1 Ecole Normale Supérieure de Lyon, Laboratoire de Biologie
Moléculaire de la Cellule, CNRS-UMR5161/INRA-UMR1237, 46, allée
d'Italie, 69364 Lyon Cedex 07, France
2 Marine Biology Research Division, Scripps Institution of Oceanography,
University of California San Diego, La Jolla, CA 92093, USA
* Author for correspondence (e-mail: lzholland{at}ucsd.edu)
Accepted 22 October 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Anterior/posterior patterning, Endoderm, RA signaling pathway, Gene cascade, Evolution, Lancelet, Branchiostoma floridae
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Throughout the chordates, RA signaling specifies anterior/posterior
position of pharyngeal structures such as the gill slits (aquatic chordates)
or pharyngeal arches and pouches (non-aquatic chordates). Excess RA prevents
formation of the gill slits (branchial basket) in tunicates
(Hinman and Degnan, 2000). In
amphioxus the pharynx is absent; the mouth (thought to be a modified gill
slit) and gill slits do not form and the pharyngeal endoderm remains thin
(Escriva et al., 2002
;
Holland and Holland, 1996
).
Conversely, in embryos treated with a RA antagonist, the pharynx with its
thickened endoderm is expanded posteriorly
(Escriva et al., 2002
).
Similarly, in vertebrates, excess RA prevents pharyngeal development in
lampreys and causes fusion of the first two branchial arches in gnathostomes
(Kuratani et al., 1998
;
Lee et al., 1995
;
Mulder et al., 1998
), while
decreased RA signaling has the opposite effect, expanding pharyngeal
structures posteriorly. Consequently, in vertebrates with reduced RA
signaling, the first pharyngeal pouch and the first two pharyngeal arches are
normal, the second pouch is expanded posteriorly and the remaining pouches do
not form (Niederreither et al.,
2003
; Quinlan et al.,
2002
; Wendling et al.,
2000
). Similarly, in amphioxus, the mouth is enlarged and the gill
slit primordia are either elongated or absent altogether; presumably a low
level of RA signaling is essential for gill slit penetration
(Escriva et al., 2002
).
RA signaling in chordates is directly mediated by the RA receptors (RARs),
that heterodimerize with the retinoid X receptors (RXRs)
(Laudet and Gronemeyer, 2001)
In general, vertebrates have three RARs and three RXRs, whereas amphioxus and
tunicates have one each (Bertrand et al.,
2004
; Nagatomo et al.,
2003
). In chordates other than tunicates
(Ishibashi et al., 2003
), RARs
are autoregulated, and their expression generally reflects the level of RA
signaling. In vertebrates, RAR gene expression is generally high in the
foregut endoderm (Matt et al.,
2003
; Smith,
1994
), while in amphioxus, AmphiRAR is most intensely
expressed in the middle third of the endoderm, just posterior to the mouth and
the first three gill slits (Escriva et
al., 2002
).
The effects of altered RA signaling on endodermal expression of RARs are
similar in amphioxus and vertebrates. For example, in the mouse, treatment
with an RA agonist induces ectopic expression of RARß in the
first two pharyngeal pouches (Matt et al.,
2003). Correspondingly, in amphioxus, excess RA expands expression
of AmphiRAR into the anteriormost pharyngeal endoderm, while
treatment with an RA antagonist downregulates RAR
(Escriva et al., 2002
;
Wendling et al., 2000
).
However, little is known of the molecular mechanisms downstream of RAR/RXR
that underlie pharyngeal patterning. In addition to RARs, only a few genes are
known that exhibit altered expression in the pharyngeal endoderm in response
to altered levels of RA signaling. In vertebrates, these include
Hoxa1 and Hoxb1, expressed in the caudal pharynx,
Pax1 and Pax9 expressed in pharyngeal pouches 1-3 and 1-4,
respectively, and Fgf3 and Fgf8 expressed in the endoderm of
the pharyngeal arches and caudal-lateral pharynx respectively
(Neubüser et al., 1995
;
Wallin et al., 1996
;
Wendling et al., 2000
).
Expression of the single Pax1/9 gene in amphioxus is also affected by
increased RA (Holland and Holland,
1996
), while in tunicates, expression of Otx in the
pharynx is decreased by RA treatment
(Hinman and Degnan, 2000
).
Hox1 genes in both vertebrates and amphioxus are direct targets of RA
signaling (Arcioni et al.,
1992; Balmer and Blomhoff,
2002
; Manzanares et al.,
2000
; Ogura and Evans,
1995
). Expression of Hoxa1 and Hoxb1 in the
pharyngeal endoderm of vertebrates is expanded by treatment with RA or an RA
agonist, while treatment with an RA antagonist or mutation of the RA response
element (RARE) markedly decreases Hoxa1 expression and eliminates
that of Hoxb1 (Alexandre et al.,
1996
; Li and Lufkin,
2000
). Ectopic expression of Hoxa1 results in a similar
phenotype to that found with application of RA. However, since loss of
Hoxa1 and Hoxb1 affects hindbrain patterning and migration
of neural crest into the pharyngeal arches
(Gavalas et al., 1998
;
McClintock et al., 2002
;
Pasqualetti et al., 2001
;
Rossel and Capecchi, 1999
),
some authors reasoned that the effects of altered expression of these genes on
pharyngeal patterning are probably due primarily to abnormal neural crest
(Gavalas et al., 1998
;
Rossel and Capecchi, 1999
).
Others, however, have emphasized a more direct role of Hoxa1 and
Hoxb1 in mediating RA signaling in the pharyngeal endoderm
(Matt et al., 2003
;
Wendling et al., 2000
).
Amphioxus is particularly useful for deciphering the molecular mechanism whereby RA signaling in the endoderm regulates anterior/posterior patterning of the pharynx, because it lacks neural crest and has single genes for RAR, Hox1 and most other endodermal markers. Moreover, at the neurula stage, the pharynx of the small, transparent embryos consists of only two cell layers - an inner endoderm and an outer ectoderm. The pharynx is asymmetrical with the first three gill slits forming ventrally on the right in an anterior/posterior series and the mouth, thought to be a modified gill slit, on the left (Fig. 1). Metamorphosis, resulting in a bilaterally symmetrical adult, occurs at 9-11 gill slits.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridization, light microscopy and photography
In situ hybridizations were performed as previously described
(Holland et al., 1996). Clones
used as templates for riboprobes were as follows: AmphiPax1/9
(U20167) (Holland et al.,
1995
); AmphiNotch (Y12539)
(Holland et al., 2001
);
AmphiWnt3 (AF361013) (Schubert et
al., 2001
); AmphiNodal (AY083838)
(Yu et al., 2002
);
AmphiHox1 (AB028206) and AmphiOtx (AF043740) (both provided
by J. Garcia-Fernàndez and P. W. H. Holland); AmphiIslet
(AF226616) (provided by W. R. Jackman); AmphiFoxA2
(HNF3ß) (Y09236), AmphiPitx (AJ438768) and amphioxus
hedgehog (AmphiHh) (Y13858) (all three provided by Sebastian
M. Shimeld). After in situ hybridization, the embryos were first photographed
as whole mounts and subsequently counterstained in Ponceau S, embedded in
Spurr's resin and prepared as sections for light microscopy
(Holland et al., 1996
).
Microinjection of antisense morpholino-oligonucleotides
Microinjection of amphioxus eggs was as described by
(Holland and Yu, 2004).
Unfertilized eggs were injected with either the control morpholino
(5'-CCTCTTACCTCAGTTACAATTTATA-3') or one specific for
AmphiHox1 from B. floridae
(5'-ATTCTTGCCGTGTCCATTTGCTCCA-3') (Gene Tools, Philomath, OR,
USA). Approximately 2 pl of a solution containing 15% glycerol, 2 mg/ml Texas
Red dextran (Molecular Probes, Eugene, OR, USA) and 500 µM morpholino was
injected. The morpholinos were heated to 65°C for 5 min prior to use.
After injection, the eggs were fertilized and fixed at either the late neurula
stage (24-30 hours) or the early larval stage (36-40 hours). Fixed embryos
showing clear fluorescence of the Texas Red dextran were analyzed by in situ
hybridization (Holland et al.,
1996
).
In vitro translation assay
For in vitro translation, the AmphiHox1 coding region cDNA was
amplified by PCR and cloned into the pCS2+ vector
(Rupp et al., 1994;
Turner and Weintraub, 1994
).
In vitro translation was with the TnT Quick Coupled Transcription/Translation
System. 200 ng of plasmid DNA containing the AmphiHox1 coding region
was assayed together with different amounts of control or
AmphiHox1-specific morpholino (100 ng, 500 ng, 1000 ng or 5000 ng).
After the reactions, the samples were subjected to electrophoresis on a 12%
polyacrylamide gel and transferred to a nitrocellulose membrane. AmphiHox1
protein was detected by the Transcend Non-Radioactive Translation Detection
Systems (Promega, Madison, WI, USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
Early response genes: AmphiWnt3, AmphiPax1/9, AmphiPitx and AmphiNotch respond to RA signaling by the mid-neurula stage
At the mid-neurula stage, AmphiWnt3 transcription begins in the
ventral endoderm just posterior to the pharynx
(Fig. 2M). The onset of
expression is later than that of either AmphiRAR or
AmphiHox1 and appears to be earlier in RA-treated embryos and a
little later in BMS009 embryos than in controls
(Fig. 2M,O,Q). The anterior
limit of AmphiWnt3 expression (38% at 20 hours) is similar to that of
AmphiHox1 (40% at 20 hours) and is also shifted anteriorly by RA (to
20% at 20 hours) and slightly posteriorly by BMS009 (to 43% at 20 hours). In
addition, BMS009 broadens the domain posteriorly
(Fig. 2O-R). However, by 30
hours of development, AmphiWnt3 is almost completely downregulated in
the endoderm of RA-treated larvae (Fig.
2P), although whether it is a direct target of RA signaling
remains to be determined.
AmphiPax1/9 and AmphiPitx are expressed in the pharyngeal
endoderm with posterior limits at the mid-neurula stage (51% and 54%
respectively) overlapping the anterior limits of AmphiHox1 and
AmphiRAR (compare Fig.
2A-D with Fig.
3A,B,G,H; Fig. 7)
(Holland et al., 1995;
Yasui et al., 2000
).
Expression of both genes is reduced where the first gill slit will form
(Fig. 3A,B,G,H). Expression of
AmphiPax1/9 does not change throughout the neurula and early larval
stages (Fig. 3B). After 36
hours, expression of AmphiPitx becomes limited to the mouth and to
Hatschek's anterior left diverticulum, the precursor of Hatschek's pit, the
homolog of the adenohypophysis (Fig.
3I, double arrowhead). At the midneurula stage,
AmphiNotch is broadly expressed in the pharyngeal endoderm with a
posterior limit (35%) somewhat rostral to those of AmphiPax1/9 and
AmphiPitx (Fig. 3P).
Expression of AmphiNotch is reduced where the first gill slit will
form (Fig. 3P,Q)
(Holland et al., 2001
). By the
early larval stage (30 hours), the posterior limits of AmphiNotch,
AmphiPax1/9 and AmphiPitx are approximately the same (44-49%;
compare Fig. 3Q with
Fig. 3B,H).
Treatment with RA at the gastrula stage shifts the posterior limit of all three genes anteriorly in the endoderm. This shift is not marked at 18-20 hours; the posterior limits shift only from 54% in controls to 50% with RA for AmphiPax1/9, 51% to 40% for AmphiPitx and 35% to 30% for AmphiNotch. However, by 30 hours, the difference is obvious with the posterior limits of AmphiPax1/9 and AmphiNotch shifting to 17% in RA-treated larvae and that of AmphiPitx to 0%. RA-treatment also eliminates the zones of reduced expression where the gill slits would normally have formed (Fig. 3C,D,J,R,S). BMS009 has the opposite effect - the pharyngeal expression domains are expanded posteriorly (to about 71% for AmphiPax1/9, 60% for AmphiPitx and 53% for AmphiNotch at 20 hours; Fig. 3E,F,M-O,T,U). Expression of AmphiPitx in Hatschek's anterior left diverticulum is not affected by changes in RA signaling (Fig. 3I,L,O). Although the level of expression of AmphiPax1/9 is not obviously affected by levels of RA signaling (Fig. 3C-F), AmphiPitx and AmphiNotch appear to be downregulated by RA and upregulated by BMS009 (Fig. 3J-O,R-U).
The relatively early response of these three genes to altered levels of RA signaling that together with AmphiWnt3, they are comparatively high up in the hierarchy of the RA signaling pathway. Their expression patterns and response to altered levels of RA signaling indicate that that high levels of RA signaling suppress AmphiPax1/9, AmphiPitx and AmphiNotch expression in the middle third of the endoderm. Moreover, the posterior limits of AmphiPax1/9 and AmphiPitx are just posterior to the anterior limits of AmphiRAR and AmphiHox1, suggesting that AmphiRAR acting via AmphiHox1 (see below) may set the posterior limit of expression of these genes as well as the anterior/posterior extent of the endodermal domain of AmphiWnt3.
Late response genes: the posterior limit of AmphiNodal and AmphiOtx is not affected by altered RA signaling until the late neurula/early larval stage
During amphioxus development, AmphiNodal and AmphiOtx are
normally expressed throughout all or most of the length of the endoderm at the
early to mid-neurula stage (Fig.
4A-C,M). For both genes, expression is reduced ventrally in the
primordia of the first two gill slits (Fig.
4A,C,M). By the late neurula (24 hours), expression of
AmphiNodal becomes largely restricted to the anterior endoderm
(posterior limit at 42%), although there is still weak expression in the
midgut and hindgut (Fig. 4C).
Expression of AmphiOtx similarly becomes restricted to the pharyngeal
endoderm, but later than that of AmphiNodal. By the early larval
stage (30 hours), AmphiOtx remains expressed in the pharyngeal
endoderm (Fig. 4N), but
AmphiNodal is largely downregulated
(Fig. 4D)
(Williams and Holland, 1996;
Williams and Holland, 1998
;
Yu et al., 2002
). RA treatment
at the gastrula stage has little effect on endodermal expression of either
gene at the mid-neurula stage (Fig.
4E,O). However, by 24 hours, RA treatment restricts the endodermal
expression of AmphiNodal to the anterior pharynx (posterior limit at
37%; Fig. 4G). Expression is
downregulated somewhat sooner in RA-treated embryos than in controls
(Fig. 4D,H). The effect of RA
on the posterior limit of AmphiOtx is not apparent until the early
larva. At 30 hours, the pharyngeal expression domain of AmphiOtx is
reduced and the posterior limit is shifted anteriorly compared to controls
(posterior limit at 26%; Fig.
4N,P). BMS009 has the opposite effect. The posterior limits of the
strong pharyngeal expression domains of both AmphiNodal and
AmphiOtx are shifted posteriorly at the late neurula and early larval
stages respectively (Fig.
4I,K,L,Q,R), and downregulation of AmphiNodal in the
endoderm is delayed (Fig. 4L).
Since, unlike AmphiPax1/9, the anterior/posterior extent of
expression of both genes in the endoderm is not regionalized at the
mid-neurula stage and is affected rather late by RA and BMS009 treatments, it
is likely that they are farther downstream than AmphiPax1/9 in the
hierarchy of RA signaling.
AmphiNodal and AmphiPitx together with AmphiHh
are the only known amphioxus genes with pharyngeal expression limited to the
left side of the endoderm (Fig.
4B) (Shimeld,
1999; Yasui et al.,
2000
; Yu et al.,
2002
). However, neither RA nor BMS009 induces expression of
AmphiNodal (Fig.
4F,J), AmphiPitx or AmphiHh (data not shown) on
the right side of the endoderm in amphioxus. We conclude that RA signaling
does not control left/right asymmetry of the amphioxus pharynx.
The posterior limits of endodermal expression of AmphiIslet, AmphiFoxA2 (HNF3ß) and AmphiHh are not substantially changed by levels of RA signaling
The endodermal expression domain of amphioxus AmphiIslet at the
mid-neurula stage is similar to that of AmphiOtx but does not extend
as far posteriorly (74% versus 91%; Fig.
5A) (Jackman et al.,
2000). However, unlike AmphiOtx, AmphiIslet remains
expressed along much of the length of the endoderm through the early larval
stage (Fig. 5B,C). As with
AmphiOtx, RA treatment inhibits downregulation of AmphiIslet
where the first gill slit would normally have formed
(Fig. 5D-F). However, neither
RA- (Fig. 5D,F) nor BMS009
treatment (Fig. 5G-I) has a
marked effect on the posterior limit of expression.
In control embryos, AmphiFoxA2 is expressed throughout the pharynx
at the mid- to late-neurula stages (Fig.
5J) (Shimeld,
1997). Unlike AmphiOtx and AmphiIslet,
AmphiFoxA2 remains expressed where the first gill slit will form,
although expression is reduced anteriorly in the pharyngeal endoderm
(Fig. 5J-L). The only evident
effect of altered RA signaling on endodermal expression of AmphiFoxA2
is that in RA-treated embryos and larvae, it is not downregulated where the
gill slits and mouth would have formed
(Fig. 5M-O), while in larvae
treated with BMS009, AmphiFoxA2 is largely downregulated in an
expanded region of the anterior endoderm
(Fig. 5P-R).
AmphiHh is weakly expressed throughout the length of the endoderm
on the left side (Fig. 5S,T)
(Shimeld, 1999). Expression is
particularly high anterior to the mouth
(Fig. 5S). As development
proceeds, AmphiHh becomes upregulated around the first gill slit
(Fig. 5T). Altered RA signaling
does not substantially affect either the left/right asymmetry or the
anterior/posterior extent of endodermal expression. However, treatment with RA
reduces the size of the strong expression domain in the anteriormost
pharyngeal endoderm at the mid-neurula and almost completely downregulates
endodermal expression by the early larval stage
(Fig. 5U,V). In contrast, while
BMS009 does not alter the expression domain of AmphiHh substantially,
it does appear to upregulate the gene somewhat
(Fig. 5W,X). Failure of altered
levels of RA signaling to change the posterior limit of expression of
AmphiIslet, AmphiFoxA2 and AmphiHh suggests that they are
involved in specification of posterior foregut/midgut structures as well as
pharyngeal structures. This is not surprising in light of the roles of their
homologs in specification of the foregut and its derivatives in vertebrates
(Chen et al., 2004
;
Gauthier et al., 2002
;
Yuan and Schoenwolf,
2000
).
Injection of an AmphiHox1-specific morpholino mimics the effect of BMS009 treatments in setting the posterior limit of the pharynx
To test whether AmphiHox1 mediates RA signaling in setting the
posterior limit of the pharynx, we knocked-down AmphiHox1 function by
injection of an antisense morpholino oligonucleotide. In vitro translation
showed that the AmphiHox1-specific morpholino effectively blocks
translation of AmphiHox1 mRNA (see Fig. S2 in the supplementary
material). Injected embryos were fixed at late neurula and early larval stages
and hybridized with riboprobes for three genes: AmphiHox1,
AmphiPax1/9 and AmphiOtx. Although the
AmphiHox1-specific morpholino does not affect expression of
AmphiHox1 in the nerve cord (where expression of AmphiOtx is
expanded posteriorly), expression of AmphiHox1 in the endoderm is
shifted somewhat posteriorly as in animals treated with the RA antagonist
BMS009 (anterior limit shifted from 30% to 36%;
Fig. 6A,B). In addition,
pharyngeal expression of both AmphiPax1/9
(Fig. 6C,D) and
AmphiOtx (Fig. 6E-H) is expanded posteriorly in embryos and larvae injected with the
AmphiHox1-specific morpholino (posterior limits changed from 37% to
67% and 43% to 55%, respectively), showing that both genes are downstream of
AmphiHox1 in the RA signaling hierarchy. However, gill slits form
normally in embryos injected with the AmphiHox1-specific morpholino
(Fig. 6D,H). These results
indicate that like RA antagonist treatment, the injection of an
AmphiHox1-specific morpholino expands the pharyngeal region
posteriorly. We conclude that AmphiHox1 probably mediates RA signaling in the
amphioxus endoderm to establish the posterior limit of the pharynx, but
probably does not mediate the role of RA in gill slit penetration.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Similarly, the AmphiHox1 gene contains a RA response element
(RARE) 3' of the coding region
(Manzanares et al., 2000) to
which the AmphiRAR/AmphiRXR heterodimer can bind in vitro (H. Escriva, H. Wada
and V. Laudet, unpublished). In addition, the effects of altered RA signaling
on expression of AmphiHox1 are similar to those on vertebrate
Hoxa1 and Hoxb1, which are also expressed in the posterior
part of the pharyngeal endoderm (Matt et
al., 2003
; Wendling et al.,
2000
). These genes also contain RAREs, required to direct
expression of Hoxa1 and Hoxb1 reporter constructs to the
foregut (Huang et al., 1998
).
Not surprisingly, as with amphioxus, treatment of mouse embryos with a pan-RAR
antagonist eliminates or greatly reduces pharyngeal expression of Hox1 genes
(Matt et al., 2003
).
Conversely, treatment with RA or RAR agonists results in an anterior shift of
Hox1 gene expression in the pharynx
(Escriva et al., 2002
;
Wendling et al., 2000
). Taken
together these data clearly suggest that the general shape of the RAR-Hox1
hierarchy is conserved in vertebrates, but has become more complex due to gene
duplications early in vertebrate evolution that resulted in three RAR and
three Hox1 paralogs.
AmphiHox1 mediates the effect of RA signaling in setting the posterior limit of the amphioxus pharynx
Our results show that blocking function of AmphiHox1 expands the
amphioxus pharynx to the same extent as inhibiting RA signaling and
demonstrate an approximate hierarchy of downstream genes
(Fig. 7). In our model
(Fig. 8), RA signaling
activates AmphiHox1, which is co-expressed with the RA receptor
AmphiRAR in the middle third of the endoderm. AmphiHox1 in
turn represses AmphiPax1/9 and AmphiOtx expression posterior
to the pharynx.
|
The posterior limit of the amphioxus and vertebrate pharynx may be established by a similar suite of genes
Comparison with vertebrates suggests that the model in
Fig. 8 may also apply to
anterior/posterior patterning of the pharyngeal endoderm in vertebrates. In
vertebrates, as in amphioxus, Hox1 genes (Hoxa1 and Hoxb1)
are expressed in endoderm of the foregut and extreme caudal end of the pharynx
(Frasch et al., 1995;
Huang et al., 1998
;
Wendling et al., 2000
).
Pharyngeal expression of both genes is severely decreased by treatment with an
RA antagonist (Wendling et al.,
2000
). Moreover, as in amphioxus embryos treated with RA,
treatment of mouse embryos with an RA agonist induces strong ectopic
expression of both Hoxa1 and Hoxb1 in the anterior
pharyngeal endoderm (Matt et al.,
2003
). This suggests that in vertebrates as well as amphioxus,
Hox1 genes mediate the effect of RA in establishing the posterior limit of the
pharyngeal endoderm.
The targets of Hox1 genes in the pharyngeal endoderm of vertebrates have
not been described. However, they are probably very much the same as in
amphioxus since the pharyngeal endoderm of both amphioxus and vertebrates
expresses similar suites of genes. Vertebrates have two homologs of
AmphiPax1/9, Pax1 and Pax9. Both are expressed throughout
the length of the pharyngeal endoderm that will give rise to the pharyngeal
pouches and later in the endoderm of the definitive pharyngeal pouches
themselves (Müller et al.,
1996; Ogasawara et al.,
2000
). As in amphioxus embryos treated with BMS009, reduced RA
signaling extends expression of Pax1 posteriorly
(Dupé et al., 1999
;
Quinlan et al., 2002
;
Wendling et al., 2000
).
Conversely, RA treatment reduces Pax1 expression in the endoderm of
the third pharyngeal pouch in the mouse in connection with a greatly reduced
third pharyngeal arch or fusion of the third and fourth arches
(Mulder et al., 1998
). Altered
levels of RA signaling have similar effects on Pax9. In the mouse,
the domain of Pax9 expression in the second pouch is expanded in
embryos treated with a pan-RA antagonist, and expression where the third pouch
would normally form is nearly eliminated
(Wendling et al., 2000
). Mouse
knockouts of both RAR
and RARß have a somewhat
less severe phenotype, but even so, Pax1 expression in the third
pouch is reduced (Dupé et al.,
1999
). Together, these results suggest that in vertebrates, as in
amphioxus, high levels of RA signaling may activate RAR and Hox1 expression in
the endoderm and that Hox1 expression in turn represses, directly or
indirectly, transcription of Pax1/9 genes in the endoderm posterior
to the pharynx.
Similarly, expression of Otx genes in the pharyngeal endoderm is common to
tunicates and vertebrates as well as amphioxus. The effects of loss of Hox1
gene function on Otx expression in chordates other than amphioxus has
not been studied. However, RA treatment has a similar effect on Otx
expression in these organisms as in amphioxus. In ascidian tunicates,
reduction of the pharynx in RA-treated embryos correlates with reduced
expression of Otx in the pharynx
(Hinman and Degnan, 2000). In
addition, in vertebrates, expression of Otx genes in the anterior mesendoderm
and later in the pharyngeal endoderm of the first pharyngeal pouch
(Blitz and Cho, 1995
;
Tomsa and Langeland, 1999
) is
lost in embryos treated with RA (Bally-Cuif
et al., 1995
; Simeone et al.,
1995
).
Homologs of the remaining pharyngeal markers we have identified with their
posterior limits set by a high level of RA signaling are also expressed in the
pharyngeal endoderm of vertebrates and other chordates, although the effects
of RA signaling on expression of these genes in vertebrates is not known. For
example, expression of AmphiPitx in the endoderm around the mouth and
Hatschek's anterior left diverticulum, the homolog of the adenohypophysis
(Yasui et al., 2000), is
comparable to that of Pitx2 in the pituitary and Pitx1 in
the stomodaeum and rostral foregut endoderm in the mouse and chick
(Lanctot et al., 1997
).
Similarly, in the lamprey, Pitx genes are expressed in the stomodaeum,
neurohypophyseal duct and pharyngeal endoderm among other locations
(Boorman and Shimeld, 2002a
).
In larval tunicates, Pitx is also expressed in the nascent pharynx
(Boorman and Shimeld,
2002b
).
Vertebrate Notch genes are expressed in the pharynx as in amphioxus,
although their roles in pharyngeal development are not well understood. For
example, in the mouse, Notch2 is expressed in the anterior part of
the first branchial arch (Williams et al.,
1995), but it is not known if this is in the endodermal portion or
not. Notch1 is also expressed in the epibranchial placodes associated
with branchial arches 1-3, near the fourth arch, in neural crest migrating
into first and second arches (Williams et
al., 1995
) and in the thymus
(Weinmaster et al., 1991
;
Weinmaster et al., 1992
).
Notch genes are also expressed in the developing pancreas and the lung, which
are both endodermal derivatives (Kim and
Hebrok, 2001
; Lammert et al.,
2000
; Post et al.,
2000
). In the zebrafish blastula, Notch signaling appears to
regulate the number of endodermal cells; overexpression reduces the number of
cells expressing the endodermal marker foxa2
(Kikuchi et al., 2004
).
However, whether Notch genes have a later role in development of the
pharyngeal endoderm is unknown.
Expression of Nodal in amphioxus and vertebrates is also similar.
In amphioxus, AmphiNodal is expressed at the dorsal lip of the
blastopore in the early gastrula and throughout the length of the endoderm at
the mid-neurula stage, subsequently becoming restricted to the pharyngeal
endoderm (Yu et al., 2002). In
vertebrates, Nodal expression in mesendodermal precursors is required
for endoderm formation, in particular for the foregut endoderm where it is
upstream of Pitx2 (Faucourt et
al., 2001
; Tam et al.,
2003
). Taken together, these data suggest that the gene networks
specifying anterior endoderm are similar in amphioxus and vertebrates, and
that in both, a RAR/Hox1 signaling cascade determines fore/midgut identity and
restricts expression of anterior endodermal genes to the pharynx. However, in
vertebrates, extensive gene duplications have evidently conferred added
complexity on these gene networks.
The RA and WNT signaling cascades may interact during regionalization of the amphioxus and vertebrate endoderm
AmphiWnt3 is expressed ventrally in the endoderm just posterior to
the pharynx with anterior/posterior limits coinciding approximately with those
of AmphiRAR and AmphiHox1. Like AmphiRAR and
AmphiHox1, AmphiWnt3 expression is shifted anteriorly by RA, and
expanded posteriorly by BMS009. AmphiWnt3 is expressed relatively
late in the gut and is therefore probably acting downstream of RA signaling.
In vertebrates, Wnt3a is expressed in the vertebrate foregut endoderm
of the chick (Theodosiou and Tabin,
2003), and is downregulated by RA in an embryonic carcinoma cell
line (Katoh, 2002
) as well as
in the tail bud (Shum et al.,
1999
). This suggests that a role of Wnt3a in
regionalization of the gut in the chick may have its antecedents in an
amphioxus-like ancestor. However, whether there is cross-talk between RA
signaling and WNT signaling in patterning the vertebrate foregut remains to be
demonstrated.
Left/right asymmetry in amphioxus involves the same genes (Hh, Nodal and Pitx) as in vertebrates, but is independent of RA signaling
Specification of left/right position involves the evolutionarily conserved
series of Shh, Nodal and Pitx2
(Cooke, 2004). In the
vertebrates, high concentrations of RA randomize heart looping and can induce
bilateral expression of Nodal and Pitx2 on the right side
(Chazaud et al., 1999
;
Smith et al., 1997
;
Wasiak and Lohnes, 1999
).
However, expression of Shh is not affected
(Smith et al., 1997
), and it
appears to act either in parallel to or downstream of RA signaling
(Tsuki et al., 1999
). In
amphioxus, AmphiHh, AmphiNodal and AmphiPitx are all
expressed on the left side of the pharyngeal endoderm. Expression of these
genes on the left side of the body is not affected by altering the levels of
RA signaling. Thus RA signaling is not required for establishment of
left/right asymmetry in amphioxus, or apparently, in tunicates
(Hinman and Degnan, 1998
), and
its role in left/right asymmetry may be a vertebrate innovation.
Conclusions
Our results show that amphioxus is particularly advantageous for
understanding developmental mechanisms and that it can serve as a simplified
model for comparable patterning in vertebrate embryos. Because many amphioxus
genes are present in single copies (including RAR and the Hox genes),
functional knockdowns are relatively easy to interpret. Moreover, since
amphioxus lacks definitive neural crest, the model we present for patterning
of the pharynx applies unequivocally to the endoderm, thereby giving insights
into the separate roles of the endoderm and neural crest in pharyngeal
patterning of vertebrates. It is likely that similar regulatory cascades
involving Hox1-mediated RA signaling help to direct pharyngeal patterning in
both amphioxus and vertebrates. In addition, in vertebrates, the evolution of
neural crest evidently led to the elaboration of novel pharyngeal structures,
which were superimposed on the already existing pharyngeal patterning
intrinsic to the endoderm.
A role for Hox genes in patterning the endoderm is widespread in the animal
kingdom (Brunschwig et al.,
1999; Irvine and Martindale,
2000
; Marty et al.,
2001
). Recent evidence suggests that the RAR genes may be more
ancient than previously thought, having been secondarily lost in
Drosophila and nematodes (Bertrand
et al., 2004
). Thus, endodermal patterning by RAR/Hox1 may not be
limited to chordates, and the model we present here for regionalization of the
amphioxus endoderm may provide a framework for understanding endodermal
patterning in a wide spectrum of bilaterian animals.
![]() |
Supplementary material |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexandre, D., Clarke, J. D. W., Oxtoby, E., Yan, Y.-L., Jowett,
T. and Holder, N. (1996). Ectopic expression of
Hoxa-1 in the zebrafish alters the fate of the mandibular arch neural
crest and phenocopies a retinoic acid-induced phenotype.
Development 122,735
-746.
Allan, D., Houle, M., Bouchard, N., Meyer, B. I., Gruss, P. and
Lohnes, D. (2001). RAR and Cdx1 interactionsin
vertebral patterning. Dev. Biol.
240, 46-60.[CrossRef][Medline]
Arcioni, L., Simeone, A., Guazzi, S., Zappavigna, V., Boncinelli, E. and Mavilio, F. (1992). The upstream region of the human homeobox gene HOX3D is a target for regulation by retinoic acid and Hox homeoproteins. EMBO J. 11,265 -277.[Abstract]
Bally-Cuif, L., Gulisano, M., Broccoli, V. and Boncinelli, E. (1995). c-otx2 is expressed in two different phases of gastrulation and is sensitive to retinoic acid treatment in chick embryo. Mech. Dev. 49,49 -63.[CrossRef][Medline]
Balmer, J. E. and Blomhoff, R. (2002). Gene
expression regulation by retinoic acid. J. Lipid Res.
43,1773
-1808.
Bertrand, S., Brunet, F. G., Escriva, H., Parmentier, G.,
Laudet, V. and Robinson-Rechavi, M. (2004). Evolutionary
genomics of nuclear receptors: from 25 ancestral genes to derived endocrine
systems. Mol. Biol. Evol.
21,1923
-1937.
Blitz, I. L. and Cho, K. W. Y. (1995). Anterior
neruectoderm is progressively induced during gastrulation: the role of the
Xenopus homeobox gene orthodenticle.
Development 121,993
-1004.
Blumberg, B., Bolado, J., Moreno, T. A., Kintner, C., Evans, R.
M. and Papalopulu, N. (1997). An essential role for retinoid
signaling in anteroposterior neural patterning.
Development 124,373
-379.
Boorman, C. J. and Shimeld, S. M. (2002a). Cloning and expression of a Pitx homeobox gene from the lamprey, a jawless vertebrate. Dev. Genes Evol. 212,349 -353.[CrossRef][Medline]
Boorman, C. J. and Shimeld, S. M. (2002b). Pitx homeobox genes in Ciona and amphioxus show left-right asymmetry is a conserved chordate character and define the ascidian adenohypophysis. Evol. Dev. 4, 354-365.[CrossRef][Medline]
Brunschwig, K., Wittmann, C., Schnabel, R., Burglin, T., Tobler,
H. and Muller, F. (1999). Anterior organization of the
Caenorhabditis elegans embryo by the labial-like Hox gene ceh-13.
Development 126,1537
-1546.
Chazaud, C., Chambon, P. and Dollé, P.
(1999). Retinoic acid is required in the mouse embryo for
left-right asymmetry determination and heart morphogenesis.
Development 126,2589
-2596.
Chen, Y., Pan, F. C., Brandes, N., Afelik, S., Sölter, M. and Pieler, T. (2004). Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus. Dev. Biol. 271,144 -160.[CrossRef][Medline]
Cooke, J. (2004). Developmental mechanism and evolutionary origin of vertebrate left/right asymmetries. Biol. Rev. Camb. Philos. Soc. 79,377 -407.[CrossRef][Medline]
Couly, G., Creuzet, S., Bennaceur, S., Vincent, C. and le Douarin, N. M. (2002). Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development 129,1061 -1073.[Medline]
Dupé, V. and Lumsden, A. (2001). Hindbrain patterning involves graded responses to retinoic acid signalling. Development 128,2199 -2208.[Medline]
Dupé, V., Ghyselinck, N. B., Wendling, O., Chambon, P.
and Mark, M. (1999). Key roles of retinoic acid receptors
alpha and beta in the patterning of the caudal hindbrain, pharyngeal arches
and otocyst in the mouse. Development
126,5051
-5059.
Escriva, H., Holland, N. D., Gronemeyer, H., Laudet, V. and Holland, L. Z. (2002). The retinoic acid signaling pathway regulates anterior/posterior patterning in the nerve cord and pharynx of amphioxus, a chordate lacking neural crest. Development 129,2905 -2916.[Medline]
Faucourt, M., Houliston, E., Besnardeau, L., Kimelman, D. and Lepage, T. (2001). The Pitx2 homeobox protein is required early for endoderm formation and Nodal signaling. Dev. Biol. 229,287 -306.[CrossRef][Medline]
Frasch, M., Chen, X. and Lufkin, T. (1995).
Evolutionary-conserved enhancers direct region-specific expression of the
murine Hoxa-1 and Hoxa-2 loci in both mice and
Drosophila. Development
121,957
-974.
Gauthier, B. R., Schwitzgebel, V. M., Zaiko, M., Mamin, A.,
Ritz-Laser, B. and Philippe, J. (2002). Hepatic Nuclear
Factor-3 (HNF-3 or Foxa2) regulates glucagon gene transcription by binding to
the G1 and G2 promoter elements. Mol. Endocrinol.
16,170
-183.
Gavalas, A. and Krumlauf, R. (2000). Retinoid signalling and hindbrain patterning. Curr. Opin. Genet. Dev. 10,380 -386.[CrossRef][Medline]
Gavalas, A., Studer, M., Lumsden, A., Rijli, F. M., Krumlauf, R.
and Chambon, P. (1998). Hoxa1 and Hoxb1
synergize in patterning the hindbrain, cranial nerves and second pharyngeal
arch. Development 125,1123
-1136.
Graham, A. (2003). Development of the pharyngeal arches. Am. J. Med. Genet. 119,251 -256.
Grandel, H., Lun, K., Rauch, G.-J., Rhinn, M., Piotrowski, T., Houart, C., Sordino, P., Küchler, A. M., Schulte-Merker, S., Geisler, R. et al. (2002). Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development 129,2851 -2865.[Medline]
Hinman, V. F. and Degnan, B. M. (1998). Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae. Dev. Genes Evol. 208,336 -345.[CrossRef][Medline]
Hinman, V. F. and Degnan, B. M. (2000). Retinoic acid perturbs Otx gene expression in the ascidian pharynx. Dev. Genes Evol. 210,129 -139.[CrossRef][Medline]
Holland, L. Z. and Holland, N. D. (1996).
Expression of AmphiHox-1 and AmphiPax-1 in amphioxus embryos
treated with retinoic acid: insights into evolution and patterning of the
chordate nerve cord and pharynx. Development
122,1829
-1838.
Holland, L. Z. and Yu, J. K. (2004). Cephalochordate (amphioxus) embryos; procurement, culture and basic methods. In Methods in Cell Biology, Vol.74 (ed. C. Ettensohn, G. Wessel and G. Wray), pp.195 -215. New York: Elsevier, Academic Press.[Medline]
Holland, N. D., Holland, L. Z. and Kozmik, Z. (1995). An amphioxus Pax gene, AmphiPax-1, expressed in embryonic endoderm, but not in mesoderm: implications for the evolution of class I paired box genes. Mol. Marine Biol. Biotechnol. 4,206 -214.[Medline]
Holland, L. Z., Holland, P. W. H. and Holland, N. D. (1996). Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. In Molecular Zoology. Advances, strategies, and protocols (ed. J. D. Ferraris and S. R. Palumbi), pp. 267-282; 473-483. New York, NY: Wiley-Liss.
Holland, L. Z., Abi Rached, L., Tamme, R., Holland, N. D., Kortschak, D., Inoko, H., Shiina, T., Burgtorf, C. and Lardelli, M. (2001). Characterization and developmental expression of the amphioxus homolog of Notch (AmphiNotch): evolutionary conservation of multiple expression domains in amphioxus and vertebrates. Dev. Biol. 232,493 -507.[CrossRef][Medline]
Huang, D., Chen, S. W., Langston, A. W. and Gudas, L. J.
(1998). A conserved retinoic acid responsive element in the
murine Hoxb-1 gene is required for expression in the developing gut.
Development 125,3235
-3246.
Irvine, S. Q. and Martindale, M. Q. (2000). Expression patterns of anterior Hox genes in the Polychaete Chaetopterus: correlation with morphological boundaries. Dev. Biol. 217,333 -351.[CrossRef][Medline]
Ishibashi, T., Nakazawa, M., Ono, H., Satoh, N., Gojobori, T. and Fujiwara, S. (2003). Microarray analysis of embryonic retinoic acid target genes in the ascidian Ciona intestinalis. Dev. Growth Differ. 45,249 -259.[CrossRef][Medline]
Jackman, W. R., Langeland, J. A. and Kimmel, C. B. (2000). Islet reveals segmentation in the amphioxus hindbrain homolog. Dev. Biol. 220, 16-26.[CrossRef][Medline]
Katoh, M. (2002). Regulation of WNT signaling molecules by retinoic acid during neuronal differentiation in NT2 cells: threshold model of WNT action. Int. J. Mol. Med. 10,683 -687.[Medline]
Kikuchi, Y., Verkade, H., Reiter, J. F., Kim, C.-H., Chitnis, A. B., Kuroiwa, A. and Stainier, D. Y. R. (2004). Notch signaling can regulate endoderm formation in zebrafish. Dev. Dyn. 229,756 -762.[CrossRef][Medline]
Kim, S. K. and Hebrok, M. (2001). Intercellular
signals regulating pancreas development and function. Genes
Dev. 15,111
-127.
Kuratani, S., Ueki, T., Hirano, S. and Aizawa, S. (1998). Rostral truncation of a cyclostome, Lampetra japonica, induced by all-trans retinoic acid defines the head/trunk interface of the vertebrate body. Dev. Dyn. 211, 35-51.[CrossRef][Medline]
Lammert, E., Brown, J. and Melton, D. A. (2000). Notch gene expression during pancreatic organogenesis. Mech. Dev. 94,199 -203.[CrossRef][Medline]
Lanctot, C., Lamolet, B. and Drouin, J. (1997).
The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the
embryo and differentiates posterior from anterior lateral mesoderm.
Development 124,2807
-2817.
Laudet, V. and Gronemeyer, H. (2001). The Nuclear Receptor Facts Book. San Diego, CA: Academic Press.
Le Douarin, N. M. (1982). The neural crest. Cambridge, UK: Cambridge University Press.
Lee, Y., Osumi-Yamashita, N., Ninomiya, Y., Moon, C., Eriksson,
U. and Eto, K. (1995). Retinoic acid stage-dependently alters
the migration pattern and identity of hindbrain neural crest cells.
Development 121,825
-837.
Li, X. and Lufkin, T. (2000). Cre recombinase expression in the floorplate, notochord and gut epithelium in transgenic embryos driven by the Hoxa-1 enhancer III. Genesis 26,121 -122.[CrossRef][Medline]
Manzanares, M., Wada, H., Itasaki, N., Trainor, P. A., Krumlauf, R. and Holland, P. W. (2000). Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head. Nature 408,854 -857.[CrossRef][Medline]
Mark, M., Ghyselinck, N. B. and Chambon, P. (2004). Retinoic acid signalling in the development of branchial arches. Curr. Opin. Genet. Dev. 14,591 -598.[CrossRef][Medline]
Marty, T., Vigano, M. A., Ribeiro, C., Nussbaumer, U., Grieder, N. C. and Affolter, M. (2001). A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer. Development 128,2833 -2845.[Medline]
Matt, N., Ghyselinck, N. B., Wendling, O., Chambon, P. and Mark,
M. (2003). Retinoic acid-induced developmental defects are
mediated by RARß/RXR heterodimers in the pharyngeal endoderm.
Development 130,2083
-2093.
McClintock, J. M., Kheirbek, M. A. and Prince, V. E. (2002). Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention. Development 129,2339 -2354.[Medline]
Mollard, R., Viville, S., Ward, S. J., Décimo, D., Chambon, P. and Dollé, P. (2000). Tissue-specific expression of retinoic acid receptor isoform transcripts in the mouse embryo. Mech. Dev. 94,223 -232.[CrossRef][Medline]
Mulder, G. B., Manley, N. R. and Maggio-Price, L. (1998). Retinoic acid-induced thymic abnormalities in the mouse are associated with altered pharyngeal morphology, thymocyte maturation defects, and altered expression of Hoxa3 and Pax1. Teratology 58,263 -275.[CrossRef][Medline]
Müller, T. S., Ebensperger, C., Neubuser, A., Koseki, H., Balling, R., Christ, B. and Wilting, J. (1996). Expression of avian Pax1 and Pax9 is intrinsically regulated in the pharyngeal endoderm, but depends on environmental influences in the paraxial mesoderm. Dev. Biol. 178,403 -417.[CrossRef][Medline]
Nagatomo, K.-I., Ishibashi, T., Satou, Y., Satoh, N. and Fujiwara, S. (2003). Retinoic acid affects gene expression and morphogenesis without upregulating the retinoic acid receptor in the ascidian Ciona intestinalis. Mech. Dev. 120,363 -372.[CrossRef][Medline]
Neubüser, A., Koseki, H. and Balling, R. (1995). Characterization and developmental expression of Pax9, a paired-box-containing gene related to Pax1. Dev. Biol. 170,701 -716.[CrossRef][Medline]
Niederreither, K., Vermot, J., le Roux, I., Schuhbaur, B.,
Chambon, P. and Dollé, P. (2003). The regional pattern
of retinoic acid synthesis by RALDH2 is essential for the development of
posterior pharyngeal arches and the enteric nervous system.
Development 130,2525
-2534.
Ogasawara, M., Shigetani, Y., Hirano, S., Satoh, N. and Kuratani, S. (2000). Pax1/Pax9-related genes in an agnathan vertebrate, Lampetra japonica: expression pattern of LjPax9 implies sequential evolutionary events toward the gnathostome body plan. Dev. Biol. 223,399 -410.[CrossRef][Medline]
Ogura, T. and Evans, R. M. (1995). A retinoic acid-triggered cascade of HoxB1 gene activation. Proc. Natl. Acad. Sci. USA 92,387 -391.[Abstract]
Pasqualetti, M., Neun, R., Davenne, M. and Rijli, F. M. (2001). Retinoic acid rescues inner ear defects in Hoxa1 deficient mice. Nat. Genet. 29, 34-39.[CrossRef][Medline]
Piotrowski, T. and Nüsslein-Volhard, C. (2000). The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio). Dev. Biol. 225,339 -356.[CrossRef][Medline]
Piotrowski, T., Ahn, D.-G., Schilling, T. F., Nair, S.,
Ruvinsky, I., Geisler, R., Rauch, G.-J., Haffter, P., Zon, L. I., Zhou, Y. et
al. (2003). The zebrafish van gogh mutation disrupts tbx1,
which is involved in the DiGeorge deletion syndrome in humans.
Development 130,5043
-5052.
Post, L. C., Ternet, M. and Hogan, B. L. M. (2000). Notch/Delta expression in the developing mouse lung. Mech. Dev. 98,95 -98.[CrossRef][Medline]
Quinlan, R., Gale, E., Maden, M. and Graham, A. (2002). Deficits in the posterior pharyngeal endoderm in the absence of retinoids. Dev. Dyn. 225, 54-60.[CrossRef][Medline]
Rossel, M. and Capecchi, M. (1999). Mice mutant
for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and
defects in craniofacial development. Development
126,5027
-5040.
Rupp, R. A., Snider, L. and Weintraub, H. (1994). Xenopus embryos regulate the nuclear localization of XMyoD. Genes Dev. 8,1311 -1323.[Abstract]
Schubert, M., Holland, L. Z., Stokes, M. D. and Holland, N. D. (2001). Three amphioxus Wnt genes (AmphiWnt3, AmphiWnt5, and AmphiWnt6) associated with the tail bud: the evolution of somitogenesis in chordates. Dev. Biol. 240,262 -273.[CrossRef][Medline]
Schubert, M., Holland, N. D., Escriva, H., Holland, L. Z. and
Laudet, V. (2004). Retinoic acid influences anteroposterior
positioning of epidermal sensory neurons and their gene expression in a
developing chordate (amphioxus). Proc. Natl. Acad. Sci.
USA 101,10320
-10325.
Shimeld, S. M. (1997). Characterisation of amphioxus HNF-3 genes: conserved expression in the notochord and floor plate. Dev. Biol. 183,74 -85.[CrossRef][Medline]
Shimeld, S. M. (1999). The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog. Dev. Genes Evol. 209,40 -47.[CrossRef][Medline]
Shum, A. S. W., Poon, L. L. M., Tang, W. W. T., Koide, T., Chan, B. W. H., Leung, Y.-C. G., Shiroishi, T. and Copp, A. J. (1999). Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo. Mech. Dev. 84,17 -30.[CrossRef][Medline]
Simeone, A., Avantaggiato, V., Moroni, M. C., Mavilio, F., Arra, C., Cotelli, F., Nigro, V. and Acampora, D. (1995). Retinoic acid induces stage-specific antero-posterior transformation of rostral central nervous system. Mech. Dev. 51, 83-98.[CrossRef][Medline]
Smith, S. M. (1994). Retinoic acid receptor isoform B2 is an early marker for alimentary tract and central nervous system positional specification in the chicken. Dev. Dyn. 200, 14-25.[Medline]
Smith, S. M., Dickman, E. D., Thompson, R. P., Sinning, A. R., Wunsch, A. M. and Markwald, R. R. (1997). Retinoic acid directs cardiac laterality and the expression of early markers of precardiac asymmetry. Dev. Biol. 182,162 -171.[CrossRef][Medline]
Sucov, H. M., Murakami, K. K. and Evans, R. M. (1990). Characterization of an autoregulated response element in the mouse retinoic acid receptor type ß gene. Proc. Natl. Acad. Sci. USA 87,5392 -5396.[Abstract]
Tam, P. P. L., Kanai-Azuma, M. and Kanai, Y. (2003). Early endoderm development in vertebrates: lineage differentiation and mrophogenetic function. Curr. Opin. Genet. Dev. 13,393 -400.[CrossRef][Medline]
Theodosiou, N. A. and Tabin, C. J. (2003). Wnt signaling during development of the gastrointestinal tract. Dev. Biol. 259,258 -271.[CrossRef][Medline]
Tomsa, J. M. and Langeland, J. A. (1999). Otx expression during lamprey embryogenesis provides insights into the evolution of the vertebrate head and jaw. Dev. Biol. 207,26 -37.[CrossRef][Medline]
Tsuki, T., Capdevila, J., Tamura, K., Ruiz-Lozano, P.,
Esteban-Rodriguez, C., Yonei-Tamura, S., Magallón, J., Chandraratna, R.
A. S., Chien, K., Blumberg, B. et al. (1999). Multiple
left-right asymmetry defects in Shh-/- mutant mice unveil
a convergence of the Shh and retinoic acid pathways in the control of
Lefty-1. Proc. Natl. Acad. Sci. USA
96,11376
-11381.
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
Veitch, E., Begbie, J., Schilling, T. F., Smith, M. M. and Graham, A. (1999). Pharyngeal arch patterning in the absence of neural crest. Curr. Biol. 9,1481 -1484.[CrossRef][Medline]
Wada, H., Garcia-Fernàndez, J. and Holland, P. W. H. (1999). Colinear and segmental expression of amphioxus Hox genes. Dev. Biol. 213,131 -141.[CrossRef][Medline]
Wallin, J., Eibel, H., Neubuser, A., Wilting, J., Koseki, H. and
Balling, R. (1996). Pax1 is expressed during development of
the thymus epithelium and is required for normal T-cell maturation.
Development 122,23
-30.
Wasiak, S. and Lohnes, D. (1999). Retinoic acid affects left-right patterning. Dev. Biol. 215,332 -342.[CrossRef][Medline]
Weinmaster, G., Roberts, V. J. and Lemke, G. (1991). A homolog of Drosophila Notch expressed during mammalian development. Development 113,199 -205.[Abstract]
Weinmaster, G., Roberts, V. J. and Lemke, G.
(1992). Notch2: a second mammalian Notch gene.
Development 116,931
-941.
Wendling, O., Dennefeld, C., Chambon, P. and Mark, M.
(2000). Retinoid signaling is essential for patterning the
endoderm of the third and fourth pharyngeal arches.
Development 127,1553
-1562.
Williams, N. A. and Holland, P. W. H. (1996). Old head on young shoulders. Nature 383, 490.[CrossRef]
Williams, N. A. and Holland, P. W. H. (1998). Molecular evolution of the brain of chordates. Brain Behav. Evol. 52,177 -185.[CrossRef][Medline]
Williams, R., Lehndahl, U. and Lardelli, M. (1995). Complementary and combinatorial patterns of Notch gene family expression during early muse development. Mech. Dev. 53,357 -368.[CrossRef][Medline]
Yasui, K., Zhang, S. C., Uemura, M. and Saiga, H.
(2000). Left-right asymmetric expression of BbPtx, a Ptx-related
gene, in a lancelet species and the developmental left-sidedness of
deuterostomes. Development
127,187
-195.
Yu, J. K., Holland, L. Z. and Holland, N. D. (2002). An amphioxus nodal gene (AmphiNodal) with early symmetrical expression in the organizer and mesoderm and later asymmetrical expression associated with left-right axis formation. Evol. Dev. 4,418 -425.[CrossRef][Medline]
Yuan, S. and Schoenwolf, G. C. (2000). Islet-1 marks the early heart rudiments and is asymmetrically expressed during early rotation of the foregut in the chick embryo. Anat. Rec. 260,204 -207.[CrossRef][Medline]