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Mechanism of hyperthermia effects on CNS development Rostral gene expression domains remain despite severe head truncation and the hindbrainotocyst relationship is altered

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TERATOLOGY 59:139–147 (1999)
Mechanism of Hyperthermia Effects on
CNS Development: Rostral Gene Expression
Domains Remain, Despite Severe Head
Truncation; and the Hindbrain/Otocyst
Relationship Is Altered
of Experimental Medicine, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic
2Department of Anatomy and Developmental Biology, St. George’s Hospital Medical School, London SW17 ORE,
United Kingdom
To study the mechanism of hyperthermia on the development of the rostral neural tube,
we used a model in which closely-staged presomite
9.5-day rat embryos were exposed in culture to 437C
for 13 min, and then cultured further for 12–48 hr. This
treatment had little effect on the development of the
rest of the embryo, but resulted in a spectrum of brain
defects, the most severe being a lack of all forebrain
and midbrain structures. Whole-mount in situ hybridisation was used to monitor the expression domains of
Otx2, Emx2, Krox20, and hoxb1. These showed that
there were no ectopic expression patterns, for any
gene at any stage examined. Even in those embryos
which apparently lacked all forebrain and midbrain
structures, there were expression domains of Otx2 and
Emx2 in the most rostral neural tissue, and these
retained their nested dorso-ventral boundaries, showing that cells fated to form rostral brain were not wholly
eliminated. Thus, heat-induced rostral neural tube truncation is of a quite different mechanism from the
respecification proposed for retinoic acid, despite their
very similar phenotypes. In the hindbrain region of
treated embryos, we observed decreased intensity of
Krox20, staining and an abnormal relationship developed between the position of hoxb1 expression and the
otocyst and pharyngeal arches. In the most extreme
cases, this domain was shifted to be more caudal than
the rostral edge of the otocyst, while the otocyst
retained its normal position relative to the pharyngeal
arches. We interpret this as a growth imbalance between neuroepithelium and overlying tissues, perhaps
due to a disruption of signals from the midbrain/
hindbrain boundary. Teratology 59:139–147, 1999.
r 1999 Wiley-Liss, Inc.
Hyperthermia can be teratogenic in all experimental
species tested, and is probably also teratogenic in
humans (reviewed by Edwards et al., ’95). The development of the brain is particularly sensitive to hyperthermia. In rats, the most sensitive period for the induction
of head defects is during late gastrulation, between
9–10 days of embryonic age (Webster et al., ’85). Lack of
development of eyes from the forebrain, and open
neural tube in the midbrain region were the most
commonly observed defects at term. This sensitivity of
the rostral neural tube is also seen when gastrulating
rat embryos are exposed to hyperthermia in culture
(Walsh et al., ’87, ’97). When observed at about the
25-somite stage, the most severely affected embryos
appeared to lack the whole of the fore- and midbrain
(Walsh et al., ’87, ’97).
The cellular response to heat is increasingly welldocumented (reviewed by Nover and Scharf, ’97), but
the role of heat shock proteins and other aspects of the
response in the mechanism of developmental disruption remain obscure, despite much study (see Thayer
and Mirkes, ’97). Recent research on the cellular response in heat-exposed embryos has concentrated on
the pathway leading to apoptosis (e.g., Mirkes et al.,
’97). It is well-known that hyperthermia induces cell
death in embryos (see Walsh et al., ’97), but again, the
mechanism of induction is unknown, and the role in
teratogenesis is unclear.
The induction, by heat, of embryos that lack rostral
neural structures is reminiscent both of retinoic acid
(RA) treatment, and of the targeted mutation of genes
required for rostral development. When gastrulating
vertebrate embryos are exposed to high levels of exogenous RA, the development of the rostral neural tube
can be severely affected or truncated (Durston et al.,
’89; Conlon and Rossant, ’92; Simeone et al., ’95;
Avantaggiato et al., ’96). This is generally thought to be
because RA causes a rostral expansion of the expression
Grant sponsor: Grant Agency of the Czech Republic; Grant numbers:
304/1716/1994, 304/0909/1996; Grant sponsor: Biotech; Grant number: BIO2 CT-930107; Grant sponsor: UK Medical Research Council.
*Correspondence to: Daniela Buckiová, Institute of Experimental
Medicine, Academy of Sciences of the Czech Republic, Vı́deòská 1083,
142 20 Prague 4, Czech Republic. E-mail: bucki@
Received 15 July 1997; Accepted 24 September 1998
domains of certain specifying genes, which results in
the transformation of rostral cells to a more caudal
phenotype (Simeone et al., ’95; Avantaggiato et al., ’96).
Similarly, the rostral neural tube does not develop in
mice with targeted mutations of Lim1 or Otx2, transcription factors required in head specification (Shawlot and
Behringer, ’95; Acampora et al., ’95; Matsuo et al., ’95;
Ang et al., ’96).
Hyperthermia is known to affect gene and protein
expression, both as general repression in response to
heat shock, and in more specific ways: e.g., the protein
product of the fushi tarazu gene in Drosophila is
stabilized by heat shock, resulting in an overexpression
phenotype (Welte et al., ’95). To investigate the possibility that hyperthermia causes abnormalities of the
rostral neural tube by altering the expression of developmental control genes, we monitored the temporal and
spatial expression of key genes in heat-treated rat
embryos. We utilized whole-embryo culture to allow
precise staging of embryos at the time of hyperthermia,
and used whole-mount in situ hybridization to detect
the expression of: Emx2, which is expressed in dorsal
forebrain (Simeone et al., ’92); Oxt2, which is expressed
throughout the fore- and midbrain (Simeone et al., ’93);
Krox20, which is expressed in rhombomeres 3 and 5 of
the hindbrain (Wilkinson et al., ’89); and hoxb1, which
is restricted to rhombomere 4 in cranial expression
(Murphy et al., ’89). Each of these genes encodes a
transcription factor, of the homeobox or zinc finger
(Krox20) type. Expression of receptor tyrosine kinase
gene EphA4 (Sek-1) is restricted to rhombomeres 3 and
5 after the segmentation of hindbrain (Irwing et al.,
bottles were briefly cooled, returned to the 38°C incubator, and cultured for 12, 24, or 48 hr.
Twenty-four hours following heat treatment
Whole-embryo culture
Forty control and 98 treated embryos were morphologically examined. After 24 hr of culture, untreated
embryos had developed 10–12 pairs of somites (Fig.
2A–D). The head folds were closed, but the caudal
neuropore was wide open. The otic pit and optic cup
were marked, and the first pharyngeal arch was visible.
Embryos had almost completed turning, to be dorsally
convex. Heat-treated embryos fell into two distinct
groups. About half the embryos were not markedly
affected (Fig. 2E–H). In these, the head folds had closed
normally. Some were slightly retarded, in that the otic
pit was less indented and turning was incomplete (Fig.
2E,F). Also, the optic cup may have been of a slightly
different shape compared to the controls in some embryos (e.g., Fig. 2F), but objective measures were not
taken. There were irregularly shaped somites in a
proportion of embryos (Fig. 2H). In the remaining
heat-treated embryos, the head folds were not fused
(Fig. 2I–K). In some embryos the folds were completely
open (Fig. 2I,J), while in others the folds were fused in
some segments of the brain (Fig. 2K, for an example of
open hindbrain). In those embryos with wholly unfused
head folds (Fig. 2L) , the fore- and midbrain region was
Embryos were cultured as described previously
(Brown et al., ’91). Briefly, Wistar rats were mated
overnight and conceptuses were explanted in the afternoon of day 10 of gestation (plug ⫽ day 1). Embryos
were staged using criteria previously described (Fujinaga et al., ’92), and presomite embryos of late allantoic
bud, early head fold, and late head fold stages (nomenclature of Downs and Davies, ’93) were selected. These
were cultured (4 conceptuses per 4 ml medium) in
5O-ml bottles, on rollers, at 38°C. The culture medium
was 75% immediately centrifuged, heat-inactivated,
0.2-µm filtered rat serum, and 25% Eagle’s minimal
essential medium with Earle’s salts. Embryos were
gassed initially with 5% 02, changed to 20% at 16 hr and
40% at 26 hr, with 5% CO2 and the balance N2.
Exposure to heat
Bottles containing embryos were placed in a 38°C
incubator and rotated for 30 min–1 hr to equilibrate.
Bottles were then placed upright in a water bath for 13
min at 43°C, or 38°C for controls. After treatment,
Whole-mount in situ hybridization
Embryos harvested at 12, 24, or 48 hr following
treatment were rinsed in phosphate-buffered saline
and then fixed in 4% (w/v) paraformaldehyde overnight.
Whole-mount in situ hybridization was performed essentially as described by Wilkinson and Green (’90). Digoxigenin-labeled antisense RNA probes were synthesized
from cDNA fragments of the following lengths, cloned
into the EcoR1 site of pKS: Otx2, 260 bp; Emx2, 600 bp;
Krox20, 700 bp; hoxb1, 800 bp; and EphA4, 1,500 bp.
Twelve hours following heat treatment
The sample sizes, i.e., numbers of heat-exposed and
control embryos morphologically examined, were 44
controls and 82 exposed. After 12 hr of culture, untreated embryos had developed, on average, six pairs of
somites (Fig. 1A–D). The head folds were elevated, but
not opposed, and embryos had not begun body turning.
Compared to controls, heat-treated embryos were not of
markedly different morphology (Fig. 1E,F). Some embryos were retarded, and in some cases this appeared
more pronounced in the most rostral portion of the head
(e.g., Fig. 1F), but this was not dramatic in any embryo.
The neural groove in the caudal portion of the embryo
was irregular in a small proportion of embryos (e.g.,
Fig. 1H). Although there was some variability in morphology of the rostral neural tubes, embryos did not fall
into obviously different groups.
Fig. 1. Embryos of 4–6 somites, 12 hr following heat treatment.
Whole-mount in situ hybridization of Otx2 (A, E), Emx2 (B, F),
Krox20 (C, G), and hoxb1 (D, H) in control (A–D) and treated (E–H)
embryos. A, B, E, F: Right lateral views. C, D, G, H: Dorsal views. The
rostral (arrow) and caudal (arrowhead) margins of Otx2 and Emx2
expression are marked. Note the diminished expression of Krox20 in
rhombomere 3 of the treated embryo (double arrowhead, G), and the
irregular neural groove (H).
small, the optic cup was usually not visible, and the otic
placode was not indented (Fig. 2I,J). Appearance of
growth retardation was not dependent on embryonic
stage at time of treatment, as seen in comparison of 16
controls and 38 treated embryos. The ratio of retarded
and fully developed embryos (evaluation based on level
of open anterior neural plate) was 1.0.
about 30% of embryos the head region was grossly
normal (Fig. 3D). In contrast, there was a spectrum of
head defects in the remaining 70% of treated embryos.
In the most severe cases, the whole of the fore- and
midbrain region appeared to be missing (Fig. 3J,K).
Obviously, there was no optic primordium in these
embryos, but the otocyst had developed. The optic
primordium was also not visible in slightly less affected
embryos, which had a neural tube rostral to the hindbrain, but without any of the morphological features of
the mid- or forebrain (Fig. 3G,L). Still less affected
embryos had an optic primordium, but reduced foreand midbrain regions, which were unfused in some
cases (Fig. 3F, for an example of unfused midbrain).
Treated embryos were significantly retarded in any
Forty-eight hours following heat treatment
After 48 hr of culture, untreated embryos (n ⫽ 40)
had developed 22–24 pairs of somites (Fig. 3A–C). The
optic stalk was closing, the otocyst was closed, and
three pharyngeal arches were visible. In all heattreated embryos (n ⫽ 136), the heart and structures
more caudal were overtly normal (not shown), and in
Fig. 2. Embryos of 10–12 somites, 24 hr following heat treatment.
Whole-mount in situ hybridization of Otx2 (A, E, I), Emx2 (B, F, J),
Krox20 (C, G, K), and hoxb1 (D, H, L) in control embryos (A–D),
treated embryos with mild phenotype with closed head folds (E–H),
and treated embryos with severe phenotype with open head folds
(I–L). Right lateral views. The rostral (arrows) and caudal (arrowheads) margins of Otx2 and Emx2 expression are marked. Note the
expression of Krox20 in migrating crest cells in the control embryo
(arrow in C) and the diminished expression of Krox20 in rhombomere 3
of the treated embryos (arrow in K).
tested parameters by morphological scoring system
(yolk sac diameter, crown-rump, head length, or somites)
(Brown, ’90).
The proportions of embryos with differing phenotypes were compared with the exact embryonic stage at
the time of explantation and heat treatment (Table 1).
Embryos were categorized into three groups, based on
rostral brain morphology: grossly normal; reduced; or
truncated/missing (no optic primordium). All control
embryos were normal. Each phenotype was found following treatment at late allantoic bud, early head fold, or
late head fold stages. The proportions were similar
following exposure at late allantoic bud, and early head
fold stages, with 70–75% of embryos affected, most of
Fig. 3. Cranial portions of embryos of 22–24 somites, 48 hr following
heat treatment. Whole-mount in situ hybridization of Otx2 (A, D, G,
J), Emx2 (B, E, K), and hoxb1 (C, F, H, L) in control embryos (A–C)
and treated embryos (D–L) of various phenotypes. Right lateral views,
except for ventral views in J and K. The rostral (black arrowheads)
and caudal (black arrows) margins of expression are marked. For the
hoxb1 embryos, the rostral edge of the otocyst (white arrowheads) and
the middle of the first pharyngeal arch are also marked (white arrow).
TABLE 1. Stage variation in the proportions
of phenotypes 48 hr after exposure of presomite
rat embryos to hyperthermia in culture*
Fore/midbrain phenotype
embryos Normal Reduced Truncated
Stage at
Late allantoic bud
Early head fold
Late head fold
*The truncated phenotype was defined as absence of the optic
primordium, while the reduced phenotype had no telencephalic evaginations.
which had missing or severely truncated fore- and
midbrain regions. Following treatment at the late head
fold stage, fewer embryos were affected (about 60%),
less than half of which had missing or truncated rostral
brain structures. Because the phenotypes did not differ
markedly between the different treatment stages, these
are pooled in the following descriptions of gene expression patterns. Finally, it is worth noting that there were
no overt signs of necrosis, observable as opaque areas
following some chemical treatments, in any heattreated embryo.
Gene expression patterns
Twelve hours following heat treatment. In 4–6somite embryos 12 hr following heat treatment, there
was no obvious difference in the expression domains of
Otx2, Emx2, or hoxb1 compared to control embryos.
Otx2 was expressed from the most rostral tip of the
neural tube, including the ventral portion of the forebrain, to a caudal margin just rostral to the preotic
sulcus (Fig. 1A,E). The domain of Emx2 was smaller,
did not include the ventral lip of the forebrain, and had
a distinctly more rostral caudal margin than Otx2 (Fig.
1B,F). Hoxb1 was expressed in a band just caudal to the
preotic sulcus, representing rhombomere 4, throughout
the latero-medial extent of the neural tube (Fig. 1D,H).
In control embryos, Krox20 was expressed in the characteristic pattern of two bands, representing rhombomeres 3 and 5, again throughout the latero-medial
extent of the neural tube (Fig. 1C). In some embryos, a
population of expressing cells was observed emanating
from the neural crest at the caudal edge of the rhombomere 5 tube (Fig. 1C). In many treated embryos, the
Krox20 expression domain in r3 was very faint (double
arrow in Fig. 1G) or absent, and that in rhombomere 5
was reduced in the latero-medial plane (Fig. 1G).
However, the rostro-caudal dimension of the two domains, and the gap between them, as well as their
location relative to the preotic sulcus, appeared normal.
Twenty-four hours following heat treatment.
In 10–12-somite control embryos, Otx2 was expressed
throughout the dorsal and ventral forebrain and midbrain, with a caudal border in the region of the midbrain/
hindbrain junction (arrowhead in Fig. 2A). Emx2 expres-
sion was restricted to the dorsal forebrain, and was also
seen in the lateral ridge, adjacent to the most caudal
somites (Fig. 2B). In the brain, the caudal margin of
expression was just caudal to the forebrain/midbrain
sulcus (arrowhead in Fig. 2B). As before, Krox20 was
observed in rhombomeres 3 and 5, which overlay the
rostral two thirds of the otic pit, and in migrating
neural crest cells (arrow in Fig. 2C). Hoxb1 was expressed in rhombomere 4, rostral to the otic pit, and in
the primitive streak (Fig. 2D). In those heat-treated
embryos in which the rostral neural tube was closed,
the expression patterns of Otx2, Emx2, and hoxb1 were
not noticeably different from controls (Fig. 2E,F,H). In
those embryos with smaller fore- and midbrains, compared to controls, domains were correspondingly
smaller, but not ectopic (see Fig. 2F for Emx2). However, in these embryos, as at 12 hr, Krox20 was markedly fainter, particularly in rhombomere 3 (Fig. 2G).
In those heat-treated embryos in which the rostral
neural tube was open and truncated, the domains of
Otx2 and Emx2 were abnormally shaped, but this
appeared to be in register with the abnormal morphology (Fig. 2I,J). In some cases, Otx2 expression appeared
to be stronger in the ventral neural tube (although still
with a caudal margin at the presumed midbrain/
hindbrain junction, Fig. 2I) but this seemed to be
characteristic of the elevated neural fold stage, and was
seen in control embryos in this phase of neurulation
(not shown). Even in embryos with a severely reduced
rostral head, Emx2 remained restricted to the most
rostral region, and was excluded from the ventral
portion (Fig. 2J). Krox20 was reduced in the more
severely affected embryos, as before (Fig. 2K), and
again hoxb1 was not affected (Fig. 2L).
Forty-eight hours following heat treatment. In
22–24-somite embryos, the patterns of Otx2 and Emx2
in control and heat-treated embryos were of the same
profile as observed in the 10–12-somite embryos (Fig.
3). The one exception was some heat-treated embryos
with fused head folds, in which Otx2 expression was
less intense in the fore- and midbrain (arrow in Fig.
3D), but which had staining in the roof of the hindbrain
(Fig. 3D). The hindbrain staining was considered to be
unspecific, although this was not observed with other
probes. The reduced staining more rostrally is of unknown significance, since other embryos in this group
had overtly normal levels of expression (not shown).
The most striking finding was that Otx2 and Emx2
domains were seen even in those embryos which apparently lacked fore- and midbrain structures (Fig. 3J,K).
In these cases, Otx2 was expressed in the rostralmost
portion of the truncated head, in a strip from the dorsal
side of the neural tube (arrow in Fig. 3J), to the most
ventral part, adjacent to the pharyngeal cavity (arrowhead in Fig. 3J). Emx2 was also expressed in the most
rostral portion, and from the dorsal edge of the truncated head (arrow in Fig. 3K), but in contrast to Otx2,
expression stopped about halfway to the pharyngeal
cavity (arrowhead in Fig. 3K), leaving a ventral region
without staining.
Krox20 is no longer expressed in the hindbrain at this
stage (not shown). A hoxb1 domain of normal rostrocaudal width was observed in all heat-treated embryos,
but in the more severely affected embryos, this was
reduced in the latero-medial plane (Fig. 3H,L). Strikingly, the position of the hoxb1 domain relative to the
otocyst was shifted in heat-treated embryos. In control
embryos, hoxb1 expression, representing rhombomere
4, lies immediately dorsal to the first pharyngeal arch
(white arrow in Fig. 3C), with a caudal boundary that is
more rostral than the rostral edge of the otocyst (white
arrowhead in Fig. 3C). There was a relative caudal shift
of hoxb1 expression in heat-treated embryos, which was
graded in proportion to the severity of head truncation.
In mildly affected embryos, the expression domain just
overlapped the rostral edge of the otocyst, and was
dorsal to the first pharyngeal arch (Fig. 3F). In more
severely affected embryos, the domain overlapped the
otocyst, and was dorsal to the second pharyngeal arch
(Fig. 3H,L).
As we reported previously (Williams et al., ’97), and is
to be expected, the expression patterns we observed in
cultured rat embryos are indistinguishable from those
described for mouse embryos in vivo. Also, the morphological effects of heat exposure that we observed in
cultured rat embryos are very similar to those previously described (Walsh et al., ’87, ’97). The brief heatshock at immediately presomite stages induced a spectrum of rostral truncation of the neural tube, ranging
from apparent absence of the whole of the fore- and
midbrain, to overtly normal heads. The reason for this
variability is unclear, but is not simply stage-dependence. Similarly, the cause of the particular sensitivity
of the rostral neural tube, more caudal structures being
essentially unaffected, remains obscure.
The major objective of this study was to investigate
the possibility that respecification of tissue, due to
ectopic expression of control genes, might be responsible for rostral truncation. We found no evidence to
support this theory. For all of the genes examined, and
at all stages, there was no evidence for anterior expansion of expression domains, nor any other ectopic
expression. This contrasts with retinoic acid-induced
head truncation, in which marked shifts in both Otx2,
Emx2, and other genes have been reported (Durston et
al., ’89; Conlon and Rossant, ’92; Simeone et al., ’95;
Avantaggiato et al., ’96). In some of these studies,
retinoic acid also caused a relative expansion of hindbrain tissues, which we did not observe. It is notable
that we found regions of Otx2 and Emx2 expression
even in those embryos that appeared to lack all foreand midbrain tissue. In addition, the spatial relationships of these genes were unaltered, despite the grossly
abnormal structures in which they were located. Thus,
Otx2 was expressed throughout the dorso-ventral extent of the most rostral tissue, while Emx2 was excluded from the most ventral portion. This suggests
that a very small fraction of the cells normally fated to
become fore- and midbrain remain in these embryos. It
was not possible to reliably compare the relative caudal
boundaries of Otx2 and Emx2, so it is possible that the
midbrain was eliminated completely, but this was not
the case in slightly less affected embryos (e.g., Fig. 3G).
The very severe reduction in the numbers of fore- and
midbrain cells could be due to induced cell death, or
impaired proliferation, or a combination of both. We
have no direct evidence in either regard, but the
relative lack of phenotypic effects at 4–6 somites, 12 hr
following exposure, suggests that the action of heat was
not an immediate large-scale induction of cell death.
That heads could appear relatively normal at this
stage, yet be severely truncated later, is probably
explained by the differential growth of the brain regions
over this phase. In the period following 4–6 somites
there is a very rapid expansion in the forebrain (MorrissKay and Tuckett, ’87).
The very severe reduction in the numbers of fore- and
midbrain cells could be due to induced cell death,
impaired proliferation, or a combination of both. By the
20-somite stage, the most affected embryos showed a
domain of hoxb1 expression that was significantly more
caudal than normal, relative to both the pharyngeal
arches and the otocyst. In contrast, there was no
indication of a change in the position of the otocyst,
relative to the pharyngeal arches. This is another effect
of heat that contrasts with effects of exogenous retinoic
acid, for which a rostral shift in the position of the
otocyst relative to the pharyngeal arches is well-known
(Webster et al., ’86). The distance of the heat-induced
caudal shift in the relative position of the hoxb1 domain
was, by 20 somites, equivalent to 1 or 2 rhombomeres,
from the normal position where the caudal margin of
expression is rostral to the otocyst, to one in which the
rostral margin is registered with the rostral edge of the
Caudal shift of hoxb1 expression is the only finding of
ectopic expression in this study. We assume that the
expressed domain still represents rhomobomere 4, because hoxb1 (Gavalas et al., ’98; Studer et al., ’98) is
responsible for the establishment and maintenance of
this segment. Expression of other spatially restrictedspecific genes, e.g., Krox20 and EphA4, have been
downregulated at 20 somites embryos.
There was no apparent shift in the positions of either
hoxb1 or Krox20 at 10–12 somites, 24 hr after exposure.
Also, EphA4 (Sek-1) expression that was segmentally
restricted in rhombomere 3 and rhombomere 5 in the
stages (Irwing et al., ’96); confirmed the unchanged
identity of the area (not shown).
However, reductions in the intensity of expression of
Krox20 in rhombomere 3, and in the latero-medial
extent of expression in rhombomere 5, were consis-
tently observed at both 4–6- and 10–12-somite stages.
The exact significance of this is not clear, but may
reflect an effect on proliferation in these regions. Whatever the significance, there was not an equivalent
latero-medial reduction in hoxb1 expression in the
interposed rhombomere 4 at the 4–6- and 10–12-somite
stages, but this was observed at 22–24 somites.
It is possible that the reduction in the midbrain
population, as shown by the much reduced Otx2 domain, may have affected signals from the midbrain/
hindbrain border region. Although the caudal limit of
Otx2 expression is at the midbrain/hindbrain boundary
(Millet et al., ’96), null mutants of Otx2 also lack
rhombomeres 1 and 2 (Acampora et al., ’95; Matsuo et
al., ’95; Ang et al., ’96). This has been suggested to be an
effect on the early precaudal plate, but an alternative
explanation is an action of the isthmus-organizing
region located at the midbrain/hindbrain boundary,
which provides instructive signals that pattern the
adjacent areas (Ang, ’96). Further insight may be
gained by study of Fgf8, a major signal from this region.
Endogenous retinoic acid also plays a role in patterning
the hindbrain, but current evidence does not support a
role for alterations in retinoid signaling in heat-induced
defects. Exogenous retinoic acid can cause ectopic expression, predominantly anteriorization, and transformation in rhombomeres 1–4 (Morris-Kay et al., ’91;
Conlan and Rossant, ’92; Marshall et al., ’92), while
retinoid deficiency, at least in the quail, causes loss of
rhombomeres 4–8 (Maden et al., ’96). Neither of these
responses is similar to our observations of heat-induced
We are grateful to E. Boncinelli, R. Krumlauf, and D.
Wilkinson for the gift of probes. We thank Fiona Mann
and Ian Andrew for technical help and valuable advice.
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