very little
change between the early neurula stage and the tailbud stage (not
and induced expression of some Hox genes, confirming
previous findings that RA posteriorizes noggin explants
(Papalopulu and Kintner, 1996).
All Hox genes expressed predominantly in the hindbrain (Hoxb-1, Hoxb-3, Hoxb-4) were strongly inducible
in noggin explants by continuous RA treatment. Hoxb-5,
which is expressed in the posterior hindbrain as well as
the spinal cord, was also inducible. Two other 58 posterior Hox genes were far less inducible. Hoxb-7 was not
induced at all, and Hoxb-9 was induced at a low level.
Intriguingly, the apparent cut-off point in inducibility
found here, between Hoxb-5 and Hoxb-7, coincides with
the division between the Antennapedia and Bithorax
Fig. 5. RA-induced activation of Hoxb gene expression in noggininjected animal caps. RNase protection assays were used to analyze the
expression of otx-2 (Otx), neural cell adhesion molecule (NCAM), and six
of the Hoxb genes: Hoxb-1 (b-1), Hoxb-3 (b-3), Hoxb-4 (b-4), Hoxb-5
(b-5), Hoxb-7 (b-7), and Hoxb-9 (b-9) in noggin-injected (Noggin AC) and
control (NIC AC) animal caps in the absence of RA (C) or following
addition of 1026 M RA (6), 1027 M RA (7), or 1028 M RA (8) at the early
gastrula stage (stage 10). Animal caps were dissected out at stage 10,
and the caps and control whole embryos (WE) were harvested at stage
23–25 for analysis. The data presented were obtained from a number of
experiments. Expression of each gene was tested in at least two
experiments, and the results obtained were consistent. Otx-2 was
expressed in noggin-injected animal caps and was also detectable in
control caps (see also Lamb and Harland, 1995; Itoh and Sokol, 1997),
and, in both cases, this expression was down-regulated by RA. No Hoxb
gene expression was detectable in non-RA-treated animal caps. However, expression of Hoxb-1, Hoxb-3, Hoxb-4, and Hoxb-5, but not of
Hoxb-7 or Hoxb-9, was detectable following exposure of noggin-induced
caps to 1028 M RA. The expression levels of the induced genes increased
with exposure to increasing concentrations of RA. Hoxb-4 expression was
induced to levels several-fold higher than expression of the other
RA-inducible genes. Low levels of Hoxb-9 expression were detectable in
noggin-induced animal caps that were treated with high concentrations of
RA. Activation of Hox gene expression also occurred in control animal
caps in response to RA (see also Kolm and Sive, 1995). Hoxb-1 through
Hoxb-5 were expressed in animal caps that were treated with 1026 M RA.
Only noggin-injected animal caps expressed the neural marker N-CAM,
and expression levels were increased by the addition of RA.
complexes in Drosophila (Akam, 1989; Duboule and
Dollé, 1989; Kappen and Ruddle, 1993).
The five RA-inducible hindbrain Hox genes showed a
colinear, anterior-to-posterior sequence of concentra-
Fig. 6. A–Q: RA-mediated transformation in vitro. Wholemount in situ
hybridization analysis of animal cap explants. Control, whole, stage 26
embryos were hybridized with antisense probes for Otx-2 (A), En-2 (B),
and Hoxb-4 (C). D–O: Animal cap explants taken at stage 10 from
noggin-injected, fertilized eggs and cultured to stage 26. D–F: Expression
of Otx-2 (D), en-2 (E), and Hoxb-4 (F) in explants cultured in the absence
of RA. Only Otx-2 transcripts were detected in the noggin-induced
explants. The level of Otx-2 expression was very variable, but a high
percentage of explants (22/30; 73%) contained detectable Otx-2 transcripts. In D, only the explant marked with an asterisk was unstained. G–I:
Expression of Otx-2 (G), en-2 (H), and Hoxb-4 (I) in noggin-expressing
explants that were treated with 1027 M RA from stage 10 onward.
Expression of Otx-2 was down-regulated in many explants (only 9/24
showed detectable Otx-2 expression; 37%), but no induction of en-2 or
Hoxb-4 was evident. We also assayed for Hoxb-3 and Hoxb-9 expression
in RA-treated explants. However, no expression was detected at any of
the RA concentrations tested. The in situ hybridization technique appears
to be less sensitive than RNAse protection assays, in which 38 Hoxb gene
expression was already detectable in explants treated with low concentrations of RA. J–L: Expression of Otx-2 (J), en-2 (K), and Hoxb-4 (L)
following treatment with 5 3 1027 M RA. There was a further decrease in
the number of Otx-2-expressing explants at this RA concentration to 3/23
(13%), and there was an induction of Hoxb-4 expression in a low
percentage of cases (4/22; 18%). In L, Hoxb-4 expression is visible
(arrow) in one of the explants. M–O: Expression of Otx-2 (M), en-2 (N),
and Hoxb-4 (O) following treatment with 1026 M RA. Only Hoxb-4 is
expressed detectably. A very high percentage of explants were Hoxb-4
positive (21/23; 91%). The expression in many of the explants appears to
be at a much higher level than in the CNS of control embryos. This
expression was apparently unpatterned in contrast to the effects of
fibroblast growth factor (FGF) on noggin-treated, anterior neural tissue
(Cox and Hemmati-Brivanlou, 1995; Lamb and Harland, 1995). P: Animal
caps dissected from uninjected embryos and hybridized to an antisense
Otx-2 probe. No transcripts were detectable. Q: Animal caps dissected
from uninjected embryos and treated with 1026 M RA did not hybridize to a
Hoxb-4 probe at detectable levels.
Fig. 7. Hoxb-1 through Hoxb-5
are induced colinearly by RA in noggin-injected animal caps: Hoxb-1 is
induced to 50% of its maximal expression level by a lower concentration of RA than are successively
more 58 Hoxb genes. Hoxb gene
transcript levels were determined
relative to those of the internal control, Xom 62/9, by phosphorimager
analysis of RNase protection assay
gels (see Fig. 5). Expression levels
induced by 1026 M RA were taken to
represent 100% expression. Expression levels of Hoxb-7 and Hoxb-9
were undetectable and very low, respectively, and are not included here.
Fig. 8. RA concentration-dependent induction of Hoxb gene expression in noggin-injected animal caps.
Phosphorimager analysis of RNase
protection gels (see Fig. 5) was used
to determine the expression levels of
six Hoxb genes relative to the expression of the internal standard, Xom
62/9. The total expression units are
arbitrary. Hoxb-7 and Hoxb-9 show
very little response to the RA treatment. However, there is a partial
colinearity in the degree of induction
by 1026 M RA of the more 38 Hoxb
genes. Hoxb-1 is induced the least,
and progressively more 58 genes are
induced to progressively higher levels, with the exception that Hoxb-4 is
induced to much higher levels than
all of the other Hoxb genes tested.
tion dependences for induction. Hoxb-1 (r4) was induced to 50% maximal expression by 2.6 3 1028 M RA,
Hoxb-3 (r5 and r6) was induced by 2.0 3 1027 M, Hoxb-4
(r7 and r8) was induced by 2.1 3 1027 M, and Hoxb-5
(r8; spinal cord) was induced by 3.5 3 1027 M (Fig. 7).
Besides colinearity in the RA-concentration-dependent half maximal induction of Hox genes, we observed
that the maximal levels of induction were successively
greater for successively more 58 RA-inducible Hoxb
genes. Hoxb-4 expression could be induced to levels
several fold higher than expression of the other induced
Hoxb genes (Fig. 8).
It is not yet known how different combinations and
expression levels of Hox genes affect a-p-specification.
Our findings suggest, however, that an integral, RAsensitive mechanism patterns the hindbrain.
Toward a Model for Retinoid Patterning of the
The data described above show that a retinoid may be
involved in an early subdivision of the CNS, transforming presumptive anterior CNS into neural tissue with
hindbrain specification. Subsequently, a gradient of a
retinoid morphogen may be involved in providing an
informational code for a-p level in the developing
hindbrain, because different hindbrain patterning genes
are induced and repressed by different RA concentrations that correlate with their axial expression levels.
The data suggest the existence of sequentially higher
RA concentration windows for induction of sequentially
more posterior hindbrain genes, resembling the induction of successively more dorsoanterior markers by
successively higher concentration windows of activin
(Green et al., 1992, 1994).
Retinoid patterning activity in the hindbrain is likely
to be mediated at least in part through direct interactions of retinoid receptors with Hox genes. RA response
elements have been found in the vicinity of several
paralogue group 1 and group 4 Hox genes (Langston
and Gudas, 1992; Pöpperl and Featherstone, 1993;
Marshall et al., 1994; Studer et al., 1994; Frasch et al.,
1995; Ogura and Evans, 1995a,b; Doerksen et al., 1996;
Morrison et al., 1996; Dupé et al., 1997; Langston et al.,
The question arises whether a gradient of endogenous active retinoid that could pattern the developing
hindbrain is available in vivo during embryogenesis. At
the gastrula and early neurula stages, when neural Hox
gene expression patterns are established, Xenopus embryos contain active retinoids at physiologically relevant concentrations (Chen et al., 1992, 1994; Pijnappel
et al., 1993; Creech Kraft et al., 1994; Blumberg et al.,
1996). It has also been reported that the late (posterior)
organizer regions of vertebrate embryos (chicken,
mouse, and Xenopus) are sources of active retinoids
(Chen et al., 1992, 1994; Hogan et al., 1992) and that
Xenopus embryos contain a tenfold posterior-to-anterior concentration gradient of an active retinoid at the
early neurula stage (Chen et al., 1994).
The RA concentrations at which different hindbrain
patterning genes were induced or repressed in Xenopus
embryos or explants in this study (1028 to 1026 M) are
high, suggesting that RA itself is not the morphogen in
vivo. However, there are selectively active retinoids
that are available in early Xenopus embryos and that
posteriorize the early embryo with very high efficiency
(Pijnappel et al., 1993; unpublished observations).
ously (Godsave et al., 1994). RA (Acros) was prepared as
a 1022 M stock in dimethyl sulfoxide (DMSO) and
stored at 280°C in small aliquots. Twenty to thirty
embryos were exposed to RA continuously from stage 10
onward in the dark in 7 ml tap water containing 25
(g/ml gentamycin and a range of concentrations of RA
(from 1028 M to 1026 M). Concentrations higher than
1026 M were toxic. Continuous exposure to RA was used
to ensure that the treatment lasted for the entire period
of a-p patterning in the CNS. In most cases, the effects
on gene expression were analyzed at tailbud stage
29–31, when morphological landmarks are available to
facilitate identification of gene expression zones. Embryos were demembranated manually and fixed for
wholemount in situ hybridization in MEMFA (Harland,
1991) for 2 hours. They were washed once in 100%
methanol and stored at 220°C in fresh methanol.
Embryos used for RNAse protections were frozen in
liquid nitrogen and stored at 280°C.
General Conclusions
In Situ Hybridization
In conclusion, our data show that RA can differentially regulate genes of the Hoxb cluster in neural
tissue. RA concentration-dependent induction of Hoxb
and other a-p patterning genes suggest a mechanism
whereby retinoids can contribute to setting up a series
of expression zones of genes involved in providing a-p
positional information. It is clear that patterning of the
main body axis occurs in several steps and that retinoids may be involved at several stages. It is also clear
that other signals are required to generate the complete
range of axial pattern. The Xenopus embryo is an
excellent model system for further investigations in
this area.
The in situ hybridization probes used were Otx-2, a
gene that is expressed in the forebrain and midbrain
(Fig. 1A; Blitz and Cho, 1995; Pannese et al., 1995);
en-2, a marker of the midbrain-hindbrain border (Fig.
1D; Hemmati-Brivanlou et al., 1991; Eizema et al.,
1994); Krox-20, which is expressed in r3 and r5 of the
hindbrain (Fig. 1G; Bradley et al., 1992); and six of the
Hoxb genes, Hoxb-1, Hoxb-3, Hoxb-4, Hoxb-5, Hoxb-7,
and Hoxb-9 (Fig. 2A,E,I,M,P,S; Godsave et al., 1994;
Godsave and Durston, 1997). Hoxb-1 through Hoxb-4
are expressed in sequential zones along the hindbrain.
The rhombomeres in which expression of these Hoxb
genes is strongest are r4 for Hoxb-1, r5 and r6 for
Hoxb-3, and r7 and r8 for Hoxb-4. The anterior expression boundaries of Hoxb-5 and Hoxb-7 are near the
posterior end of the hindbrain, and expression extends
along the spinal cord, whereas Hoxb-9 expression is
restricted to the spinal cord.
Embryos and RA treatments
Xenopus embryos were obtained by in vitro fertilization, dejellied, cultured, and staged as described previ-
Fertilized eggs were dejellied, transferred into 3%
ficoll in 25% MMR solution (Newport and Kirschner,
1982), and microinjected in the animal hemisphere
with 150 pg noggin D58 mRNA (Smith and Harland,
1992). At the early blastula stage, embryos were transferred into 20% MMR and cultured overnight at 14°C to
stage 10. The resulting early gastrulae were placed in
Flickinger medium (Flickinger, 1949) containing 25
µg/ml gentamycin, the vitelline membranes were removed by using sharpened forceps, and animal caps
were dissected out by using electrolytically sharpened
tungsten needles. Explants were cultured individually
in 1% agarose-coated wells of 96-well plates, in 100 µl
Flickinger medium containing a range of concentrations of retinoic acid, at 20°C until control embryos
reached stage 23–27. They were then fixed for in situ
hybridization as for whole embryos or frozen in liquid
nitrogen for RNAse protection analysis.
Digoxigenin-labeled probes were generated by in
vitro transcription of linearized templates, incorporating digoxigenin-11-UTP according to the manufacturer’s instructions (Boehringer-Mannheim, Indianapolis,
IN). Antisense probes for Otx-2, En-2, Hoxb-3, Hoxb-4,
and Hoxb-9 were as described in Godsave and Durston
(1997). Antisense Krox-20 probe was as described by
Bradley et al. (1992). Antisense Hoxb-1 probe was a
900-nucleotide (nt) sequence recognizing a part of the
homeobox and more 58 coding sequence. The antisense
Hoxb-5 probe was a 1,200-nt sequence complimentary
to the complete coding region of the Hoxb-5 gene. The
Hoxb-7 probe recognized a 300-nt coding sequence
upstream from the homeobox. A subclone of XlHbox2
cDNA (Wright et al., 1987) was used as the template. A
sense Hoxb-3 probe was used as control for nonspecific
in situ hybridization staining (results not shown). The
in situ hybridization procedure was as described by
Harland (1991), with slight modifications as described
by Godsave and Durston (1997). Pigmented embryos
and explants were bleached following staining and
fixation in MEMFA (see Godsave and Durston, 1997).
RNase Protection Analysis
RNA was isolated from embryonic material by using
proteinase K, as described by Ruiz i Altaba (1993).
RNAse protection assays were performed as described
by Dekker et al. (1992a) by using a32P (UTP) to label
the antisense RNA probes. The Otx-2 probe was that
described by Pannese et al. (1995), the neural cell
adhesion molecule (N-CAM) probe was that described
by Mayor et al. (1995), and the Hoxb probes were those
described by Dekker et al. (1992a).
We are grateful to Richard Morgan and Pim Pijnappel for critical reading of the paper. We thank Richard
Harland for the noggin-expression clone; Ken Cho and
Maria Pannese for otx-2 in situ and RNAse protection
clones, respectively; David Wilkinson for the Krox-20
clone; and Richard Morgan for the N-CAM clone. S.F.G.
and J.v.d.W. were funded by Netherlands Science Organization (NWO) Life Sciences research grants 805–33–
021 and 417442, respectively. A.J.D. thanks the EU
Biotech Programme (contract ERBBIO4CT960378) for
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