Download Ascidian embryogenesis and the origins of the chordate body plan

457
Ascidian embryogenesis
body plan
Anna Di Gregorio*
For more than a century,
system
for classic
be introduced
and Michael Levine
ascidians
embryological
simple, well-defined
development
have been a widely
studies.
cell-lineages,
and world-wide
Ascidians
compact
distribution.
into developing
embryos
genomes,
methods.
The ascidian
chordate
body plan and provides
the molecular
and differentiation
larva represents
pathways
the
a useful
underlying
of the notochord
the
and neural
tube.
Addresses
Division
of Genetics,
Department
of Molecular
Koshland Hall, University of California,
*e-mail: [email protected]
Correspondence:
Berkeley,
and Cell Biology,
California
94720,
21
USA
Anna Di Gregorio
Current Opinion in Genetics & Development
1998, 8:457-463
http://biomednet.com/elecref/0959437X00600457
ccjCurrent Biology Publications ISSN 0959-437X
Abbreviations
bHLH
basic helix-loop-helix
central nervous system
CNS
GFP
green fluorescent protein
pem
Su(H)
posterior
Suppressor
ad\~nccs
in determining
the genetic blueprint
for the formation of the ascidian tadpole.
responsible
rapid
DNA can
usmg simple
most simplified
morphogenesis
used
possess
Transgenic
electroporation
model for studying
and the origins of the chordate
end mark
of Hairless
Introduction
Ascidians,
or tunicates,
belong
to the
subphylum
ITrochordata and are generally considered
to rcprescnt
the
simplest
chordates.
Ascidian tadpoles
contain under 2000
cells but nonetheless
exhibit a basic chordate body plan that
includes II dorsal neural tube, an axial notochord flanked by
muscles and a ventral cndodermal
strand. This group of
organisms offers a number of favorable attrihutcs for analyzing gene networks responsible
for the specification
of chordate tissues
and for exploring
the transition
between
protochordate
and vertebrate
body plans. Ascidians possess
small, compact genomes
which are comparable
in size to
t host of Ijln.wp/iik~~ and Cilrt~~~~~ccIN/irisd~is
rlqy~s.
Ascid ian
embryos possess well-&find
cell lineages that arc similar in
detail to those &scribed
in 6’. &u~.c. Klectroporation
and
microinjection
methods permit the efficient incorporation
of
transgcnic DNA and RNA into de\ eloping embryos. hlosaic
incorporation
is not ~1prohlcm in this system because of the
small number
of clca\~ges
involved
in the formation
of
adwnccd-stage
embryos.
‘liansgenesis
has been wed to
characterize
&rcgulatory
I)NA elements
that mediate tissue-specific
and lineage-specific
patterns of gene expression
and to ~producc mutant phenotypes
aicl ectopic expression of
regulatory genes. Antisensc
methods ha\‘e dso been used to
disrupt gene function.
Here, \ve summarize
some recent
Rcccnt studies on the cmbryogencsis
of solitary ascidians
have focused
on
the
molecular
mechanisms
underlying
tail morphogenesis.
Four different
genera
arc being
i nves t igatcd: HuIw~~nthiu, Ciwro, PhrlllMsio and 11l~/gu/u;
most molecular
studies
ha1.e focus4
on the former two.
Hnbq~nthicr is a food crop in Japan and is therefore
readily a\.ailable in Iargc quantities
for experimental
purposes
and in terms of molecular
analyses,
it is presently
the
most cxtensivcly
studied
ascidian.
HuIqnthia
possesses
large, easily injectable
eggs (300 microns
in diameter)
and large blastomeres,
which
have been
extensiveI)
manipulated
in a variety of lineage-tracing
studies
[l-3].
Ciom/ has smaller eggs (-170 microns
in diameter)
but
offers a key d\.antage
for molecular
studies:
it is a uhiquitous species and its synchronously
de\,cloping
can be easily elcctroporatcd
PN IIIUSSP with
I)NA after chemical
dechorionation.
‘I\vo related
species
of ,lfr&uI~
possess
embryos
transgenic
radically
distinct
tadpoles:
LlI. fmht~~ has a normal tail, whereas ‘11. 0(7d~~
lacks one. If wc assume that tails represent
the ancestral or
basal condition
in ascidians. it is rcasonahlc
to hypothesize
that .\I. ouultcl has cithcr lost critical gents required for tail
de~elopmcnt
or, alternatively,
has acquired
one or more
repressors
that suppress
tail dwelopmcnt.
A combination
of subtracti\.e
hybridization
assays and antisense-mediated
gene disruption
methods
identified
3 potential
regulatory
gene, .VNNS. 3s ;I key component
in this morphological
transition
[A-.5]. ‘I’he tailless species, , II. omdtu. appears to
Jo\+,n-rcgiilate
,Ilcu-ls expression
during critical periods of
tailbud formation.
:\ cross-section
of the tail rewals
the basic chordate
fcaturcs of the ascidian
tadpole
(Figure
1). It possesses
a
prominent,
central notochord
that is composed
of just 40
cells. hlost of these cells, 32 of the 40, correspond
to the
primary
notochord
lineage,
lvhich arises from the h3.1
hlastomeres
of S-cell embryos (Figure la). In contrast, the
remaining
8 sccondar)- notochord
cells arise from the R1.l
blastomere,
\vhich also forms most of the tail muscles.
These
latter cells are located on either side of the notochord, in lateral locations that arc reminiscent
of the positions of the somitic mesodcrm
in vcrtehratcs
(Figure Id).
The specification
of the tail muscles
by the 134.1 hlastomcre represents
one of the classic examples
of a prclocalizcd qtoplasmic
determinant
(e.g. see 161).
‘I’he endoderm
of a tadpole contains
-500 cells [7] that
include ;I ‘pharynx’, located in the trunk, and an cndodermal
458
Pattern
formation
and developmental
mechanisms
tube contains
just
4 non-neuronal,
ciliated
‘cpcndymul’
cells (Figure
Id). There is uidencc
that the dorsal-most
ependymal
cell dots not deri\:e from the primary in\ agim-
Figure 1
tion of the neural tube but, rather. is recruited
after the
completion
of neurulation
from the dorwl epidermis
[X9].
Innervation
of the tail muscles invol\cs
the projection
of
non-myelinatcd
axons from the posterior
regions of the
cerebral \.esicle; the axon tracts run between
the surfaces
of the ependymal
cells. Hence,
e\.en though some p-cwmptive
motoneurons
have been tentatively
identified
in
its most proximal part [la]. the spinal cord is not ;I source
of autonomous
ncuronal
acti\ it). Despite
its simplicit!;
recent
studies
suggest
that the ascidian
neural ttlbc is
indeed
homologous
to the \,ertebrate
neural
tube
[1.3*,1-l’].
Although
Halotqnthiu, Ciotlu, .Mo[quI~ and l’hu/IN~k~ reprcsent evolutionarily
diverse
ascidians,
they show similar
Current Op~mon I” Geneks
& Developmen
Lineages of the basic chordate tissues in the ascldian tadpole.
Diagram of an iScell embryo. The uppermost cells in the animal
embryonic
lineages
and the same
larval body plan.
(:onsequently,
it is possible to apply the lessons learned in
one system to another.
Relow, \ve briefly summarize
the
existing information
regarding
the molecular
basis for tail
morphogenesis,
with a particulru emphwis
on the specification of the notochord.
tail muscles, and neural tklbc.
(a)
hemisphere, b4.2 and a4.2, give rise to the epidermis (green). The
vegetal regions of the B4.1 and A4.1 blastomeres form the endoderm
(yellow).
The lateral region of the B4.1 blastomere,
vegetal-most
along with the
region of b4.2, gives nse to the tail muscles
(orange)
to the mesenchyme
(light blue). The lateral region of A4.1 forms
notochord
(red), while the region spanning the junction between
and
the
A4.1 and a4.2 blastomeres
gives rise to the neural tube (blue).
Redrawn after [7]. (b) Lateral view of a tadpole-stage
embryo: the
same color code is used as in (a). The CNS is composed
of a cerebral
vesicle in the trunk and the neural tube in the tail (blue). The cerebral
vesicle contains
and ocellus
are shown
the two naturally
pigmented
sensory
organs,
otolith
(black). In the trunk region, some of the mesenchyme
(light blue). The endoderm
consists
of the pharynx
cells
in the
trunk and the endodermal strand in the tall (yellow). The notochord
runs along the middle of the tail (red). (c) A superficial lateral view of
the tadpole.
groups
This angle reveals one of the two bilaterally
of muscle
cells (orange),
which
situated
in Ciona are 36 and in
Halocynthia
42. Only the trunk epidermis (green) is shown. (d) Cross
section through the midtail region of the tadpole (see line in b). The
notochord
is located
in the center
situated
on either side (orange).
situated
just below
located
dorsally
of the tail (red). The tail muscles
The endodermal
(ventral) the notochord,
(blue). The tail epidermis
strand
(yellow)
;1 scclucnce-specific
transcriptional
acti\‘ator that is essential
for the differentiation
of the notochord in zebrafish,
frogs, chicken,
and mice [1.5-10). In
vertebrates,
this gene exhibits
t\vo distinct
phases
of
expression:
during
early stages of embryogcncsis,
it is
expressed
throughout
the presumptive
mesodcrm:
at I;ktcr
stages, after the subdivision
of the mesodcrm
into :lxial
and p2mixial lineages, its expression
becomes
rcstrictcd
to
axial regions that form the notochord
[ZO]. ‘I’his biphwic
expression
pattern has thwarted
attempts
to identify gcncs
acting downstream
of I?I.N(/I)‘NIJ~
that are rcsponsiblc
for
notochord
differentiation.
‘i’hc situation
in ascidians
is
simpler and should f:lvor future efforts
chord-specific
R~c4~~ur:y target genes.
to identify
noto-
are
is
while the neural tube is
is shown
Notochord
Hr-crr4yuql
encodes
in green.
strand, which runs along the length of the tail (81 (Figure 1 b).
The endodermal
strand of ascidians is peculiar in that it does
not play a role in digestion, as ascidian tadpoles 3re non-fecding larvae. Morphologically,
it shows some similarities to the
hypochord
of fish and amphibians,
thus suggesting
that the
primordial
role of the endodermal
derivatives
beneath
the
notochord is to provide further structural support to the tail.
Although
neurulation
appears to be highly conserved
in
ascidians and vertebrates
[9,10], the fully developed
larval
CNS is composed
of only -330 cells [ll]. It includes ~1sensory vesicle that contains
the pigmented
sensory organs,
otolith and ocellus,
and a caudal neural tube, or spinal cord,
which is extremely
simplified.
In cross-section
the neural
Studies in HoLoqdio
first rcvcaled the strict lineagc-spccific regulation
of an ascidian counterpart
of the nlouse
Hrdyury
(1”) gcnc, lls-7’(21]. This gent is activated :It the
Wcell
stage solely in the progenitors
of the primary notochord cells. ‘I’his onset of expression
coincides
prcciscl)
clonal
restriction.
with
time
of
notochord
the
Disaggregated
blastomeres
f:Gl to form notochord
cells
when they deri\;e from AZ-cell stage embryos but can form
notochord
when
isolated
from Wcell
embryos
[221.
Rrochyury and As-‘l’ are members
of the ‘Ilbox f:lmil? of
transcription
factors (reviewed
in [23]). In Hnl~~cy~~thio,:I
second ‘1%0x gent. As-f “2_, has been sho\vn to be exprcsscd
in the tail muscles [24]. It has been proposed that the composite expression
patterns
of As-l and As-‘/‘_’ in ascidians
represent
the biphasic expression
pattern observed
for the
vertebrate
Bru&ur~~ gene. ‘I‘hus. the \wtebrate
gene ma?
contain separate
enhancers
that direct expression
in the
early mesoderm
and notochord,
while separate
genes
Ascidian embryogenesis
and the origins of the chordate body plan Di Gregorio
and Levine
459
Figure 2
Current Op~mon tn Genetics& Development
ESpression of GFP transgenes in electroporated Ciona embryos. (a,b) Brightfield and fluorescence
photographs,
respectively, of tadpole-stage
E:mbtyos that were electroporated
with the 3.5 kb C/Bra 5’ flanking region attached to a GFP reporter gene. The transgene is expressed efficiently
most of the notochords
of these different
animals. (c,d) Fluorescence
electroporated
with a 5.6 kb region of the Ci-fkh 5’ flanking sequence
and endodermal strand (yellow in panel d), as well as in the notochord
photograph
and diagram,
respectively,
of a tadpole-stage
in
embryo that was
attached to a GFP reporter gene. There is bright staining In the trunk endoderm
(red) and spinal cord (blue). Mesenchyme cells are shown in light blue.
these tasks in ascidians. This situation appears
to represent
a rare example
of 3 common
ancestral
gcnc
duplication
in ascidians.
In general,
ascidians,
as with
Dr-oso~M~ and (;: f+gu~.s, seem to contain single copies of
genes and gene complexes
that arc duplicated
in vertebrates; for example,
a single HflM/Hos cluster seems to IX
present [ZS,Zh].
accomplish
Experimental
studies in Hdoqnthiu suggest that the presumptive endodcrm
is a source of an inductive signal, possibly bF(;F,
that triggers
notochord
specification
at the
transition from the ?&cell to &L-cell stage of embryogcnesis
[Z]. (:urrent
studies
attempting
to identify
in both H~loryn~hk and Ciorm are
and characterize
the &-regulator)
DNA that directs the notochord-specific
patterns
of As-T
and C&o
Hwt.hyq
(Ci-B/u) expression,
respectively.
‘I’hcse studies have been facilitated
by the demonstration
that transgenic
DNA can bc cfticiently
introduced
into
developing
(;i’oNn embryos \,ia electroporation
[27’]. In the
experiments
shown in Figure 2 (a and b), the 5’ flanking
sequence
from the Ci-Bru gene was attached
to a GFP
(green
fluorescent
protein)
reporter
gene and the 0
Br-&FP
fusion gene was mixed with fertilized, chemically
dcchorionatcd
eggs, which were then subjected
to a brief
460
Pattern formation and developmental
clcctrical
pulse. ‘I’he electroporated
mechanisms
embryos
fallowed
bwe
tadpoles; the fluorescent
labeling
indicates
efficient
incorporation
of the transgcnc.
In
I;igurc Zc, the 5’ flanking sequence from the Chow homolog
of_fM/HYF.?P (C’QM) has been used to direct GFP expresto
develop
into
sion not only
free-swimming
in the
notochord
but
also
in the
neural
tube
[ 1.3’1.
and the endoderm
presumptive
cndodcrm,
neighboring
wlidate
axial
or
In
summar!;
gcnic
electroporation
detailed
procedure
characterization
has
of
the
permitted
a rapid
minimal
and
tie
this
enhancer
the
hy
of
in ascidians.
homologous,
in
is currently
pates
in this process, ‘I’hc issue
the enhancer
can be activated
in
entiation
has
implicated
&NI)/)N.s
lineages,
the tail muscles,
notochord,
and
mesenchyme. by a combination
of Suppressor of
Hairless
(Su[H])
and
bfII,II
activators
[27’]. ‘I’hc
cnhanccr appears to bc rcpresscd
in the tail m~~sclcs and
mesenchymc by a C;otu/.wuil homolog (CL.CNN)[2X’]. Snail
\vorks
can
Ci-H/n
enhancer
rcportcr
gcncs
is a zinc-finger repressor that establishes an early
houndar)
enhancers
between
ectoderm
all
mesodermal
trunk
embryonic
mesoderm
in the I~fv.sf~iO~i/ff embryo
w/i/
gcnc
thcsc.
family
.wu/,
dcrm
[29,30].
are found
is expressed
of zebrafish
and
neurogenic
hlultiple
specifically
during
members
in vertebrates
the
of the
[31-M]:
in the
subdivision
one
paraxial
of the
of
meso-
notochord
and somitic lineages [?A].
Ci-SIIN
gene
notochord
binding
is exprcsscd
specification
sites
mutations
exist
in
and
in
the
in t\vo of these
the
tail
434
bp
sites
maternal
muscles
[-Il.&_?]
(Ii-Sna
determinants
cnhanccr,
othcrwisc
normal
to the
isolation
known
as
in\,ol\,ed in
‘I’hc cctopic cuprcssion of C-.stu
in the
[16,47].
the
sequences,
USC of
C-Kin
repression
porarcd
of
5
embryos.
repressor
regulatory
notochord-specific
that
‘I’hus,
results
transgenes
C-.snt/
subdivides
the
in
the
as a
co function
mcsoderm
into
separate
and muscle lincagcs [E-Y]. It is concei\ablc that
\.crtcbrate SW/ genes play a similar
role in the subdivision
notochord
of the
axial
and
paraxial
‘I’hc identification of Su(H)-binding
cnhanccr [27’] raises the possibility
pathway
is
important
for
sites in the Ci-H/a
that
notochord
differentiation
in
St](H) has been shown to function
downstream
of
;I wriet);
of de\.clopmental
processeh
in both
J~msophih and vertebrate systems ([35-371; see revieu. b)
G \Vcinmaster, pp 336332);
a Su(H) homolog
may also
function downstrum
of the linl2 rcccptor in C,‘.&gow
\\,hcrhcr
the
exprcs\ion
of
embryos.
the
cxperiniental
cytoplasm
~vhich
mcthods
been
nianipula-
of
ascidian
specify
both
antero-l”)stcrior
used
eggs
the
axis
tail
[4.3.-Lll.
isolation
ofcan-
to idcntifh
lhc\c
and the
in an effort
haw ltd. for instance,
approwhes
of maternal
specification
of
genes
in the
and
genes.
collcctiwl!~
of
in
I’i<hl--I
(::I
asi\
in the
the
po\-
so-c:lllcd
cells of the dc\ clap-
I\vo poswrior
the
of
cells
/w/t/
of
\ i;l
the
IiN!\
of the antci-ior-
adheyi\,e
orl?;;m. ccrct~ral
cells. ‘I’he pm/ gcncs cncodc 11
pigment
putati\rc
egg.
the dc\,clopnicnt
\2riety of disparate proteins:
a nuclear localization
signal
whcrcas
the
O\~erexprcssion
including
and sensory
;Intcro-l)ostcrior
csprcsscd
postcriormost
suppresses
tissues,
the
arc first
ultimately
strand.
larval
and three
[-Ml.
prrtici-
conscr\,cd gcnc nct-
of
and
cl toplasm
microinjection
domain,
a :Va+~fi signaling
and
then
embryo,
\aicle,
mcsoderm.
signaling
dctcrmining
\egetal
of these
cndodcrmal
most
tracings
the
of a group
terior-\,egetal
ing
mcsodcrm)
,LsA
determinants
the
\Iost
myoplasm,
in co-clccrro-
appcus
tail
through
carly
thepo.st~~jol-ur/n/t/N/;I’
(/xw) gents, \vhich could bc
Jlr~//mZ transgencs to be misexpressed in the tail m11rclcs.
notochord,
bl;(;l:
Jlrmhy~q
the
that
Ciom
[-Ci]. ‘These
G
although
of
the
diffcr-
transgcnic
have
and
example.
notochord-specific
hybridization
Follr
whcthcr
notochord
mice. (:on\crscly, it \ho11ltl
to cwaminc vcrtcbratc notochord-spccifi~
in
that
genes
is unclear
rpccify
is Iit-
wrtchratcs
the
by
directs
contains
didate
in
thcrc
and
regulation
approached
suggest
during
C-W/z
cause
least
Tail muscles
A variety of lineage
muscles
diffcrcntiation.
(at
the
in electroporated
Subtractive
‘I’he
in
no cl,idcncc
bc
bc possible
Cons
that
also conserved. For
arc
hccn
networks
tr;in+
tlnderstanding
Although
it
gcnc
that
genetic
\\.hcthcr
~iiitl
for
of ascidians
there
evidence
upstream
to the
cirhcr
clcmcnts.
experimental
is emerging
required
onlv
should
dctcrmining
a framework
for the notochord-specific expression of
GI;I’ and IocZ reporter genes. A 434 bp enhancer from the
C-KIZ 5’ flanking region is sufficient to direct an apparcntly normal notochord-specific expression pattern. ‘I’herc
IlKi\
stlidicr
hl:(;I;-rcsponsi\.c
notochords
arc evolutionarily
<is-regulatory
model
has provided
that
(7 its action
ITuturc
contains
specification
doubt
limitin,
a combination
studies
notochord
‘I’he
disprwe
the CLb’rz
thus
mesoderm.
for example,
and
has
12-type
a
1’1~~1-1 contains
an src-homology
nuclear
3 (St 1.3)
localization
zinc-finger
signal
motifs.
C,‘;ONN.
;Vot&
in
[.3X]. bF(;l:
is
able
Ha/oc,'nthio 1391.
and
bF(;F
cation
contacts
intimate,
restricts
each
the
of
may
Prior
the
range
notochord
for
dictarcd
which
hF(;F
of
notochord
formation.
notochord
endoderm
formation
combination
to norochord
contact
over
bc critical
primary
presumptive
cell-cell
the
induct
suggesting that a
signaling
in ascidians.
embryos,
to
directly.
by
the
in
:l’otc/l
spccifiin .32-ccl1
precursor
Perhaps
:Vot/h
can diffuse
cells
the
path\va)
from
the
One
of
the
outstanding
cmbryogenesis
minants
that
specify
thcrc
arc 36 tail
H-l.1
hlastomcrc
hecn
identified
expression
of the
problems
in
is t-he identification
field
of asciriian
maternal
(ITigure
that
1a.c).
exhibit
early
numb
of
muscle-specific
stages
regulator)
X
dcter-
In the C,'iom tadpole.
muscles.
muscle cells, and 2X of thcsc arise
during
myogcnic
the tail
the
of the
of dc\~clopnicnt.
gent .lJyoJl
from
gcneh
patwrns
the
ha\-c
of
1lomolo~:s
ha\c been
isolarcd
and Ci’iow [4X,49].
‘I‘hcse genes are not maternalI> cxprcsscd and do not
exhibit any of the propertics cxpcctcd
of an asciclian mliscle determinant.
II/ sit// hybridia1tion assa)-s rc\uI that the
and
characterized
in
both
Hu/oqt,hio
Ascidian embryogenesis
gents
are actiwted
esis,
after
the
at relatively
X-cell
stage.
actin genes are expressed
contrast,
muscle-specific
[.W]. These
results
tiatcd
to the onset of .lJyo/I cxprcssion.
prior
that
there
arc
observed
of
in
in vcrtcbrates,
the
that muscle
problems
,WyoD
gene
that
late stages of embryogcnIn
at the onset of the 32-cell
suggest
and the origins of the chordate body plan Di Gregorio and Levine
which
family)
is ini-
It is unlikely
studies
redundancy
(as
is
possess
multiple
members
because
both
C~ONO and
associated
during
ha\e identified
with
a common
the regulatory
early stagcs
regions
of myogenesis.
gene is acti\,ated in the primary
systems.
teins
(e.g. see [.X3,54]).
these
izcd
R-boxcs
an unusual
nizcd
contain
~~‘i-.vm
B-I.1 enhancer
‘AC’
E-boxes
mary
muscle.
number
of
suggests
this
actin
&-regulatory
gene
muscles
13 bp scquencc
that includes
motif.
Xloreo\u,
early
these
in the prifound
that
[.55]. One
The
mediate
of these
the muscle-specific
expression
gents
that
contain
are acti-
HI:MA.T
multiple
\l,hcreas those that are cspresscd
is a
E-box
Hr;M.-l4h and
the
tww
elements
a copy of the A(:-core
(CL’;-SIIN and
in a
genes.
Hn/oc:yt/&Jcontains
in
HNIIM~N~ZI~NI
muscle actin gents)
this motif
with
that
is dso
ascidian
elements
in the primary
\atcd
motif
muscle-specific
copies of
later, at the 64-
cell stage (SIIC~ as the Hr:ll,~l/~ gene), contain just a single
copy of the AGcorc
E-box clement.
Future studies 11ill
attempt
this
to identify
sequcncc
niatcrnally
muscle
the twru-acting
motif
and
cxpresscd
factor(s)
to dcterminc
and segregate
that binds
w,hethcr
3s predicted
the\;
to
arc
for classic
dercrminants.
plified
in
to designate
forerunner
of the vcrtehrate
gle gene expression
IXonrthelcss.
talizcd
tht: ascidian
in
[.%I’]).
The
of die verrebratc
gents.
such as
regions
the dorsal
of the neural
as dug,
~<J.Y-.?
and
neural
crc’;t cells
junction
between
sal ccrodcrm.
placodc-like
Scparatc
/‘(Jx-
7,
and form
the
ventral
regulator):
rhe tloorplatc
induces
regularory
the dorsal
genes such
to form rhe dorsal roof and the
These
latter
cells
arise
ar the
neural plate :md the dor-
cells do nor exist
in (:i(wJ.
inducts
ecmderm
the prospccti\,e
structures
seen in 1 ertcbrates
tube to express
tube to express
[SX,.W].
Such
studies
notochord
neural
sim-
ha1.c been
it ma); be a bit
cpendymal
floorplatc
Future
cell 3s LI
on the basis of a sin-
studies
\vill determine
in the vertcbratc
identified
floorplate,
in the vcntrnl
ependymal
cells of rhe ascid-
Pm and mui/ homologs
are expressed
cells that arise from
invaginating
ncuroepithelium
named
during
pattern
\crtebratc
homolog
is reminiscent
neuriilation
is expressed
eral ependymd
progenitors
neurula&n
[6.5,66].
in another
Similarly,
tube,
during
neurulation
sm/(;FP fusion
suggest
neural
that
in embryos
gene [13’].
dorso~entral
tochordates.
Keccnt
cephalochordate)
of \ertebratcs
-
cells. expressing
these
the
results
vertcbratc
that
pro-
0111 in amphioxus
the cIoscst
some
litring
migrating
(a
rclati\.e
epidcrmal
Zhtcrl-ks homolog, A~~~phiIN/,
could be
precursors
will
studies
carried
Ci-
\vith a
together,
of
during
in detail
in the mosr simplified
considered
suggest
a
cells
electroporated
patterning
stiidies
-
transicntl)
cpend>-mal
‘litken
the lat-
Ph~~/INsio
mew
has been followed
is already present
tube
considered
staining
its
;I Paw5
possibly
ascidian,
of rhe lnrcral
but this
A
in
seen dur-
tt/i/h/u[67]. The Ci-.snogent is only expressed
in the
of
assays shocv that
of the situation
in the neural
cells.
region
ncurulation.
H~Pu..2--.~7. has been isolated
HN/qN~hiu, and i?/ .rit// hybridization
expression
in the lateral
the dorsal-most
of the vertebrate
determine
fcattlres
the key innovations
neural
Lvhether
of the neural
in vertebrate
crest
ascidians
crest. which
[6X].
contain
is one of
dc\.elopment.
counterparts.
that it is compartmen-
to that
jidhd/HA~F-.?p,
[.57]. In contrast,
tube is highly
\ ertebrarc
suggest
similar
regions
neural
its
rcccnt studies
dorso-\.entrally,
(reviewed
to
although
the
is rcminis-
tube.
I’LJ_Y.~/~homolog.
ing
to rhe vcntralcontact with
pattern
ventral
tube.
tSwzic/Imdgdq(621,pitched [Ml, and nvfrin [6-t], are
ian neural
the
the
cxpres-
and neural
is restricted
expression
pattern.
other genes
also expressed
Roth
extensive
strand.
in vertebrates,
premature
any emergent
comparison
tissue
Id). This
cent of what is seen
Future
Neural tube
As discussed abo\c,
(or
have been isolated
cells, that are in closest
(Figure
ependymal
of miitage-
Ci-vu expression
for
Interestingly
H~;M,l4~ muscle
essential
transgenes
a-c essential
other
3 E-boxes
analysis
a rudimentary
genes exhibit
endodermal
in the latter
notochord
such as
scqtrenccs;
The
core sequence.
B4.1 -enhancer///d
latter
contains
Expression
most epcndymal
at the 32[.52]. This
3s sho\vn in
‘CX’ core
contains
of~l~~rtr//lHi~~~-.?~
‘rhesc
sion in the notochord.
lvhether
lineage
enhancer
elements:
homologs
the Ci-sm
sequences
arc targets for bHLH procharactcrS ome of the previously
(CANNTG)
ho\ve\.cr, the
motif
of genes expressed
muscle
cell stage by a 500 bp B1.1-specific
enhancer
contains 7 different
E-box
other
sequence
For cxamplc.
Ascidian
tube
[ 13’,60’,61].
and characterized.
Hu/my~d’N,seem to contain single topics of this gene family as seen in lhv.ro~hi/~1.511.
Keccnt
neural
floorplate
stage
specification
genetic
the ascidian
vestigial)
461
in urochordates
identified
Ho~oqy?~t~ia, and ,1fo&d~
but
[11’,56’].
suggest
Conclusions
An outline
and future prospects
of the gene nct\+orks
go\uning
tion of the ascidian tadpole tissues
Various
methods
ha\.e been
advantages of these simple
the
&regulatory
identification
morphogenesis.
the
boundary
ascidians
evolutionary
Recxuse
between
should
provide
origins
of their
in transgenic
a short-cut
rlnderlying
phylogenetic
in\.crtebrarcs
important
of the chordate
unique
that
in Hu/ofy?lt/id
providing
networks
to cmergc.
the
It is conceivable
expression
thereby
of the gent
to exploit
identified
Cimw nill direct appropriate
and other wrtcbrates,
is beginning
used
chordates.
modules
the specifica-
and
cliics
and
mice
for the
\wtehrate
location
at
L ertebratcs.
regarding
body plan.
the
462
Pattern formation
and developmental
mechanisms
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