distinct expression and shared activities of members of ... · 1b). the high degree of identity...
TRANSCRIPT
INTRODUCTION
The hedgehog family of secreted signaling proteins representsa recent addition to the known repertoire of moleculesemployed by vertebrates in the establishment of embryonicpattern. Originally identified by Nüsslein-Volhard andWieschaus (1980), the hedgehog gene functions in Drosophilato help coordinate the identities of cells within embryonicsegments (reviewed by Hooper and Scott, 1992; see alsoHeemskerk and DiNardo, 1994), and later in the patterning ofadult precursors including the appendages and the eye (Mohler,1988; Ma et al., 1993; Heberlein et al., 1993; Tabata andKornberg, 1994; Basler and Struhl, 1994). Whereas only singlehedgehog genes have been found in Drosophila and severalother invertebrate species (Chang et al., 1994), hedgehog genesin vertebrates constitute a multi-gene family (Echelard et al.,1993; Krauss et al., 1993; Chang et al., 1994), with the sonichedgehog class (shh; also referred to as vhh-1 or Hhg-1)receiving the greatest degree of experimental attention. Studiesof embryonic expression and function suggest that shh plays arole in dorsoventral patterning of the neural tube and somites(Echelard et al. 1993; Krauss et al., 1993; Roelink et al., 1994;Fan and Tessier-Levigne, 1994; Johnson et al., 1994) and inanteroposterior patterning of the developing limb (Riddle et al.,
1993; Chang et al., 1994; Niswander et al., 1994; Laufer et al.,1994). Since the shh class of hedgehog genes has been themajor focus of experimental effort in vertebrates, little isknown regarding the expression and functions of other ver-tebrate hedgehog family members. In particular, it is unclearwhether distinct hedgehog genes have related activities andwhether hedgehog genes play early roles in such processes asmesodermal or neural induction and patterning.
Since Xenopus remains the vertebrate most amenable toexamination of embryonic signaling processes that areinvolved in mesodermal and neural induction (reviewed byHarland, 1994), analyses of Xenopus hh genes (X-hh) providethe opportunity to place this newly described class of signalingmolecules within the context of well studied signalingpathways. We report here the isolation of four X-hh familymembers, which constitute three distinct classes of X-hhproteins. One of these represents the Xenopus homologue ofthe previously reported sonic hedgehog (X-shh) class. Theother two, banded hedgehog (X-bhh) and cephalic hedgehog(X-chh), display novel patterns of hh expression. Since highlevel expression of all four family members induces ectopiccement gland formation in animal cap explants, suggestingqualitatively similar activities for these proteins, we focusedon the activities of X-bhh as representative of X-hh activities
2337Development 121, 2337-2347 (1995)Printed in Great Britain © The Company of Biologists Limited 1995
The hedgehog family of signaling proteins is associatedwith a variety of spatial patterning activities in insects andvertebrates. Here we show that new members of this familyisolated from Xenopus laevis are expressed embryonicallyin patterns suggestive of roles in patterning in theectoderm, nervous system and somites. Banded hedgehog isexpressed throughout the neural plate and subsequently inboth the nervous system and in the dermatome of somites.Cephalic hedgehog is expressed in anterior ectoderm andendodermal structures, and sonic hedgehog is expressed inpatterns which parallel those in other species. Injection ofRNAs encoding Xenopus hedgehogs induces ectopic cement
gland formation in embryos. Similar to reported activitiesof noggin and follistatin, Xenopus hedgehogs share acommon ability to induce cement glands in animal capexplants. However, hedgehog activities in naive ectodermappear capable of acting independently of noggin and fol-listatin since, although all three are induced by activin inanimal cap explants, X-hh expression does not inducenoggin or follistatin.
Key words: hedgehog, neural induction, cement gland, follistatin,noggin, Xenopus laevis
SUMMARY
Distinct expression and shared activities of members of the hedgehog gene
family of Xenopus laevis
Stephen C. Ekker1,*, L. Lynn McGrew2,*, Cheng-Jung Lai2,*, John J. Lee1,*, Doris P. von Kessler1,Randall T. Moon2,† and Philip A. Beachy1,†
1Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine and Howard Hughes MedicalInstitute, Baltimore, MD 21205, USA2Department of Pharmacology, University of Washington School of Medicine and Howard Hughes Medical Institute, Seattle, WA 98195, USA
*Contributed equally to this work†Authors for correspondence (e-mail: [email protected])
2338
in general. High level expression in whole embryos and inanimal cap explants leads to direct induction of cement gland,which is also a reported activity of the neural inducers noggin(Lamb et al., 1993) and follistatin (Hemmati-Brivanlou et al.,1994). X-hh inducing activities can act independently ofnoggin and follistatin since, although all three are induced byactivin, X-bhh expression in animal cap explants is neitherinduced by nor induces noggin and follistatin.
MATERIALS AND METHODS
Isolation of X-hh cDNAsFragments of X-hh sequences were isolated from genomic DNA usingdegenerate primers in PCR reactions as described (Chang et al., 1994).Using these fragments as probes, four distinct full-length cDNAs wereobtained from a Xenopus laevis stage 24 developmental library(Richter et al., 1988). One of the initial X-hh fragments isolated byPCR amplification did not yield a cDNA in this screen. The regionsencoding the predicted open reading frames (Fig. 1) were sequencedon both strands as recommended (US Biochemicals). All four openreading frames are closed upstream of the initiating methionine (datanot shown). Part of the X-shh open reading frame was confirmedthrough the isolation of the corresponding region from genomic DNA(data not shown).
Expression of X-hh RNAs during development and RT-PCR analysesNorthern analyses were performed using poly(A)-containing RNAisolated from various developmental stages (McGrew et al., 1992).Aliquots of total RNA were digested with RNAse-free DNAse(Promega) and approximately 5 µm was used as template to generatefirst strand cDNA according to manufacturer’s instructions (LifeSciences, Inc.); 1/20th of this cDNA was used as template in subse-quent RT-PCR analyses. Control tubes contained everything exceptreverse transcriptase and were run in parallel with the experimentalreactions, and were negative in each case. Detection of X-bhh wasdone using primers C1 (5′-TACGGAATGCTGGCTAGG-3′) and C2(5′-CCCACTGTTGTCCATCGC-3′) in standard RT-PCR conditionswith the following reaction profile: 94˚C for 1 minute, 58˚C for 1minute, and 72˚C for 1 minute, for 30 cycles. The resulting productwas sequenced directly to confirm its identity. Detection of histoneH4 was as described by Niehrs et al. (1994).
Relative levels of transcripts encoding EF-1α and XAG-1 wereassayed by RT-PCR as described in the accompanying paper (Lai etal., 1995), and noggin and follistatin transcripts were monitored byRT-PCR as described (Hemmati-Brivanlou et al., 1994).
Injection constructsThe expression construct pT7TS-X-bhh was made by insertion of theX-bhh open reading frame into the SpeI site of the vector pT7TS (giftfrom A. Johnson and P. Kreig, University of Texas at Austin). Theopen reading frame fragment was generated by PCR using cDNA astemplate with primers bhh-U (5′-GGACTAGTCACCATGCAGT-TGCCCAAGGT-3′) and bhh-D (5′-AGCATACTAGTAGGCTCA-GCTTTCCAACTG-3′). Similarly, expression construct pT7TS-X-shhwas made by insertion of the X-shh open reading frame into the BglIIsite of pT7TS using primers shh-U (5′-ATATGGATCCCGAGAT-GCTGGTTGCGACTCA-3′) and shh-D (5′-GCGCGGATCCTTT-TTCAACTGGATTTCGTTG-3′) in PCR reactions and subsequentBamHI digestion of the resulting PCR product. Construct pT7TS-X-shhfs contains a single base pair insertion after nucleotide 117; theresulting open reading frame encodes the first 39 amino acids of X-shh and continues for 6 more amino acids in the alternate readingframe before terminating. Expression constructs pSP64-X-chh and
pSP64-X-hh4 were made by insertion of the open reading frames intoBglII site of plasmid pSP64T. Open reading frames were generatedusing primers chh-U (5′-ATATGGATCCGGACATGCCAGC-AGTCCGGATT-3′) and chh-D (5′-CGCGGGATCCTAAATT-AAGGCATATCCCACCA-3′) for X-chh and primers hh4-U (5′-ATATGGATCCGGACATGCCAGCAGTCCGGATT-3′) and hh4-D(5′-CGCGGGATCCTAATTTAAGGCATATCCCACCA-3′) for X-hh4 in PCR reactions and subsequent BamHI digestion.
A noggin expression construct in the vector pSP64T was generatedby PCR of the coding region of the reported noggin sequence, andwas also provided by Richard Harland (University of California,Berkeley). A follistatin expression construct in the vector pT7TS wasgenerated by PCR of the coding region of the reported Xenopus fol-listatin coding sequence (Hemmati-Brivanlou et al., 1994), using theforward primer 5′-GAAGATCTCCCAGCACTGAGGATG-3′ andthe reverse primer 5′-GGACTGGTCACTTACAGTTGCAAGATC-3′. The resulting construct was sequenced.
In situ hybridization and immunohistochemistryWhole-mount in situ hybridizations were performed according toHarland (1991). For histological examination embryos wereembedded in paraffin and 8-10 µm sections were prepared (Kelly etal., 1991). Detection of the monoclonal antibody 12/101 (Kintner andBrockes, 1984) was according to the method of Klymkowsky andHanken (1991). Detection of β-galactosidase activity after co-injection of lacZ and X-bhh RNAs was performed according to themethod of Westerfield et al. (1992) using 6-chloro-3-indolyl-β-galac-topyranoside (Biosynth AG, Switzerland) as substrate, followed by insitu hybridization for XAG-1.
Embryo manipulationsRNA was synthesized from X-hh pT7TS-based constructs andinjected by standard methods (Moon and Christian, 1989). Embryoswere injected with the indicated amounts of synthetic mRNA in theanimal pole of each blastomere at the 2-cell stage, then cultured(Moon and Christian, 1989) and analyzed as indicated. In experimentsinvolving animal caps, the explants were made from stage 8 blastulasas in the accompanying paper (Lai et al., 1995). After further incu-bation to the stages indicated for sibling embryos, explants weresubjected to in situ hybridization as described above or to RT-PCRreactions as described above.
RESULTS
Isolation of a family of Xenopus hedgehoghomologuesDegenerate primers were used in PCR reactions with genomicDNA as template as described (Chang et al., 1994), and theresulting X-hh gene fragments were used as probes to screen astage 24 cDNA library (Richter et al., 1988). Four distinctfamily members were isolated, and the predicted proteinproducts are shown in Fig. 1A. Xenopus cephalic hh (X-chh)and X-hh4 are closely related (93% amino acid identity) whilethe other two X-hh family members are more divergent (Fig.1B). The high degree of identity between X-chh and X-hh4relative to the other X-hh family members suggests a recentgene duplication event in the Xenopus lineage and our analysisconsequently focused primarily on one of these two, X-chh.Like all other known vertebrate hh genes, these X-hh genescontain an amino-terminal signal sequence indicating probablesecretion of X-hh protein products in vivo. Comparison of X-hh genes with murine hh genes indicates that Xenopus bandedhedgehog (X-bhh) is most similar to mouse Indian hh (70%
S. C. Ekker and others
2339hedgehog gene family of Xenopus
amino acid identity) and X-chh and X-hh4 are most similar tomouse Desert hh (64% and 63% amino acid identities, respec-tively). Based upon its expression pattern (see below) and highsequence identity to mouse sonic hh (78%), X-shh appears torepresent the Xenopus homologue of sonic/vhh-1/Hhg-1. Thespatial patterns of expression for X-bhh and X-chh (see below)are novel among reported vertebrate hh genes and thus definetwo new classes of vertebrate hh gene expression during devel-opment.
Expression of X-hh transcripts is temporally andspatially restricted Steady-state transcript levels for X-bhh, X-shh and X-chh (Fig.1C-E) indicate a peak of gene expression during neuralinduction and early organogenesis. Expression of X-bhh wasmonitored by RT-PCR since it is expressed at lower levels thanthe other two genes. Additional cycles of PCR begin to showX-bhh bands in the minus RT control, but longer exposure of
films and independent experiments detect specific amplifica-tion of X-bhh transcript by stage 8 (data not shown).
Transcript localization by in situ hybridization showed thattranscripts from all three of these genes are localized to thesub-epithelial layer of the marginal zone with no dorsal orventral bias at early gastrulation (Fig. 2A,E,I). Differences inX-hh expression become apparent at or before stage 14, withX-bhh expression observed in peripheral regions of the neuralplate (arrows, Fig. 2B), but not in the presumptive midline(arrowhead, Fig. 2B; see Hausen and Riebesel, 1991 fordescription of early embryogenesis). At neural tube closure, aprominent anterodorsal patch of X-bhh expression is observed(Fig. 2C, arrow) with more diffuse expression apparent in thesomitic and pre-somitic mesoderm. By the early tadpole(stages 28-30), widespread expression is observed throughoutanterior structures with highest levels in the otic vesicle, theeye, and the branchial arches (Fig. 2D; ov, e, ba). Also by thisstage, mesodermal expression of X-bhh occurs as an array of
Fig. 1. Four members of the Xenopuslaevis hedgehog gene family. (A) Predicted amino acid sequences ofX-shh, X-bhh, X-chh, and X-hh4 arealigned and given in the single lettercode. Residues identical in all foursequences are boxed, and a dashindicates a gap in the alignment. Thepredicted amino-terminal signalsequence (von Heijne 1986) indicatedby the solid black bar, ends just beforethe highly conserved CGPGRsequence. Predicted sites of N-linkedglycosylation are indicated byasterisks, and the presumed site ofautoproteolytic cleavage is indicatedby the arrow (see accompanyingarticle by Lai et al., 1995). GenBankaccession nos: Xshh, U26314; Xbhh,U26404; Xchh, U26349 and Xhh4,U26350. (B) Percentage identity ofresidues carboxy-terminal to the signalsequence. (C) Developmental profileof X-bhh mRNA expression (upperpanel) relative to control histone H4(lower panel), as determined by RT-PCR (see Materials and methods). Thedevelopmental stages, according toNieuwkoop and Faber (1967), aregiven above each lane, with lanenumbers at the bottom. X-bhhtranscripts were not detected bynorthern analysis, though inindependent RT-PCR reactions,expression was detected as early asstage 8 (data not shown). (D,E) Developmental profiles of X-shh, X-chh, and the control transcriptGαs-1 (Otte et al., 1992, bottom) asdetermined by northern analysis.
2340 S. C. Ekker and others
Fig
. 2. L
ocal
izat
ion
of X
-hh
tran
scri
pts
duri
ng e
arly
dev
elop
men
t. L
ocal
izat
ions
of
X-h
htr
ansc
ript
s du
ring
dev
elop
men
t are
rev
eale
d by
in s
itu h
ybri
diza
tion
to e
mbr
yos
with
antis
ense
pro
bes
for
X-b
hh(A
-D),
X-s
hh(E
-H),
and
X-c
hh(I
-K).
(A
) Si
de v
iew
of
anea
rly
gast
rula
em
bryo
(st
age
10),
with
ani
mal
hem
isph
ere
up. X
-bhh
tran
scri
pts
are
loca
lized
to th
e su
b-ep
ithel
ial l
ayer
of
the
entir
e m
argi
nal z
one
(arr
ow).
(B
) A
nter
ior
view
of a
neu
rula
em
bryo
(st
age
14).
X-b
hhex
pres
sion
occ
urs
thro
ugho
ut m
ost o
f th
e ne
ural
plat
e, w
ith s
tron
gest
exp
ress
ion
alon
g th
e la
tera
l bor
der
(arr
ows)
and
no
expr
essi
on a
long
the
mid
line
(arr
owhe
ad).
(C
) L
ater
al v
iew
of
a la
te n
euru
la e
mbr
yo, a
fter
neu
ral t
ube
clos
ure
(sta
ge 1
9/20
). T
he m
ost p
rom
inen
t dom
ain
of X
-bhh
expr
essi
on is
in th
e an
teri
orof
the
embr
yo (
arro
w),
with
mor
e di
ffus
e ex
pres
sion
obs
erve
d po
ster
iorl
y in
the
mes
oder
m. (
D)
Lat
eral
vie
w o
f a
tailb
ud e
mbr
yo (
stag
e 28
) w
ith a
nter
ior
tow
ard
the
left
.H
igh
leve
ls o
f X
-bhh
tran
scri
pts
are
seen
in s
peci
fic s
truc
ture
s of
the
head
incl
udin
g th
eey
e (e
), o
tic v
esic
le (
ov)
and
bran
chia
l arc
hes
(ba)
. X-b
hhtr
ansc
ript
s ar
e al
so lo
caliz
ed in
a ch
evro
n-sh
aped
ban
d w
ithin
eac
h so
mite
, pre
dom
inan
tly w
ithin
the
derm
atom
e (s
ee te
xt;
data
not
sho
wn)
. (E
) Si
de v
iew
of
an e
arly
gas
trul
a em
bryo
(st
age
10),
with
ani
mal
hem
isph
ere
up. X
-shh
tran
scri
pts
are
mos
t pro
min
ent i
n th
e su
b-ep
ithel
ial l
ayer
of
the
entir
e m
argi
nal z
one
(the
low
er a
nd u
pper
arr
ows
mar
k th
e do
rsal
and
ven
tral
sid
es o
f th
e
embr
yo, r
espe
ctiv
ely)
. With
mor
e ex
tend
ed in
cuba
tion
duri
ng th
e co
lor
reac
tion,
low
erle
vel e
xpre
ssio
n ca
n be
see
n to
ext
end
over
the
anim
al h
emis
pher
e (n
ot s
how
n).
(F)
Ant
erio
r vi
ew o
f a
neur
ula
embr
yo (
stag
e 14
). X
-shh
expr
essi
on is
res
tric
ted
to th
epr
esum
ptiv
e m
idlin
e (a
rrow
head
) an
d is
abs
ent f
rom
the
rest
of
the
neur
al p
late
(ar
row
s).
(G)
Lat
eral
vie
w o
f a
late
neu
rula
em
bryo
(st
age
19/2
0) w
ith a
nter
ior
to th
e le
ft. X
-shh
expr
essi
on is
in th
e no
toch
ord
and
vent
ral n
eura
l tub
e w
ith th
e m
ost p
rom
inen
t exp
ress
ion
obse
rved
in a
n an
teri
or d
omai
n (a
rrow
). (
H)
Lat
eral
vie
w o
f a
tailb
ud e
mbr
yo (
stag
e29
/30)
with
ant
erio
r to
war
d th
e le
ft. X
-shh
is e
xpre
ssed
exc
lusi
vely
in c
ells
of
the
noto
chor
d (n
) an
d flo
orpl
ate
(fp)
thro
ugho
ut th
e ax
is. S
tron
g ex
pres
sion
in th
e br
ain
and
othe
r an
teri
or s
truc
ture
s pe
rsis
ts. (
I) S
ide
view
of
an e
arly
gas
trul
a em
bryo
(st
age
10),
with
anim
al h
emis
pher
e up
. X-c
hhex
pres
sion
ext
ensi
vely
ove
rlap
s th
at o
f X
-bhh
and
X-s
hhan
dis
mos
t pro
min
ent i
n a
band
of
cells
with
in th
e m
argi
nal z
one
(arr
ow).
(J)
Top
vie
w o
f a
mid
-neu
rula
em
bryo
(st
age
15/1
6) w
ith a
nter
ior
to th
e le
ft. X
-chh
tran
scri
pts
are
mos
tab
unda
nt in
an
extr
eme
ante
rior
dom
ain
of th
e em
bryo
; the
ant
erio
r po
rtio
n of
the
neur
alpl
ate
is in
dica
ted
by th
e ar
row
s. (
K)
Lat
eral
vie
w o
f a
tailb
ud e
mbr
yo (
stag
e 29
/30)
with
ante
rior
to th
e le
ft. L
ow le
vels
of
X-c
hhtr
ansc
ript
s ar
e pr
esen
t in
the
phar
ynge
al c
avity
.(L
) Si
de v
iew
of
a ta
ilbud
em
bryo
hyb
ridi
zed
with
a X
-bhh
sens
e pr
obe
as a
con
trol
.
2341hedgehog gene family of Xenopus
Fig. 3. Expression of X-bhh in mesoderm and ectodermal derivatives. Whole embryos, hybridized in situ with a probe specific for X-bhhtranscripts, were embedded in paraffin and sectioned (see Materials and methods). (A) Sagittal section of a stage 14 neurula with anterior tothe left. X-bhh transcripts are most prominent in the sub-epithelial and epithelial layers of the neuroectoderm but are also present at lowerlevels in the underlying chordamesoderm (see arrows in the insert), (np) neural plate, (a) archenteron. (B) Parasagittal section through thehead of a stage 28 tailbud embryo with anterior to the left. X-bhh-expressing cells are seen in the sub-epithelial layer of the ectoderm(arrows), just dorsal to the cement gland (cg); (e) eye. (C) Transverse section of a stage 28 embryo at the level of the eye (e). X-bhhexpression is found in both the superficial and deep layers throughout the roof of the mesencephalon (me). Expression is also observedprominently in the prospective retinal layer of the eye vesicle (e). (D) Transverse section of a stage 28 embryo at the level of the otic vesicle(ov). X-bhh expression is seen in the dorsal roof of the rhombencephalon (rh), and in the epithelial layer of the ectoderm. Expression is alsoprominent in the otic vesicle (ov), (n), notochord. (E) Para-sagittal section through the head of a stage 28 embryo with anterior to the left. X-bhh transcripts are detected in the dorsal mesencephalon and rhombencephalon (arrows) and in the branchial arches (arrowheads). (F) Dorsal view of a whole tailbud embryo (stage 23) with anterior to the left. The embryo has been double-labeled by in situ hybridizationwith a probe specific for X-bhh transcripts prior to immunostaining with monoclonal antibody 12/101, specific for the myotomal portion ofthe somite (Kintner and Brockes, 1984). A block of two adjacent somites (s) is delimited by the arrows. (G) Lateral view of the embryo inF, at higher magnification, and with anterior to the left. The brown 12/101 stain highlights the myotome of each somite (area between twoarrows); the dark blue X-bhh stain is localized to a single central portion of the dermatome of each somite (see text; data not shown). (H) Glancing section in the plane of the dermatome of an embryo similar to that in F with anterior to the left. The arrows demarcate theboundaries of a single somite and the arrowhead denotes X-bhh staining.
2342
chevron-shaped bands, delineating the somites and suggestingthe name banded (Fig. 2D; arrowhead).
Expression of X-shh in the early gastrula is also localized tothe sub-epithelial layer of the marginal zone (Fig. 2E, arrows),but differs from X-bhh in showing a low level of expressionthroughout the animal pole. Expression in the early neurula islargely restricted to the midline (Fig. 2F, arrowhead), inmarked contrast to the broad distribution of X-bhh transcriptsthroughout the neural plate during the same period (Fig. 2B).By stage 20, after neural tube closure, X-shh expression islocalized to the axial mesoderm including the prechordal plateas well as the entire ventral midline of the neural tube (Fig.2G; histology not shown) and, as noted with X-bhh, expressionis elevated in anterior structures. A similar pattern ofexpression occurs throughout later development in thenotochord and floorplate (Fig. 2H; n, fp) which parallelsexpression in other species.
The expression of X-chh in the marginal zone is similar tothat of both X-bhh and X-shh during early gastrulation (Fig.2I). In the neurula X-chh displays a novel pattern of expressionthat is restricted exclusively to anterior structures, encompass-ing both neural plate and endodermal cells and providing thebasis for the name cephalic (Fig. 2J). Thereafter, expression isobserved on the inner surface of the pharynx at the earlytadpole stage (Fig. 2K), and the overall decline in signal cor-responds to the observed decline in steady-state levels of tran-script (Fig. 1E).
Expression of X-bhh was further examined by embeddingand sectioning of whole embryos after in situ hybridization. Asagittal section through a stage 14 neurula demonstrates a highlevel of X-bhh expression in both layers of the neuroectodermwith lower levels of expression in the underlying chordame-soderm (Fig. 3A and insert; see arrows). Later in embryonicdevelopment X-bhh continues to be expressed in both neuraland non-neural tissues. For example, a sagittal section througha tailbud embryo (arrows, Fig. 3B) shows expression of X-bhhin sub-epithelial ectodermal cells that border the cement glandbut not within the adjacent neuroepithelium in the prosen-cephalon. A transverse section through a similar stage embryoat the level of the eyes (Fig. 3C) demonstrates strongexpression in both the roof of the mesencephalon and in thelateral wall of the eye vesicle, but also lower levels ofexpression in the sub-epithelial layer of the epidermis. Moreposteriorly at the level of the rhombencephalon (Fig. 3D), X-bhh-expressing cells are still localized to the dorsal portion ofthe neural tube with particularly abundant expression alsoobserved in the otic vesicle that invaginates from the sub-epithelial layer of the epidermis. Fig. 3E shows a parasagittalsection that highlights the diffuse signal along the dorsal mes-encephalon and rhombencephalon (arrows) as well asexpression in the branchial arches (arrowheads, Fig. 3E).
To further characterize X-bhh expression in somiticmesoderm, early tailbud stage embryos were double-labeled(Fig. 3F) by in situ hybridization to detect X-bhh and with theantibody 12/101 (Kintner and Brockes, 1984) to highlight themyotomal portion of the somite. A lateral view of the sameembryo (Fig. 3G) indicates that X-bhh expression is localizedto the midline of each somite (arrows demarcate somite bound-aries). This is seen more clearly in a parasagittal section of asimilarly staged embryo (Fig. 3H; arrowhead denotes X-bhhexpression between somite boundaries, marked by arrows).
Transverse sections through the trunk of a slightly later stageembryo demonstrate that X-bhh-expressing cells are on theperimeter of the myotome, with greatest expression in thedermatome (data not shown), at a location reminiscent of thatreported for expression of Xwnt-11 (Ku and Melton, 1993).Embryos doubly stained for the expression of Xwnt-11, by insitu hybridization, and for the expression of X-bhh, by whole-mount immunohistochemistry with an anti-peptide antibody(Lai et al., 1995), provide the suggestion that Xwnt-11 and X-
S. C. Ekker and others
Fig. 4. Injection of X-bhh mRNA induces enlarged and ectopicanterior ectodermal and neural structures. Embryos at the two cellstage were injected with synthetic lacZ (A,C) or X-bhh mRNA andthe resulting tadpoles were subjected to whole-mount in situhybridization with probes for the XAG-1 cement gland marker (A,B)and the XANF-2 anterior pituitary gland marker (C,D) (see Materialsand methods). lacZ-injected embryos display wild-type patterns ofXAG-1 (A) and XANF-2 (C) expression; X-bhh-injected embryos, incontrast, display enlarged (white arrows in B and D) and ectopic(arrowheads in B) cement glands and an expanded region of labelingwith the anterior pituitary marker (compare dark arrows in C and D).Normal and expanded cement glands in panels C and D (whitearrows) are visible due to natural pigmentation. (E) Embryos co-injected with lacZ and X-bhh RNAs into both dorsal tier one cells ofthe 32 cell embryo develop with foci of XAG-1 expression(arrowheads) in only a subset of cells expressing β-galactosidase(red-brown stained cells beneath the bracket).
2343hedgehog gene family of Xenopus
bhh may be expressed in many of the same cells of thedermatome (data not shown).
Overexpression of X-bhh induces enlargement orectopic formation of cement gland and anteriorpituitary gland in embryos We began investigating the activities of the X-hh family ofgenes by examining the effects of high level expression of onefamily member on embryonic development. Embryos at the 2-cell stage were injected at the animal pole in both blastomereswith synthetic X-bhh or control (lacZ) RNA. In several inde-pendent experiments, 57% of X-bhh-injected embryos (n=68)showed enlarged cement glands (Fig. 4B,D, white arrows) ascompared to control embryos (Fig. 4A,C, white arrows). In situhybridization using the cement gland marker XAG-1 (Sive etal., 1989; Lamb et al., 1993) confirmed the enlargement of thecement gland and further showed that ectopic cement glandswere present in 10% of the injected embryos (n=95), usuallyclustered near the primary cement gland (Fig. 4B, arrowheads).We also used the anterior pituitary marker XANF-2 (Mathers
et al., 1995) to inquire whether structures arising fromectoderm, yet posterior to the cement gland, can be influencedby X-bhh RNA injection. The anterior pituitary gland is indeedenlarged (Fig. 4D, black arrow) in 34% of X-bhh-injectedembryos (n=35) relative to control embryos (Fig. 4C, blackarrow).
The formation of foci for ectopic cement glands in responseto injection of X-bhh RNA (Fig. 4B) suggested that not all cellsexpressing X-bhh participate in the formation of cementglands. To provide a marker to indicate which cells inheritinjected mRNAs, we injected 32 cell stage embryos with amixture of lacZ and X-bhh RNAs. After staining for β-galac-tosidase activity and for XAG-1 expression, we found that onlya subset of the cells expressing β-galactosidase also expressXAG-1 (Fig. 4E).
Expression of all four X-hh genes can inducecement gland formation in animal caps These initial experiments, which sought to identify develop-mental events and embryonic structures that are sensitive to
Fig. 5. Cement gland inductionin animal cap explants from X-bhh-injected embryos. (A) Uninjected tailbud embryostained for expression of thecement gland marker XAG-1 byin situ hybridization (whitearrow). (B) Histological sectionof a stage 25-equivalent animalcap explant from an uninjectedembryo stained for XAG-1. (C) Histological section of stage25-equivalent animal capexplant from a lacZ mRNA-injected embryo stained forXAG-1. (D) Animal caps fromX-bhh-injected embryos,cultured until siblings reachedstage 25 and stained for XAG-1.(E) Histological section of oneof the explants shown in Ddemonstrating foci of cementgland formation (whitearrowheads). (F) Histologicalsection of a X-bhh-injectedanimal cap showing the denselypacked columnar morphologycharacteristic of cement glandsecretory cells (bracket).
2344
high levels of hh, revealed that X-bhh can induce theformation of ectopic cement glands in whole embryos. Thisobservation raised the question of whether the other threemembers of the X-hh family have a similar activity andwhether this activity is able to directly divert embryonicectoderm from epidermal to cement gland fates. To addressthese questions embryos were injected with synthetic mRNAsfrom each of the four X-hh genes, or with lacZ and aframeshifted version of X-shh (X-shhfs) as controls, they werethen reared to the blastula stage and animal caps dissected andincubated until sibling embryos reached stage 25. Animal capswere assayed for the expression of the cement gland markerXAG-1 by whole-mount in situ hybridization, and other animalcaps were embedded and sectioned for light microscopy.Multiple foci of XAG-1 expression are apparent in animalcaps isolated from embryos injected with X-bhh mRNA overa 50-fold concentration range (Fig. 5D,E, arrowheads), and inanimal caps from embryos injected with X-chh, X-shh, and X-hh4 (see Table 1). Animal caps from uninjected (Fig. 5B) andlacZ-injected embryos (Fig. 5C), in contrast, do not express
XAG-1 and instead differentiate as atypical epidermis (see alsoTable 1). Histological analyses of X-bhh-injected animal capsrevealed that surface cells differentiate into densely packedcolumnar cells (Fig. 5F, bracket), which are characteristic ofthe secretory cells that comprise the pigmented, mucus-secreting cement gland (Lyerla and Pellizari, 1974; Picard,1975). Moreover, these histological analyses revealed nomesodermal cell types (data not shown), nor do these explantsshow induction of early or late mesodermal markers by RT-PCR (Lai et al., 1995). These results demonstrate that X-hhsinduce the differentation of cement gland in ectodermal tissuein the absence of mesoderm.
X-bhh is induced by activin but can actindependently of noggin and follistatinSince both noggin and follistatin (Lamb et al., 1993; Hemmati-Brivanlou et al., 1994) are able to induce cement gland andanterior neural markers in animal caps, we tested the possibil-ity that X-hh activities can induce the expression of either ofthese factors, and also whether noggin and follistatin activitiescan directly increase the expression of X-bhh. In addition, sinceactivin is capable of inducing the expression of noggin and fol-listatin (Thomsen and Melton, 1993; Hemmati-Brivanlou etal., 1994), we tested the ability of activin treatment to induceexpression of X-bhh. Two-cell embryos were injected sepa-rately with X-bhh, noggin, or follistatin mRNAs and animalcaps were analyzed by RT-PCR for the expression of the othertwo genes. Fig. 6A demonstrates that, in contrast to activin(lane 1), X-bhh does not induce either noggin or follistatin (lane2). Conversely, in explants cultured to the equivalent of stage11 (data not shown) or stage 25 (Fig. 6B), it is apparent thatneither noggin nor follistatin induce X-bhh to levels obtainedby treatment with activin. These data are most consistent withthe interpretation that X-bhh is neither directly upstream nordownstream of previously described anterior neural inducers
S. C. Ekker and others
Fig. 6. X-bhh is not induced bynor induces the expression ofnoggin or follistatin.(A) Activin-treated (lanes 1,4), X-bhh-injected (lanes 2, 5),and uninjected (lanes 3, 6)animal caps were assayed byRT-PCR for the expression ofnoggin, EF-1α, and follistatinwhen sibling embryos hadreached stage 11. To confirmthe effectiveness of X-bhhmRNA injection, remaininganimal caps were assayed forexpression of the cement glandmarker, XAG-1 (lower panel)when sibling embryos reachedstage 25. Note that activin butnot X-bhh treatments inducenoggin and follistatin. (B) Uninjected animal capexplants were cultured in thepresence (lanes 2, 7) or
absence (lanes 5, 10) of activin, and explants were also prepared from embryos injected with noggin (lanes 3, 8) and follistatin (lanes 4, 9)mRNAs. Explants were cultured until siblings reached stage 25, and were then assayed for the expression of X-bhh (upper panel), EF-1α, orXAG-1 (lower panels). Note that X-bhh expression is induced by activin but not by noggin or follistatin.
Table 1. Induction of the cement gland marker XAG-1 byX-hh in Xenopus animal caps
Dose XAG-1% RNA injected induction (n)
X-bhh 60 pg 32 (34)X-bhh 1.5 ng 75 (39)X-bhh 3 ng 79 (28)
X-chh 3 ng 59 (32)
X-hh4 3 ng 58 (19)
X-shh 3 ng 75 (8)
X-shhfs 3 ng 0 (10)X-shhfs 6 ng 0 (31)
lacZ 3 ng 0 (19)
2345hedgehog gene family of Xenopus
in Xenopus and thus represents an independent pathway forinduction of anterior ectodermal fates.
DISCUSSION
Expression of Xenopus hedgehog genes In the normal embryo, progressive induction and patterning ofdorsal ectoderm to differentiate into cement gland and neuralectoderm begins during gastrulation (Kintner and Melton,1987; Sive et al., 1989; Sharpe and Gurdon, 1990; reviewedby Saha and Grainger 1992, by Doniach 1993, and by Harland1994). It is therefore noteworthy that expression of X-hh genesis detectable by whole-mount in situ hybridization within theearly gastrula (Fig. 2A,E,I), although the general expressionobserved throughout sub-epithelial layers of the marginal zoneis not immediately suggestive of dorsoanterior specification.By the neurula stage, X-hh genes are expressed in distinctpatterns which correlate well with a role for X-hh genes inpromoting dorsoanterior fates. X-bhh (Fig. 2B,C) and X-shh(Fig. 2F,G) are expressed along most of the anterior-posteriorextent of the body axis by the late neurula stage but with themost prominent expression observed in anterior structures ofthe head and brain. This anterior bias is most pronounced forX-chh, whose expression is restricted exclusively to theanterior-most portion of the embryo from the neurula stageonward (Fig. 2J). Thus, all three of these genes fulfil the criteriaof being expressed in cells that are candidates for providingendogenous signals which are involved in the early inductionand patterning of anterodorsal ectodermal and neural struc-tures. Since all four X-hh genes display essentially indis-tinguishable activities in direct induction assays (Table 1), theoverall distribution of X-hh activity in the embryo is most rea-sonably considered as the summed expression of all genes.Given the anterior emphasis in expression of individual genesat the neurula stage, this overall sum is strongly biased towardanterior expression, supporting our proposal that X-hh activi-ties function in the induction and patterning of anterior struc-tures in the normal embryo. With subsequent development, X-bhh and X-shh are expressed in distinct patterns suggestive ofadditional roles in both the somites and the nervous system,respectively.
Induction of cement gland by X-hh activitiesOn the basis of histology and expression of a specific markergene, cement gland induction from naive animal cap ectodermoccurs through the activity of products from all four X-hhgenes. Upon titration through a 50-fold concentration range ofinjected X-bhh mRNA, this inductive activity appears to occurin response to a critical threshold of X-hh activity. Above thisthreshhold, foci for cement glands increase in number and size;below this threshold the foci for cement glands decrease infrequency, with unaffected caps forming atypical epidermisthat is histologically indistinguishable from that of uninjectedcontrols. Since multiple foci for cement glands are detected,this suggests that only a subset of cells expressing X-hh par-ticipate in formation of cement glands. Consistent with thisidea, injection of embryos with a mixture of lacZ and X-bhhRNAs reveals that many cells expressing β-galactosidase donot express a cement gland marker, XAG-1. Further histologi-cal analyses and antibody staining for the muscle marker
12/101 and a neural-specific N-CAM marker detect no addi-tional organized tissues that are induced by X-hh activities ineither animal cap explants or in embryos (data not shown).
Since cement gland formation can be induced by dorsalmesoderm (Cooke et al., 1987; Sive et al., 1989) it is notablethat animal caps from X-bhh-injected embryos form cementglands without induction of the mesodermal markers Xwnt-8,goosecoid, or Xbra, as monitored by RT-PCR (Lai et al., 1995),consistent with the idea of direct induction of cement glandsfrom ectoderm. It remains possible that X-hh activities exertinfluences on mesoderm not evident from the expression ofthese marker genes since, in amniotes, sonic hedgehogexpression appears to impose pattern upon the mesoderm oflimbs and somites (Riddle et al., 1993; Chang et al., 1994;Niswander et al., 1994; Laufer et al., 1994; Fan and Tessier-Levigne, 1994; Johnson et al., 1994).
Cement gland induction by X-bhh is independent ofinduction of noggin and follistatinnoggin (Lamb et al., 1993) and follistatin (Hemmati-Brivanlouet al., 1994) have been shown previously to be expressed incell types and to display activities consistent with potentialroles in neural induction. Like X-bhh, both noggin and follis-tatin can divert animal cap ectoderm to differentiate as cementgland, an extreme anterior cell fate. However, there are sig-nificant differences in the activities of these factors, since X-bhh has a very limited ability to directly induce neural genes(Lai et al., 1995), whereas both noggin and follistatin directlyinduce general neural markers such as N-CAM, and at least inthe case of follistatin, markers such as En-2 that are indicativeof more posterior neural fates. Thus, the direct inducingactivity of X-bhh is primarily the cement gland, which is asubset of the reported activities of noggin and follistatin.
Given this overlap in the ability of X-bhh, noggin, and fol-listatin to induce the cement gland, it seemed plausible thateither noggin or follistatin might be able to induce theexpression of X-bhh in animal cap explants or, alternatively,that X-bhh activities involve the activation of noggin or follis-tatin. We therefore undertook experiments with animal capexplants prepared from embryos previously injected withsynthetic X-bhh mRNA and demonstrated that neither follis-tatin nor noggin expression were induced, indicating that thedirect cement gland inducing activity of X-bhh in naiveectoderm can act independently of noggin and follistatin. Inlike manner, animal caps from embryos injected with syntheticfollistatin or noggin mRNA failed to show induction of X-bhh.We cannot rule out the possibility that an unidentified X-hh isinduced by either factor, but since X-bhh, noggin and follis-tatin are all induced in animal caps by activin, the simplestinterpretation is that X-bhh is neither directly upstream nordownstream of previously described neural inducers inXenopus and thus represents an independent pathway forinduction of the cement gland.
The distinct patterns of expression of X-bhh, X-chh, and X-shh and their common activity in induction of the cement glandin animal cap explants have implications for the mechanismsof action and normal functions of these genes. First, asreviewed above, during neurulation these genes are enrichedin overlapping patterns in anterior structures of the embryo andX-bhh is expressed in cells adjacent to the cement gland (Fig.3B). Given these patterns of expression and their common
2346
ability to directly induce cement glands, Xhhs meet the criteriaof being expressed in spatial patterns and with demonstrableactivities consistent with roles in specifying differentiation ofthe cement gland and other extreme anterior ectodermal fates.Xhh-expressing cells of axial structures and of the somites arenot likely to be involved in the induction of cement gland. Inthese structures Xhhs may affect patterning through modula-tion of a common signalling pathway; activation of thispathway in naive ectoderm leads to induction of the cementgland, whereas activation of this pathway in other cell typesmay have other effects. The ability of distinct members of theXhh family to induce the cement gland may provide a usefulsystem for further dissecting cellular responses to hh signals.
We wish to thank the members of our labs for helpful discussions,Janine Ptak and Carol Gavenport for oligonucleotide synthesis andDNA sequencing, Kathy Wilson for the cDNA library, John Kuo andHazel Sive for the XAG-1 primer sequences and cDNA, P. Mathersand M. Jamrich for providing the XANF-2 clone and sequence priorto publication, Richard Harland for noggin cDNA constructs, AndyJohnson and Paul Krieg for the expression vector pT7TS, Genentechfor the recombinant human activin A, and Chris Kintner for the12/101 antibody. P. A. B. and R. T. M. are investigators of the HowardHughes Medical Institute. C.-J. L. was supported by Public HealthService Award HD27525 to R. T. M.
REFERENCES
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control ofDrosophila limb pattern by the hedgehog protein. Nature 368, 208-214.
Bradley, L., Snape, A., Bhatt, S. and Wilkinson, D. (1993). The structure andexpression of the Xenopus Krox-20 gene: conserved and divergent patterns ofexpression in rhomobomeres and neural crest. Mech. Dev. 40, 73-84.
Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K.,Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994). Products,genetic linkage, and limb patterning activity of a murine hedgehog gene.Development 120, 3339-3353.
Cooke, J., Smith, J. C., Smith, E. J. and Yaqoob, M. (1987). Theorganization of mesodermal pattern in Xenopus laevis: experiments using aXenopus mesoderm-inducing factor. Development 101, 893-908.
Doniach, T. (1993). Planar and vertical induction of anteroposterior patternduring the development of the amphibian central nervous system. J.Neurobiol. 24, 1256-1275.
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J.,McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a memberof a family of putative signaling molecules, is implicated in the regulation ofCNS polarity. Cell 75, 1417-1430.
Fan, C.-M. and Tessier-Lavigne, M. (1994). Patterning of mammaliansomites by surface ectoderm and notochord: evidence for sclerotomeinduction by a hedgehog homolog. Cell 79, 1175-1186.
Harland, R. M. (1991). In situ hybridization: an improved whole-mountmethod for Xenopus embryos. Methods Cell Biol. 36, 685-695.
Harland, R. M. (1994). Neural induction in Xenopus. Current Opin. Genet.Dev.t 4, 543-549.
Hausen, P. and Riebesel, M. (1991). The Early Development of Xenopuslaevis. Springer-Verlag.
Heberlein, U., Wolff, T. and Rubin, G. M. (1993). The TGFβ homolog dppand the segment polarity gene hedgehog are required for propagation of amorphogenetic wave in the Drosophila retina. Cell 75, 913-926.
Heemskerk, J. and DiNardo, S. (1994). Drosophila hedgehog acts as amorphogen in cellular patterning. Cell 76, 449-460.
Hemmati-Brivanlou, A., Kelly, O. G. and Melton, D. A. (1994). Follistatin,an antagonist of activin, is expressed in the Spemann organizer and displaysdirect neuralizing activity. Cell 77, 283-295.
Hooper, J. E. and Scott, M. P. (1992). The molecular genetic basis ofpositional information in insect segments In Early Embryonic Developmentof Animals (ed. W. Hennig), pp.1-48. Berlin: Springer-Verlag.
Johnson, R.L., Laufer, E., Riddle, R.D. and Tabin, C. (1994). Ectopic
expression of sonic hedgehog alters dorsal-ventral patterning of somites. Cell79, 1165-1173.
Kelly, G. M., Eib, D. W. and Moon, R. T. (1991). Histological preparation ofXenopus laevis oocytes and embryos. Methods Cell Biol. 36, 389-417.
Kintner, C. R. and Brockes, J. P. (1984). Monoclonal antibodies identifyblastemal cells derived from dedifferentiating muscle in newt limbregeneration. Nature 308, 67-69.
Kintner, C. R. and Melton, D. A. (1987). Expression of Xenopus N-CAMRNA in ectoderm is an early response to neural induction. Development 99,311-325.
Klymkowsky, M. W. and Hanken, J. (1991). Whole mount staining ofXenopus and other vertebrates. Methods Cell Biol. 36, 420-439.
Krauss, S., Concordet, J.-P. and Ingham, P. W. (1993). A functionallyconserved homolog of the Drosophila segment polarity gene hh is expressedin tissues with polarizing activity in zebrafish embryos. Cell 75, 1431-1444.
Ku, M. and Melton, D. A. (1993). Xwnt-11, a novel maternally expressedXenopus Wnt gene. Development 119, 1161-1173.
Lai, C.-J., Ekker, S. C., Beachy, P. A. and Moon, R. T. (1995). Patterning ofthe neural ectoderm of Xenopus laevis by the amino-terminal product ofhedgehog autoproteolytic cleavage. Development 120, 2349-2360.
Lamb, T. M., Knecht, A. K., Smith, W. C., Stachel, S. E., Economides, A.N., Stahl, N., Yancopolous, G. D. and Harland, R. M. (1993). Neuralinduction by the secreted polypeptide Noggin. Science 262, 713-718.
Laufer, E., Nelson, C., Johnson, R. L., Morgan, B. A. and Tabin, C. (1994)Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loopto integrate growth and patterning of the developing limb bud. Cell 79, 993-1003.
Lyerla, T. A. and Pellizari, J. J. (1974). Histological development of thecement gland in Xenopus laevis: a light microscopic study. J. Morphol. 141,491-502.
Ma, C., Zhou, Y., Beachy, P. A. and Moses, K. (1993). The segment polaritygene hedgehog is required for progression of the morphogenetic furrow inthe developing Drosophila eye. Cell 75, 927-938.
Mathers, P. H., Miller, A., Doniach, T., Dirksen, M. L. and Jamrich, M.(1995). Initiation of anterior head-specific gene expression in uncommittedectoderm of Xenopus laevis by ammonium chloride. Dev. Biol. (in press).
McGrew, L. L., Otte, A. P. and Moon, R. T. (1992). Analysis of Xwnt-4 inembryos of Xenopus laevis: a Wnt family member expressed in the brain andfloor plate. Development 115, 463-473.
Mohler, J. (1988). Requirements for hedgehog, a segmental polarity gene, in patterning larval and adult cuticle of Drosophila. Genetics 120, 1061-1072.
Moon, R. T. and Christian, J. L. (1989). Microinjection and expression ofsynthetic mRNAs in Xenopus embryos. Technique 1, 76-89.
Niehrs, C., Steinbeisser, H. and De Robertis, E. M. (1994). Mesodermpatterning by a gradient of the vertebrate homeobox gene goosecoid. Science263, 817-820.
Nieuwkoop, P. D. and Faber, J. (1967). Normal Table of Xenopus laevis(Daudin). Amsterdam: Elsevier North-Holland Biomedical Press.
Niswander, L., Jeffrey, S., Martin, G. R. and Tickle, C. (1994). A positivefeedback loop coordinates growth and patterning in the vertebrate limb.Nature 371, 609-612.
Nüsslein-Volhard, C. and Wieschaus, E. (1980). Mutations affectingsegment number and polarity in Drosophila. Nature 287, 795-801.
Otte, A. P. McGrew, L. L., Nathanson, N. M., Olate, J. and Moon, R. T.(1992). Expression and potential functions of G-protein a subunits in earlyXenopus embryos. Development 116, 141-146.
Picard, J. J. (1975). Ultrastructure of the cement gland of Xenopus laevis. J.Morph. 148, 193-208.
Richter, K., Grunz, H. and Dawid, I. (1988). Gene expression in theembryonic nervous system of Xenopus laevis. Proc. Natl. Acad. Sci. USA 85,8086-8090.
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonichedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401-1416.
Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz iAltaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. and Dodd,J. (1994). Floor plate and motor neuron induction by vhh-1, a vertebratehomolog of hedgehog expressed by the notochord. Cell 76, 761-775.
Saha, M. S. and Grainger, R. M. (1992). A labile period in the determinationof the anterior-posterior axis during early neural development in Xenopus.Neuron 8, 1003-1014.
Sharp, C. R. and Gurdon, J. B. (1990). The induction of anterior and posteriorneural genes in Xenopus laevis. Development 109, 765-774.
S. C. Ekker and others
2347hedgehog gene family of Xenopus
Sive, H. L., Hattori, K. and Weintraub, H. (1989). Progressive determinationduring formation of the anteroposterior axis in Xenopus laevis. Cell 58, 171-180.
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein witha key role in patterning Drosophila imaginal discs. Cell 76, 89-102.
Thomsen, G. H. and Melton, D. A. (1993). Processed Vg1 protein is an axialmesoderm inducer in Xenopus. Cell 74, 433-441.
von Heijne, G. (1986). A new method for predicting signal sequence cleavagesites. Nucl. Acids Res. 14, 4683-4690.
Westerfield, M., Wegner, J., Jegalian, B. G., DeRobertis, E. M. andPuschel, A. W. (1992). Specific activation of mammalian Hox promoters inmosaic transgenic zebrafish. Genes Dev. 6, 591-598.
(Accepted 5 May 1995)