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Transcript 
Patterningthe vertebratebody
planll: the somitesandearly
nervoussystem
r
Somiteformation and antero-posteriorpatterning
r The role of the organizerand neuralinduction
s
*
tj
il
During and after gastrulation,the vertebrateembryo becomespatternedalong
the antero-posterior
and dorso-ventralaxes.This patterningis carriedout by a
ombination of signalsfrom variousregionsof the embryoand the interpretation
of
positional
identityby cells.Theexpression
of genesinvolvedin encodingpositionalong
the antero-posterior
by whichtheir
axisis keyto this patterning,as is the mechanism
spatialpatternsof expression
Keymorphogenetic
eventsat this
areinitiallyspecified.
'
stagearetheformationof the somites,
whichgiveriseto muscles,
skeleton,
anddermis,
by segmentation
axis.Thenervous
of block of mesodermalongthe antero-posterior
rystemis inducedin the dorsalectodermby signalsfrom adjacenttissues,particularly
|dte organizerregion,and this too raisesinterestingproblems,which includethe
r posiblesimilarities
in the four modelvertebrates.
hCrapter 3, we saw how the body axes are set up and how the three germ layers
rre initially specified in vertebrate embryos. Although amphibian, fish, chick, and
mrse embryos share some features at these stages, there are many significant
ffirences. As we approach the phylotypic stage-the embryonic stagecommon to
dlveftebrates (seeFig. 3.2)-the similarities between vertebrate embryos become
r, and so we can consider the patterning of the vertebrate body plan in a
way.
During gastrulation, the germ layers-mesoderm, endoderm, and ectodermto the positions in which they will develop into the structures of the larval
adult body. The antero-posteriorbody axis of the vertebrate embryo emerges
with the head at one end and the future tail at the other (Fig.4.1).In this
r, we focus mainly on the formation and patterning of the mesoderm that
tle somites, the blocks of cells that give rise to the skeleton and musclesof
uunk, and on the patterning of the ectoderm that will develop into the neryous
The cell movements of gastrulation and tlre action of the organizer region
qucial to establishing the vertebrate body plan and will be discussedin this
in relation to their role in the patterning processes.A detailed discussion
movements of cells and tissuesduring gastrulation is, however, deferred to
.& gastnrlation, the part of the mesoderm that comes to lie along the dorsal
of the embryo, under the ectoderm, gives rise to the notochord and
, and to head mesoderm anterior to the notochord. After gastrulation in
the internalized cells of the dorsd-most mesoderm (the organizer region)
150
.
E Dy PL AN il: T HE soMrrE s A N D E A R LYN E R V oussY srE M
4 : P A T T E R N T NrHE
G VERT EBRATBo
Frg.rLI Rearrangementof the presumptive
germ Iayersduring gastrulationand neurulation inXenopus.Themesoderm(pinkandred
bandat the
onthe left),whichisin anequatorial
blastulastage,movesinsideto giveriseto the
(redon the right),somites(orange),
notochord
(notshown).Theendoderm
andlateralmesoderm
(yellow)movesinsideto linethegut.Theneural
tube(darkblue)fonnsandtheectoderm(lightblue)
coversthe wholeembryo.Theantero-posterior
axisemerges,
withthe headat the anteriorend.
Animal
@
Vcgetal
have formed a rigid rod-like notochord along the dorsal midline, flanked on
side by mesoderm, which is beginning to form blocks of somites (seeFig.
In the chick embryo, the notochord forms in the dorsal midline anterior to
node, from the axial mesoderrn left behind as the node and primitive
regress (Fig. 4.2), and somites are formed from mesoderln on either side d
Notochord and somite formation proceed similarly in the mouse (seeFigs 3.24
3.25).Each region, such as an individual somite, now developslargely
ently, and can be considered as a developmental module, rather in the same
as the individual segments in Drosophila.The vertebrate notochord is a
structure, and its cells eventually become incorporated into the vertebrae.
As gastrulation proceeds, neurulation begins. The neural tube forms from
ectoderm overlying the notochord and the somites become positioned on
side of it. The internal structure of t};leXenopusembryo just after the end of
lation is illustrated in Fig. 4.3. The main structures that can be recognized at - stage are the neural tube, the notochord, the somites, the lateral plate mesodeq*
and the endoderm lining the gut. By this stage, different parts of the somites cm
be distinguished.
Both the mesodermal structures along the antero-posterior axis of the nrffi
and the ectodermally derived nervous system have a distinct antero-posterim
organization. Someof this antero-posterior patterning is controlled by genesczlhdm
the Hox genes, which are the vertebrate equivalent of the Hox genes responslitr
for antero-posteriorpatteming inDrosophiln(seeChapter 2). In the first part ofrtl,
chapter we describe somite development and the role of the Hox genes in tlp
antero-posteriorpatterning of the somitic mesoderm,as shown by their effectsm
the spinal column, whose vertebraederive from the somites.In the secondPart d
the chapter we consider the function of the vertebrate embryonic organizer in
establishing the antero-posterior organization of the embryo, focusing on its rrrle
in the induction of the nervous system.We will also look at the early anterepostesir
patterning of the hindbrain, in which Hox genes are again involved.
W.42 Notochordand head-foldformation
in the chick embryo,Thediagramshows
a sagittalsectionthroughthe chickembryo
(inset,dorsalview)at the stageof head-fold
nodestartsto regress.
formationasHensen's
the notochord
Asthe noderegresses,
(sometimes
calledthe headprocess
at this
stage)startsto form anteriorto it. The
mesenchyme
on either
undifferentiated
willformsomites.
sideof the notochord
SOM IT E F OR M AT ION AN D AN T ER O - POST ER IO R PA TTE R N I N G
151
tr
frtu
bL i l
I
-rl
Pft*ll
n
Fig.4.3 A cross-sectionthrough a stage22
Xenopusembryojust after gastrulationand
neurulationare completed.Thegermlayers
arenowallin placefor futuredevelopment
andorganogenesis.
Themostdorsalpartsof
the somiteshavealreadybegunto differentiate
into the dermatome,whichwill giveriseto the
dermis.Scalebar= 0.2mm.
!. 4-1ru
ilxFen's
$reek
e d in
14d
4codEry
E&d
rtu
Photogroph
P.ondRiebesell,
M.:1991.
fromHousen,
llorniteformationand antero-posteriorpatterni ng
|irftg|lll..
EE[e.
tUhiD
@f
G@l
me
@
mdj
hre
ffr
f-
ft fate maps of the various vertebrates (seeFig. 3.48) show that the notochord
,frrclops from the most dorsal region of the mesoderm, and somites from more
tnnhdateral mesoderm on either side of the midline, which is known as the
gsuial mesoderm.The somites give rise to the bone and cartilage of the trunk,
lft skeletal muscles,and the dermis of the skin on the dorsal side of the body,
d tieir patterning provides much of the body's antero-posterior organization.
&vertebrae, for example,have characteristicshapesat different positions along
tfu spine. we will first examine the development of the somites and how their
ffirent
developmental identities along the antero-posterior axis are specified.
tffi conclude our look at somite formation by discussing how individual somites
,re patterned and which structures they give rise to.
'*l somites are formed in a well defined order along the antero-posterioraxis
frmite number can be hrghly variable between different vertebrates; birds and
lfrrrnans have around 50 whereas snakes have up to several hundred. Our main
mdel organism in the discussionof somite formation will be the chick. Much of
ftwork on somite formation has been done on the chick becauseof the easewith
ffiich the process can be observed. In the chick embryo, somite formation occurs
meitler side of the notochord in the lateral mesodermanterior to the regressing
&nsen's node (Fig.4.4).Betweenthe node and the most recently formed somite,
fue is an unsegmented region-the pre-somitic mesoderm-which segmenrs
,&gether into about 12 somites in the chick embryo. changes in cell shapeand
qt'rcellular
contacts in the pre-somitic mesoderm result in the formation of
tr*inct blocks of cells-the somites. somites are formed in pairs, one on either
rflr of the notochord, with each pair of somites forming simultaneously. somite
frmati61 begins at the anterior or head end and proceedsin a posterior direction.
firomite forms every 90 minutes in the chick, every 120 minutes in the mouse,
cftry45 minutes inXenopw, and every 30 minutes in zebrafish.
152
.
PL ANII: T HE S OMITE S
A N D E A R LYN E R V OU S Y S TE M
E
4 : P A T T E R N I NTGHE VERT EBRATBODY
Fig. 4.4 Thetemporal order of somite
formation is specifiedearly in embryonic
development.Somiteformationin the chick
proceeds
direction.
in anantero-posterior
in the pre-somitic
Somitesform sequentially
somiteand
regionbetweenthe last-formed
node,whichmovesposteriorly.
Hensen's
axisof the pre-somitic
lf the antero-posterior
mesodermis rotatedthrough180', asshownby
the arrow the temporalorderof somite
formationis not altered-somite6 still develops
beforesomite10.
The actual sculpting of the somites is quite a complex Process and involw
tissue separation and cell movements, as well as integlation of cells at the anteriU
and posterior somite borders. Somite formation inXmolrus, in particular' disPq6
severalunique features.A group of cells of the unsegmentedmesoderm,whichm
oriented perpendicular to the notochord, separatesfrom the rest ofthe mesodem
by formation of an intersomitic furrow and forms a distinct block-the prospectirc
somite. The block then undergoes a 90o rotation, which involves a series d'
cellular movements and reorientations that have rarely been investigate{ fu6
which appear to depend on a cell's position within the prospective somite. Cdh
sense their locations within the block and undelgo morphological changes d
cellular rearrangements according to their Positions.
In the chick, the cells that give rise to the somites originate in the epiblast n
either side of tJreanterior primitive streak and move into it at gastrulation to fu
a population of somitogenic stem cells around Hensen's node. The stem af&
divide, and those that remain in the stem-cell region continue to be self-renerr'iry
stem cells (seeSection 1.17),but those that leave the region as the node regFeffi
form the pre-somitic mesoderm.As new cells are being added to the presomiltiir"
mesoderm at the posterior end of the chick embryo, somites are forming at '
anterior end.
The sequence of somite formation in the unsegmented region is unaffemfll
by transverse cuts in the plate of pre-somitic mesoderm, suggesting that sorniim
formation is an autonomous process and that, at this time, no extracellular sig@l
speciffing antero-posterior position or timing is involved. Even if a small piee d
the unsegmentedmesodermis rotated through 180o,each somite still forms atfu
normal time, but with the sequenceof formation running in the opposite direcfom
to normal in the inverted tissue (seeFig. 4.4).So,before somite formation begim
a molecular pattern that specifies ttre time of formation of each somite has alrear@
been laid down in the pre-somitic mesoderm, and we shall return to this patterrimg
processlater. Given the existenceofthis pattern, the prospectiveidentity 6felrtu
somite is related to the temporal order in which their cells entered the pre-somin',''
mesoderm.
P A T TE R N IN G
S OMITEFOR MA TION
A N D A N TE R O-P OS TE R IOR
5l I'
9l
5t II
s0
U[j
stl
sl I '
153
stl
5l I3
stl -II!
--
s0 I I
pre-somitic
mesoderm
I
I
5tl
sl
everygos0
s.l
minutes5-tl
f;'#i';
lq
IE
|
f('f
!-ddient
''ii't
Differentiation
of
mmiticderivatives
-
g
*,
eDithelial
somit€
6bii.iit{illiiili"
spetifiation
Segment
pre-somitic
mesoderm
somiticstem
celk
node
Hensen's
iI i=
=
=:=
lt
ll
r=
! 3 :::g
=
= = i E F :s::!;3;3;:
3 33
= :;;;3
E
3
rrililtJtJl"NLf
UUUU
!s
-E
lShouE
Posteriol
lE+ 4,5 Somiteformation in the chick,Asshownin the left-handpanel,
ffilne
from pre-somiticmesoderm,which
somitesaregeneratedsuccessively
ihderivedfrom somiticstemcellsin the primitivestreak.As pre-somiticcells
srpreleased
mesoderm,a newpairof somites
into the posteriorDre-somitic
$hukfromthe anteriorendevery90 minutes.Forclarity,in thisdiagram
qomralsomiteformationisshownfor the left-handsomiteswhilethe somites
wmthe rightshowhoweachsomitebecomesinternallypatternedduringits
furmation.Sl.the mostrecentlvformedsomite;5ll,the lastbut onesomite
arenot yet
funrred;50,somitein the process
of formation,whoseboundaries
Theborder
cellsthatwillformsomites.
@ tl, 5-ll, blocksof pre-somitic
gradient.At formation,each
Mi'een eachsomiteisspecifiedbythe FGF-8
polarity,afterwhichit canrespondto the
mrnFteacquiresantero-postedor
anddorso-ventral
axes.
signals
that patternit alongthe antero-posterior
panelshowsstagesin oneofthe cyclesof choiry1
Thetop right-hand
(blue)that sweepfrom posterior
expression
to anteriorof the pre-somitic
cell
mesoderm
every90 minutes.Duringeachcycle,a givenpre-somitic
(reddot) experiences
andwhenit
distinctphaseswhenc-hoiry1 is expressed
panelshowsthe progress
of a preisnotexpressed.
Thelowerright-hand
mesoderm
until
somiticcell(reddot)fromthe time it entersthe pre-somitic
it isincoroorated
intoa somite.Somiticcellsin the anteriorsomiteswill have
beforethey leavethe node
experienced
fewercyclesof choiry 7 expression
somites,
and
regionfor the pre-somitic
thanwillcellsin posterior
mesoderm
and
thiscoulddefinea clockthat is bothlinkedto somitesegmentation
'tells'thesomiteitspositionalongthe antero-posterior
axis.
Somiteformation is largely determined by an internal 'clock' in the pre-somitic
nsoderm. This clock is representedby periodic cyclesof gene expression,such as
tffi* of the gene c-hairy1 in the chick embryo, whose expression sweepsfrom
t& posterior to the anterior end of the pre-somitic mesoderm with a period of
9D minutes, the time it takes for a pair of somites to form. In a newly formed
rsrrnite,c-hairy1 expressionbecomesrestricted to the posterior end of the somite,
qfrcre it persists, while a new wave of c-haitt 1 expression starts at the tail end
rdthe pre-somitic mesoderm (Fig.4.5).The connection between these oscillations
rd somite formation is not yet clear, but one of the proteins whose expression
iqrcles is Lunatic fringe, which potentiates activity of the Notch-Delta signaling
gffiw-ay (Fig. 4.6). This pathway is widely involved in determining cell fate and
i&limitillg boundaries, and is involved in setting the somite boundaries. It is an
rm-mple of the transmission of signals by direct cell-cell contact, as both Notch
,M Delta are transmembraneproteins. Mice mutant for the Delta-Notch pathway
ffin do not form somites,and if they do, the somitesvary in size and are differ, nr on each side of the body. In zebrafish, expressionof genesof the hairy family
dlso oscillatesand models for the oscillation based on feedback inhibition have
hen proposed.
--_tilttil
154
.
4 : P A T T ERNT NG
T HE VERT EBRATBo
E Dy PL AN i l : TH E soMtrE s A N D E A R LYN E R V oussY srE M
Fig.4.6 Thecore Notchsignalingpathway.
Bindingofa receptorproteinofthe Notch
ligandsuchas
familyto its membrane-bound
activates
intracellular
signaling
Deltaor Serrate
from the receptor.Thisisthoughtto involvethe
tail of
cleavage
of the cytoplasmic
enzymatic
domain)andits
the receptor(Notchintracellular
to the nucleus
to bindandactivate
translocation
a transcriptionfactorof the CSLfamily.Thefinal
outcomeof this pathwayisthe activationof
genes.Notchsignaling
isveryversatile.
specific
Indifferentorganisms
andin different
activationof
developmental
circumstances,
Notchswitchesdifferentgeneson or off.
The timing and position of somite formation is determined by the interactiond
the segmentation clock with the gxowth factor FGF-8.In the chick and the mru
this forms a gmdient in both mesoderm and ectoderm with its high point at 'node. The gradient also regresses in a posterior dfuection along with the uo&r
The reason for this dynamic behavior is that FGF-8nRNA is only made in cells rinr
and around the node and is progressively degraded in cells that leave the regiou m
the node moves posteriorly. This results in a gradient in intracellular FGF-8mRlildt,
which is translated and secreted to give an extracellular head-to-tail FGF-8gadirffi
with its eventual high point in the tailbud of the embryo (Fig.a.71.Somite formatimr
occurs where the level of FGF-8is at a sufficiently low threshold. As the gradknu
moves, therefore, it successivelydefines regions in the pre-somitic mesoderur
where somites start to form (seeFig. 4.5).There is also a gradient of retinoic aclfr^,,
a small, secreted, signaling molecule derived from vitamin A, in the oppoctm
direction, which antagonizes the FGFgradient. Retinoic acid thus keeps ttre pm"
somitic region from continually getting longer. The retinoic acid is synthesizedrinr
the somites and diffi.rsesboth posteriorly and anteriorly. It is also involved in meir
taining the bilateral symmetry of somite development by, in some way not Iffi
,=$
I
FGT
gradient
X l0wtcF
ffi trighrelE intemediateFct
Flg.4.7 FGF
axisin the rrqrhelpto patterntheantero-posterior
andretinoicacidgradients
gradient
withitshighpointatthenmh
develops
intheembryo,
embryo.Anantero-posterior
of FGF
with10 pairsof somites
formed
mouse
embryo
Thisschematic
shows
thedorsal
viewof a 8.5-day
ofthefuturebrain(r1, 12,etc.,arede
andtheneural
butstillopenintheregion
tubepartlyclosed
gradbtu;
cord.TheFGF
rhombomeres
endofthefuturespinal
andattheposterior
ofthehindbrain)
FGF
mRNA,
whichisthengradually
degradec
formed
bycellsinandaround
thenodesynthesizing
ina gradienr
mesoderm.
Thisresults
uir
whencellsleave
to formthepre-somitic
theregion
anteriorly
proteinthatiscontinuously
elongrer
movingposteriorly
astheembryo
translated
FGF
andsecreted
somite,
suggs;nrq
theposition
ofthelast-formed
Thegradient
fades
outataround
inthemesoderm
Retinoic
acidsynthesier
leveldropsto somelowthreshold.
thatsomites
areformedwhentheFGF
FGF.Retinoicacidmavamq
andsecretedby the somitesformsanopposinggradientthat antagonizes
helpto patternthe embryoby switchingon the expression
of specificgenes.lt alsodiffusesin an
anteriordirectionfrom its siteof synthesis
in the somitesandmayhelpto patternthe futurehindhann
Adopted
fromDeschomps,
l. ondVonNes,
l.: 2005.
S OMITE
FOR MA TION
A N DA N TE R O-P OS TE RPIOR
ATTE R N IN G
155
Fig.4.E Thepre-somiticmesodermhas
acquireda positionalidentity beforesomite
formation. Pre-somitic
mesodermthat will give
riseto thoracicvertebraeisgraftedto ananterior
regionof a youngerembryothatwouldnormally
developinto cervicalvertebrae.Thegrafted
mesoderm
develops
according
to itsoriginal
positionandformsribsin the cervical
region.
f,d
re
rtu
&,
Lm:
r!s
wm"
ffi
iliim
ru
@r
rd.
[trP
!um"
d1n.
mderstood, buffering the somites against the signals that are setting up left-right
eqvmmetryin the lateral mesoderrn(seeChapter 3).
Somites differentiate into particular axial structures depending on their position
nbng the antero-posterioraxis. The anterior-most somites contribute to the skull,
tbse posterior to them will form cervical vertebrae,and more posterior oneswill
.dwelop as thoracic vertebrae with ribs. Specification by position has occurred
hdore somite formation begins during gastrulation: if unsegmented somitic
@esderm from, for example, the presumptive thoracic region of the chick
mbryo is grafted to replacethe presumptive mesodermof the neck region, it will
rtfll form thoracic vertebrae with ribs (Fig. 4.8). How then is the pre-somitic
lmoderm patterned so that somites acquire their identity and form particular
"mrtebrae?
*: ldentity of somitesalong the antero-posterioraxis is specified
f;rHox gene expression
l[F
rsru
T
d
d
&
mm
H
ffii{ffi
q0ru'
aEilmmr
fldl
q@ilbt
fl'
ffim'lllllllr
m"'
TlTne
antero-posteriorpatterning of the mesodermis most clearly seenin the differ,ulrcesin the vertebrae, eachvertebra having well defined anatomical characteristics
@ending on its location along the axis. The most anterior vertebrae are specialiiiiip<lfe1 attachment and articulation of the skull, while the cervical vertebrae of
fu neck are followed by the rib-bearing thoracic vertebrae and then those ofthe
ilLmnhar
region, which do not bear ribs, and finally, those ofthe sacraland caudal
mngions.
This antero-posteriorpatteming occurs early, while the pre-somitic mesofum is still unsegmented.Patteming of the skeleton along the body axis is based
,mm
the mesodermal cells acquiring a positional value that reflects their position
fug the axis and so determines their subsequentdevelopment.Mesodermalcells
tffimwill form thoracic vertebrae, for example, have different positional values from
tfue that will form ceryical vertebrae.
Patteming along the antero-posterior axis in all vertebrates involves the expresniiirr s1 a set of genes that speci[r positional identity along the axis. These are
M [Iox genes,members of the large family of homeobox genesthat are involved in
rl]ilrri-v
aspectsof development (Box aA, p. 156).The concept of positional identity,
"fl
156
.
4 : P A T T E R NIN c THE VERTEBRATE BoDY PLAN II: THE S OIVIIT ESAN D EAR LY N ER VOU S SYST EM
ilr
Box4A
TheHoxgenes
ti
inuut
1ili
The Hox genesof vertebratesbelongto a large grouP of generegion
regulatoryproteinsthat all containa similarDNA-binding
xlil '- l
The Hox gene clustersand their role in developmentaTec'
ancientorigin.The mouseand frog genesare similarto eac-
'i111
both in their codingsequence,
other and to those of Drosophilo,
ar:
and in their order on the chromosome.In both Drosophilo
vertebrates,these homeoticgenesare involvedin specifyir;
ilii{!.lll.v
by a DNAmotif of aroundI 80 basepairstermedthe honneobox,
a namethat cameoriginallyfrom the fact that this genefamily
was discoveredthrough mutationsthat producea homeotic
axis.The Hox clus'
regionalidentityalongthe antero-Posterior
arose by ger=
almost
certainly
in
Drosophilo
ters in mice and
vertebrates
an:
ancestor
of
duolication in some common
ililllllillill
transformation-a mutation in which one structure replaces
a
another.Forexample,in one homeoticmutation in Drosophilo,
segmentin the fly's body that doesnot normallybearwingsis
insects.
Mostgenesthat containa homeoboxdo not, however,belon:
to a homeotic complex,nor are they involvedin homeot:
t r a n s f o r m a t i o n sO
. t h e r s u b f a m i l i e so f h o m e o b o x g e n e s -
of around60 amino acidsknownas the horneodomain,which
motif that is characterisDNA-binding
containsa helix-turn-helix
proteins.
isencoded
The
homeodomain
tic of manyDNA-binding
transformedto resemblethe adjacentsegmentthat does bear
wings,resultingin a fly with four wings insteadof two.
Clustersof homeoticgenesinvolvedin specifyingsegment
identitywerefirst discoveredin Drosophilo.fhereis one Hoxgene
(known as HOM-C),which is organized
clusterin Drosophilo
complex
into two distinctgene complexes,the Antennapedia
and the bithoraxcomplex.Similarclustersof homeoticgenes
vertebratesincludethe Fax genes,which containa homeobc,
typical of the Drosophilagene poired.All these genes encoci
Ii1[r
celldifferentiation.
lllustrotionofter Coletta,P.,et ol.: 1994.
ill
Ir
r[T.nlilf
lIilllillltil
il
lll L
lI
,lfiil
llllillllt r
t{rlllllllllt
ilimlll1
transcription
factorswith variousfunctionsin developmentan:
lillil
I
llll|
-llllrll'
the clusters
havebeenidentifiedin manyanimals.In vertebrates,
and
known
as
the
Hox
complexes,
are
the homeoboxesof the genes are
relatedin sequenceto the homeobox
complex
of genesof the Antennapedia
in Drosophilo.In each Hox clusterthe
ll
l{lllllllllL
tiiill|IIIlllt
liilllff
I
Drosophilo
il, uililiiil
I]LIL
llLll
liliill
orderof the genesfrom 3'to 5' in the
DNA is the order in which they are
lilfll lll
expressedalong the antero-posterior
axis and specify positional identity.
lililIlillrr
l
lLlillll|u
In the mouse,there are four unlinked
Hox comp lexes, d es ignat ed Hox a,
called
Hoxb,Hoxc,and Hoxd(originally
Hoxl, Hox2,Hox3,and Hox4),located
on ch romo so mes6 , 11, 15, and 2,
Antennapedia
complex
Mouse
ililllLilililll
llililllillllil
Hoxa,
chromosome
6
respectively(see figure). The vertebrate clustershavearisenby duplication of an ancestralcluster,possibly
'lll lllllrlilil
,ii
ItNt\fllfllt
ll liiiillllllr
relatedto the singleHox clusterin the
lancelet(amphioxus),a simple chordate.All Hoxgenesthus resembleeach
Hoxc,chromosome
15
otherto someextent;the homologyis
most marked within the homeobox
Hoxd,chromosome
2
outside
and lessmarkedin sequences
it. Genesthat havearisenby duplica-
comple)(
bithorax
f|lll|ilil
rtlllllt
,,,illlIu
'rlilr
illl
I
ll llllllll
tiitl
are
withina species
tion anddivergence
knownasp*raE*gs,andthe correspon(e.9.
dinggenesin the differentclusters
Hoxa4,Hoxb4,Hoxc4,Hoxd4)are usually
known as a par*logous subgro*p. ln
the mouse there are 13 paralogous
grouPs.
lllllil
t{i'
lnY
A TTE R N IN G
50Il IITEFOR MA TION
A N DA N TE R O-P OS TE RPIOR
il
or positional value, has important implications for developmental strategy; it
funpliesthat a cell or a group of cells in the embryo acquires a unique state related
to its position at a given time, and that this determines its later development (see
Section1.15).
Homeoboxgenesthat specif,ipositional identity along the antero-posterioraxis
were originally identified inDrosophila(seeChapter 2) and it turned out that related
genes are involved in patterning the vertebrate axis. As we shall see in the final
part ofthis chapter, patterning along the antero-posterior axis by Hox genes and
other homeobox genesis not confined to mesodermal structures; the hindbrain,
for example,is also divided into distinct regions.The homeoboxgenesare the most
striking example of a widespread conservation of developmental genesin animals.
ft is widely believed that there are corlmon mechanisms underlying the develop
ment of all animals. This implies that if a gene is identified as having a central role
in tlre development of one animal, it is worth looking to seewhether it is present
in another animal and whether it has a similar function. This strategy of comparing genesby sequencehomology has proved extremely successfulin identiSring
genes involved in development in vertebrates. Numerous genes first identified in
Drosophila,in which the genetic basis of development is far better understood than
in any other animal, have proved to have counterparts involved in development in
rertebrates.
All the homeobox geneswhose functions are known encodetranscription factors.
The subsetknown as the Hox genesare the vertebrate counterparts ofa cluster of
homeobox genes in Drosophilathat are involved in speci$ring the identities of the
different segmentsof the insect body (seeChapter 2). Most vertebrateshave four
separateclusters ofHox genesthat are thought to have arisen by duplications of
ttre geneswithin a cluster, and of the clusters themselves(seeBox 4A',opposite).
The zebrafishis unusual in having sevenclusters,as a result of further duplication.
A particular feature ofHox gene expressionin both insects and vertebratesis that
tie genesin eachcluster are expressedin a temporal and spatial order that reflects
their order on the chromosome. This is a unique feature in development, as it is the
only known casewhere a spatial pattern of genes on a chromosome corresponds
to a spatial pattern in the embryo.
A simple idealized model illustrates the key featuresby which a Hox gene cluster records positional identity. Consider four genes-I, II, m, and lV-arranged
along a chromosome in that order (Fig.4.9). The genes are expressedin a correspondingorder along the antero-posterioraxis ofa tissue.Thus, geneI is expressed
throughout the tissue with its anterior boundary at the anterior end. Gene II has
its anterior boundary in a more posterior position and expression continues
posteriorly. The same principles apply to the two other genes.This pattem of
expressiondefines four distinct regions, coded for by the expressionof different
combinationsof genes.If the amount of gene product is varied within each expression domain, for example by interactions between the genes,many more regions
can be specified.
The role ofthe Hox genesin vertebrate axial patterning has been best studied in
the mouse,becauseit is possible to either knock out particular Hox genesor to alter
ttreir expression(Box48, pp. 158-159).As in all vertebrates,the Hox genesstart to
be expressedin mesoderm cells at an early stageof gastrulation when they begin
to move away from the primitive streak and towards the anterior. The 'anterior'
genesare expressedfirst. As the posterior pattern developslater, clearly defined
patterns of Hox gene expression are most easily seen in the mesoderm and
the neural tube after somite formation and neurulation, respectively (Fig. a.10).
More Hox genes are expressedas gastrulation proceeds.Tipically, the pattern of
157
Fig.4.9 Geneactivity can providepositional
values.Themodelshowshowthe patternof
geneexpression
alonga tissuecanspecifythe
distinctregionsW,X,Y,andZ. Forexample,
onlygeneI isexpressed
in regionW but allfour
genesareexpressed
in regionZ.
_,il
158
.
4 : p A T T E R N T NG
T HE vERT EBRATBo
E Dy p L AN r : T HE sor\i rrE s A N D E A R LyN E R V oussysrE M
Box 48
Genetargeting:insertionalmutagenesis
andgeneknock-out
exon
)CI$*Iffie.
cloned
gene
I
tl
It
V
drug-resistan(e
gene
ffi|lfietins
| .*on
To study the function of a gene controllingdevelopment,it is highlydesirableto be able to
introducean alteredversionof the gene into the animalto seewhat effect it has.Mice into
whichan additionalor alteredgenehasbeenintroducedare knownastransgenicmice(see
Box3E,p. 130).Twomaintechniques
for generating
transgenic
micearecurrentlyin use.One
,r
l
is to inject DNA containingthe requiredgene directly into the nucleiof fertilizedeggs;the
other isto alteror add a geneto the genomeof embryonicstem cells(EScells)in culture,and
then to inject the geneticallyalteredcellsinto the blastocyst,wherethey becomepart ofthe
lu
innercellmass.
.**
.e- .s-- 4-**
-&-@+"
€'-
+
targetgenein
cnr0m0s0me
EScellscanbe genetically
alteredbytechniques
that canbe usedto inactivatea particulargene
or introducea new one.A DNA moleculethat is introducedinto an EScellby traftsfectionwill
usuallyinsertrandomlyin the genome.Howevel it is possibleto tailor the DNAin sucha way
that it insertsat a specificpredeterminedsite by homclogous reeen'tbination;this insertion
mutation rendersthe gene non-functional.The DNA to be introducedmust containenough
sequencehomologywith the target geneto insertwithin the target genein at leasta few cells
in the culture.Theinsertionalsocarriesa drug-resistance
gene,andso cellscontainingthe insertion canbe selectedby addingthe drug,whichkillsthe othet unmodified,cells.ThemutatedE5
+
cellscanthen be introducedinto the blastocyst,producinga transgenicmousecarryinga mutation in a known gene (seefigure,left). The useof homologousrecombinationto inactivatea
geneisknownasger:eknock-cutwhenthe animalishomozygous
forthe inactivatedgene.The
,l]liil
sametechniquecan be usedto inserta novelgene.
The mutatedEScellsarethen introducedinto the cavityof an earlyblastocyst,whichis then
returnedto the uterus.They becomeincorporatedinto the inner cell massand thus into the
'"lt
embryo,wherethey cangiveriseto germ cellsandgametes.Oncethe mutant genehasentered
the germ line,strainsof miceheterozygous
for the alteredgenecan be intercrossed
to produce
1r(l
eitherviablehomozygotesor homozygouslethals,dependingon the gene involved,and the
effectof completelyinactivatingand so knocking-outthe genecan be examined.
lllrL
A techniquefor targetinga geneknock-outto a specifictissueand/ora particulartime in development is provided by the CreJoxsystem.The target gene is first 'loxed' by inserting a /oxP
sequence
of 34 basepairson eithersideofthe gene.Thesetransgenicmicearethen crossed
with
anotherline of transgenicmice carryingthe genefor the recombinase
Cre./oxPsequences
are
lLlll$
lwl
,{tii
itLlll
uttr
Illll
'i(0
recognized
by Cre,whichwill exciseallthe DNAbetweenthe two /oxPsites.In the offspring,if Cre
isexpressed
in allcells,then allcellswill excisethe 'loxed'target,causinga ubiquitousknock-outof
:l
the target gene.However,if the genefor Creis underthe controlof a tissue-specific
promoter,so
li L
that, for example,it isonlyexpressed
in hearttissue,the targetgenewill only be excisedin heart
tissue(seefigureon p. 159).lf the Cregeneis linkedto an induciblecontrolregion,it isalsopossibleto induceexcisionof the targetgeneat will by exposingthe miceto the inducingstimulus.
I
llll
Lilll
Continued
159
50I\4ITE FOR I\4A TION A N D A N TE R O-P OsTE R IOR P A TTE R
N IN C
andgeneknock-out
Box 48 (continuedl Genetargeting:insertionalmutagenesis
A significantnumber of knock-outsof a
singlegene resultin mice developingwithout
anyobviousabnormality,or with fewerand less
severeabnormalitiesthan might be expected
from the normal pattern of gene activity. A
strikingexampleis that of myoD,a key gene in
muscledifferentiation.In myoDknock-outs,the
mice are anatomicallynormal,althoughthey
do have a reduced survival rate. This could
meanthat other genescan substitutefor some
of the functions of myoD.
However,it is unlikelythat any gene is without anyvalueat allto an animal.lt is muchmore
likelythatthere
isan alteredphenotypein these
| +(re I
V
-r
Nljru,-,--E;*;;*-ffi;ffi;
ffiffi*;6;+s{,
(re
Heart-specifir
apparentlynormalanimals,which is too subtle
to be detectedunderthe artificialconditionsof
isthus probably
lifein a laboratory.Redundancy
apparentrather than real. A further complicathat, undersuchcircumtion is the possibility
stances,related genes with similar functions
may increasetheir activity to compensatefor
the mutatedaene.
,Tl
x
{T
T
nf
l
T
F,
!
ffi targetgeneexpresed
- rpressionof each gene is characterizedby a relatively sharp anterior border and,
-s-.rally,a much less well defined posterior border. Although there is considerable
:'"-erlapin expression,almost every region in the mesodermalong the antero-poste:::l axis is characterizedby a particularset ofexpressedHox genes(Fig.4.11).For
-,-ample,the most anterior somitesare characterizedby expressionof genesHoxal
and no other Hox genesare expressedin this region. By contrast, all the
--LHoxb1,
l--r genesare expressedin the most posterior regions.The Hox genesthus provide
: ;ode for regional identity. The most anterior expressionof Hox genesis in the
'rdbraiu the more anterior regions of the vertebrate body-the anterior head,
:::ebrain, and midbrain-are characterized by expression of other homeobox
:=:es such as Emcand otx, and not by Hox genes.The spatial and temporal order
:: expressionis similar in the mesoderm and the ectodermally derived nervous
i,nem, but the boundaries between the regions of gene expressionin these two
:..:n layersdo not alwayscorrespond.
ll we focus on just one set of Hox genes,those of the Hoxa complex, we find
-,:at the most anterior border of expressionin the mesoderm is that of Hoxal in
:*:e posterior head mesoderm,while Hoxall, the most posterior gene in the Hoxa
::nplex, has its anterior border ofexpressionin the sacralregion (seeFig.4.11).
-a:s exceptional correspondence,or co-linearity, between the order of the genes
:- rfie chromosomeand their order of spatial and temporal expressionalong the
i-:.ero-posterior axis, is typical of all the Hox clusters. The genes of each Hox
-::rplex are expressedin an orderly sequence,with the gene lying most 3' in the
;;i[Hw,
(re
inducible
Ubiquitous
160
'
4 : P A T T E R N T NTG
HE vERT EBRATBo
E Dy p L AN n : r HE soMrrE S A N D E A R LyN E R vous sysrE M
Hoxhl
Fig. 4.1 0 Hox gene expression in the mouse embryo after neurulation. The three panelsshon
lateralviews of 9.5 dayspost-coitumembryos immunostainedwith antibodiesspecificfor tne prcmn'
productsofthe Hoxb1,Hoxb4,and Hoxbggenes.The arrowheadsindicatethe anterior boundan oexpressionof eachgene within the neuraltube. The positionofthe three geneswithin the Hoxb og'rnr
complex is indicated(inset).Scalebar = 0.5 mm.
Photographscourtesyof A. Could.
cluster being expressedthe earliest and in the most anterior position. The correm
expression of the Hox genes is dependent on their position in the cluster, prm
anterior genesmust be expressedbefore more posterior genes.
Support for the idea that the Hox genes are involved in controlling regicmd
identity comesfrom comparing their patterns of expressionin mouse and chick rrufu
Vertebral
regions
lumbar
sacral caudal
I
I
'
Fig.4.11 Hoxgeneexpression
alongthe
antero-posterioraxisof the mouse
mesoderm.Theanteriorborderof eachgeneis
shownbythedarkredblocks.Expression
usually
extendsbackward
somedistance
but the
posterior
marginof expression
maybepoorly
defined.Thepatternof Hoxgeneexpression
couldspecify
the identityofthe tissues
at
differentpositions.
Forexample,
the patternof
expression
isquitedifferentin anteriorand
posterior
regionsof the bodyaxis.
Hoxgencs
b9
b7
Antefiol
margins
ofexpression
alO
@,:::r:r
re:::::,:
d9
d10
dtl
rer::l
-w* G a T l
re
G
dI2
dl 3
TE R N I N C
SOM IT E F O R M AT IONAN D AN T ER O - POST ER IO PAT
R
il
il
p
n
the well defined anatomical regions-cervical, thoracic, sacraland lumbar (Fig.4.12).
Eox gene expression correspondswell with the different regions' For example, even
though the number of cervical vertebrae in birds (14) is twice that of mammals, the
mterior boundaries of Hoxc5and HoxcSgene expression in both chick and mouse lie
on either side ofthe cervicafthoracic boundary. A correspondencebetween Hox gene
eryrressionand region is also similarly consewed among vertebrates at other anatoml'al boundaries.
It must be emphasized that the suurmary picture of Hox gene expression given
in Fig. 4.11 does not represent a 'snapshot' of expressionat a particular time but
rrther an integrated overall pattern of expression. Some genes are switched on
mly and are then downregulated, while others are expressed considerably later;
tte most posterior Hox genes, such as Hoxd72and Hoxd73,are expressedin the
post-anal tail, which develops later. Moreover, this summary picture reflects the
grneral expressionof the genesin embryonic regions; not all Hox genesexpressed
in a region are expressedin all the cells ofthat region. Nevertheless,the overall
nHttern suggeststhat the combination of Hox genes provides positional identity.
h the cervical region, for example, each somite, and thus each vertebra, could be
ryecified by a unique pattern of Hox gene expression.
As we saw in Fig. 4.S, grafting experiments show that the character of the
nrrites is alreadydetermined in the pre-somitic mesodermand that somitic tissue
' nT)lanted to other levels along the axis retains its original identity. This includes
frb original pattern ofHox gene expression.By contrast, transplanted lateral plate
nnoderm takes on the Hox expression pattem of its new site. Hox genesproviding
gmitional specification in somites and lateral plate seem to be separate systems,
fu€h
similar mechanisms may be involved.
mesoderm
{Id
veilebrae
somites
vertebrae
lf,i{l2
PatternsofHoxgeneexpressioninthemesodermofchickandmouseembryos,
of Hoxgenesin the
dtheir relationto regionalization.Theposteriormarginsof expression
tnsoderm varyalongthe axes.Thevertebraearederivedfrom somites,40of whichareshown.
llllhrertebraehavecharacteristic
shapesin eachof the five regions:cervical(C),thoracic(T),
differsin chickand
,llllMr (L),sacral(S),andcaudal(Ca).Whichsomitesformwhichvertebrae
startat somite20 in the chick,but at somite12 in the mouse.
thoracicvertebrae
rrruuseForexample,
']fi]lhfr'msition
so
with the patternof Hoxgeneexpression,
from oneregionto anothercorresponds
transitionin both
andthoracicvertebral
lWhd andHoxc6areexoressed
on eithersideofthe cervical
at the transitionbetweenlumbarand
rdf,ilidk
andmouse.Similarly,HoxdgandHoxd70areexpressed
mrCregions.
'ffiare"rkeA.C.:1995.
161
162
4 : P A T T ERNING
T HE VERT EBRATBODY
E
PL AN II : TH E S OMITE S
A N D E A R TYN E R V OU S Y S TE M
e.r Deletion or overexpression of Hox genes causes
changes in axiar patterri
Flg. 4.13 Homeotictransformationof
vertebraedue to deletion ofHoxcgin the
mouse.ln loss-of-function
homozygous
mutantsof Hoxc8,the first lumbarvertebrais
transformed
intoa rib-bearing
thoracicvertebra.
Themutationhasresultedin the transformation
ofthe lumbarvertebra
intoa moreantedor
structure.
If the Hox genes do provide positional values that determine
a region,s subseqw
development, then one wourd expect morphological changes
iittreir pattrrnffi
expressionis altered.This is indeed the case.In order to see
how Hox genescorul
patterning, either their expression can be prevented
by mutation, or they cu
expressedectopically, in abnormal positions. Hox gene
expression can be cin$,
nated from the developing mouse embryo by gene
knock-out techniques {n
Box 48, pp. 158-159).Experiments along these rines have
shown that the atrs.n*dfir
a given Hox gene affectspatterning in a way that accords
with the idea that Hoxgu
activity provides the cells with positionar identity. For
example, mice in whichfu
geneHoxn3has been deleted show structural defects
in the region of the headdl
thorax, where this gene is normally strongly expressed,
and tissuesderived fu
both ectoderm and mesoderm are affected. But the
Hox genes seem to spq
positional identity in rather complex ways. There
is undoubtedly some apl)frrilt
redundancy between the effects of some of the genes,
and when one gc rfu
removed, another may serve in its place. This can make
it difficult to interpret fr
results of Hox gene inactivation. There is also interaction
between the indffil
genes' and this can further complicate results.
For example, with the mrw
Hoxa3gene described above, more posterior axial structures,
where the inactiwdr
gene is also normally expressed,show no evident
defects.
This observationinustrates a general principle of Hox gene
expression,whictrh
that more posteriorly expressedHox genes tend to
inhibit the action of fr
Hox genes normally expressedanterior to them; this phenomenon
is known u
posterior dominance or posterior prevalence.
This means that a change in th
gene expression usually affects the most anterior
regions in which the gene rfu
expressed, leaving posterior structures relatively unaffected.
The effects d er
Hox gene knock-out can also be tissue specific, so that
certain tissuesin which s
Hox gene is normalry expressedappear normal, while
other tissues at tfie sm
position along the antero-posterioraxis are affected.
The apparent absenceofamr
effect may be due to redundancy,with pararogousgenes
from another comprm
being able to compensate.For exampre,HoxhTis expressed
in the same region u
Hoxal (seeFig. 4.11),and so may be rargeryabreto fulfin
the function of an abced
Hoxal gene.
Loss of Hox gene function often resurts in homeotic
transformation-fte
conversion ofone body part into another. This is the case
in a knock-out mutatlm
of HoxcS.Innormal embryos,Hoxcgis expressedin the
thoracic and more posterrm
regions of the embryo from late gastrulation onward.
Mice homozygous for muantff
Hoxc8die within a few days of birth, and have abnormalities
in patterning betweem
the seventh thoracic vertebra and the first lumbar
vertebra. The most obvim
homeotic transformations are the attachment of an
eighth pair of ribs to thp
sternum and the development of a 14th pair of ribs
on the first lumbar verteh
(Fig. 4.13).Thus, the absenceof Hoxcgmodifies the
development of some of fre
cells that would normally express it. Its absencegives
them a more anterior pomF
tional value, and they develop accordingly. In mice in
which Hoxdll is mutared,
anterior sacral vertebrae are transformed into lumbar
vertebrae. Another examph
of tJre homeotic transformation of a structure into one
normally anterior to it @r
be seenin knock-out mutations of Hoxb4.Innormal
mi ce,Hoxb4isexpressedin rhe
mesoderm that will give rise to the axis (the secondceMcar
vertebra),but not im
that giving rise to the atlas (the first cervical vertebra).
rn Hoxb4knock_out mice"
the axis is transformed into another atlas.
R
RNING
SO M IT E F OR M AT IONAN D AN T ER O- POST ER IO PATTE
By contrast, abnormal expression of Hox genesin anterior regions that normally
& not express them can result in transformations of anterior stnrctures into
tnctures that are normally more posterior. For example, when Hoxa7, whose
mmd anterior border of expression is in the thoracic region, is expressed
ftoughout the whole antero-posterior axis, the basal occipital bone of the skull is
&rnsfonned into a preatlas structure, normally the next most posterior skeletal
roffture. Overexpression of Hoxa2in the first chick branchial arch leads to transturnafion of the first arch cartilages such as the quadrate and Meckel's cartilage,
*ich is a precursor element of the lower jaw, into second arch cartilages such as
fue of the tongue skeleton.
h. mice, in the absence of all the Hox10 paralogous group genes, there are no
hrbar vertebrae and there are ribs on all posterior vertebrae; in the absence of
ilthe Hox11 paralogous group several vertebrae become lumbar. These homeotic
fimsformations do not occur if only some members of the paralogous group are
nrrt-ated, suggesting apparent redundancy. There are also synergistic interactions
hrreen Hox genes of the same paralogous group. Thus, knock-outs of mouse
ha3 do not affect the first cervical vertebra-the atlas-or the basal occipital
re of the skull to which it connects, even though Hoxa3 is expressed in the
rcoderm that gives rise to these bones. However, knock-outs of Hoxd3(which is
*o expressed in this regron) cause a homeotic transformation of the atlas into
& adjacent basal occipital bone. A double knock-out ofHoxa3 and Hoxd3 results
L omplete deletion of the atlas. The complete absence of this bone in the
*p-nce of Hox gene expression suggeststhat one target of Hox gene action is
& cell proliferation required to build such a structure from the somite cells.
very few direct targets of Hox proteins have yet been identified.
lffirtunately,
h vertebrates, unlike Drosophila,we also do not know how the pattern of Hox
[rne expression is specified. Retinoic acid has been shown experimentally to alter
t
expression of Hox genes, but whether it is involved in regulating Hox gene
qression in vivo is not clear. The gradient in retinoic acid that is present ftom
lprior to posterior along the main axis of the mouse embryo (seeFig. 4.7) could
h involved in activating Hox genes in normal antereposterior patterning.
ht
evidence suggeststhat microRNA genes may be embedded in some Hox
ruqters and these may be involved in post-transcriptional regulation of Hox gene
r4nression (seeBox 58,p.797, for how microRNAswork).
tr
Hox gene activation is related to a timing mechanism
b.all vertebrates, the Hox genesbegin to be expressedat an early stage ofgastrulah, when the mesodermal cells begin their gastrulation movements. The anteriorntst genes,which correspondto those at the 3' end of the cluster, are expressed
If an 'early' Hoxd gene is relocated to the 5' end of the Hoxd complex, for
its expressionpattem then resemblesthat of the neighboing Hoxd13.
shows that the strtrcture of the Hox complex is crucial in determining the
ofHox gene expression.
Itnfike the situation inDrosopltila,where the activation of Hox genes depends on
unequally distributed along the antero-posterior axis (see Chapter z),
mechanism of activation in vertebrates is more complex and less well underOne way the antero-posterior pattem of Hox gene exPression might be
rrablished in the somitic mesoderm is through linking gene activation to the
fue spent in the somite stem-cell region (seeFig. a.5). In the chick and mouse,
ft whole of the mesoderm alongside the dorsal midline derives from a small
153
164
.
4 : P A T T E R N TNG
T HE vERT EBRATBo
E Dy p L AN r : T HE soMrrE s A N D E A R LyN E R V oussysrE M
Fig. 4.14 Photographof quail-chickchimeric
tissue,Thequailcellsareon the left andthe
chickcellsareonthe right.
Photogroph
courtesy
of NicoleLeDouorin.
population of stem cells located in tJre anterior primitive streak and later im
the tailbud. It has been shown that genes of the Hoxb cluster follow a strio
temporal order of co-linear activation in this stem{ell region, the 3' genes treing
activatedbefore the 5'ones. Expressionofgenes that are activatedin this regionfo
maintained when the cells leave to form the pre-somitic mesoderm, and in tth
way the temporal pattenl of Hox gene activation can be converted into positidil
information as tlre cells expressing different Hox genes become distribrmd
along the antero-posterior :xis. This model is similar to that proposed for speclp
ing position along the proximo-distal axis of the vertebrate limb bud lre
Chapter 9).
We have concentrated here on the expression of Hox genesin the mesoderq h
they are also expressedin a patterned way in the neural tube after its inductiq"
and we shall return to this aspect of antero-posterior regionalization later il. th
chapter.
4.5 The fate of somite cellsis determined by signalsfrom the adjacenttissues
Fig.4.15 Thefate mapof a somitein the
chickembryo.Theventralmedialquadrant
(blue)givesriseto the sclerotome
cells,which
migrateto form the cartilageof the vertebrae.
Therestof the somlte-the dermomyotomeformsthe dermatomeandmyotome,whichgive
riseto the dermisandallthe trunkmuscles.
respectively.
Thedermomyotomealsogivesrise
to musclecellsthat migrateintothe limbbud.
We shall now return to the individual somite and see how it is patterned- Thfu
patteming process is quite independent of the global antero-posterior patterning
of the whole pre-somitic mesoderm by Hox gene expression. The somites of tln
vertebrate embryo give rise to major axial structures: the cartilage cells of rtil?
embryonic axial skeleton-the vertebrae and ribs; all the skeletal muscles, incfu&
ing those of the limbs; and much of the dermis. The fate maps for particufu
somites have been made by gxafting somites from a quail into a correspondiru
position in a chick embryo at a similar stage of development and following the f:rr.
of the quail cells. These can be distinguished from chick cells by their distincrire
nuclei, which can be detectedin histological sections(Fig.a.7al.The cells that form
the lateral (away from the midline) and medial (nearest the midline) parts of chirft
somites are of different origins, and are brought together during gastrulatim.
The medial portion comes from cells in the primitive streak close to Hensen'snodc,
whereasthe lateral portion comesfrom more posterior cells.
Cells located in the dorsal and lateral regions of a newly formed somite make qp
the dermomyotome, which expressesthe gene Pax3, a homeobox-containiry
gene of the paired family (see Box aA, p. 156). The dermomyotome is made ry
of the myotome, which gives rise to muscle cells, and the dermatome, Trn
epithelial sheet over the myotome which gives rise to the dermis. Cells from t}le
medial region of the somite form mainly axial and back muscles,and expressfu
muscle-specific transcription factor MyoD and related proteins, whereas later:dl
ffi
n
n
d
nl{
rEil
#
rd
fi&
re
r@l
ffim
4
ffi
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R N IN G
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rpnc 6iglafs to give rise to abdominal and limb muscles. The ventral part of
rffoemedial somite contains sclerotome cells that exPress tl;ie PaxT gene and
migrate ventrally to surround the notochord and develop into vertebrae and
nibs[Fig.a.1s).
lAthich cells will form cartilage, muscle, oI dermis is not yet determined at the
time of somite formation. Specification of these fates requires signals from tissues
rdjacent to the somite. This is clearly shown by experiments in which the dorsowtrtral ofientation of newly formed somites is inverted; they still develop
In the chick, determination of myotome occurs within hours of somite
fumation, whereas the future sclelotome is only determined later. Both the neural
mbe and notochord produce signals that Pattern the somite and ale required for
ib future development. If the notochord and neural tube are removed, the cells in
tlhe somites undergo apoptosis; neither vertebrae nor axial muscles develop,
rfrtough limb musculature still does.
The role of the notochord in speciffing sornitic cells has been shown by
qEriments in the chick, in which an extra notochord is implanted to one side of
ft neural tube, adjacent to the somite. This has a dramatic effect on somite
ffftrentiation, provided the operation is carried out on unsegmented Pre-somitic
lmoderm. When the somite develops, there is an almost complete conversion to
mtilage preflrrsors (Fig. a.16), suggesting that the notochold is an inducer of
wtilage. Ttre neural tube also has a cartilage-inducing effect on somites, which is
nntiated by the most venffal region of the tube, the floor plate (seeSection 10.7).
There is also evidence for a signal from the lateral plate mesoderm, which is
frmolved in speci$ring tlre lateral part of the dermomyotome, and for a signal from
th overlying ectoderm (Fig. a.771.
Some of the signals that pattern the somite have been identified. In the chick,
tle notochord and the floor plate exPress the gene Sonichedgehog,which
ffi
rlrodes a secreted protein that is a key molecule for positional signaling in a
mnrmherof developmental situations. We met Sonic hedgehog in Chapter 3 as
e signal involved in the asymmetry of stnrctures about the midline and we shall
ret it again in Chapter 9 in connection with limb development. In somite
the ventral region ofthe
the signal generatedby Sonichedgehogspecifies
xrnite and is required for sclerotome development. Signals from the dorsal neural
rffie and from the overlying non-neural ectodem speciff the dorsal region.
ttrseted signaling proteins of the Wnt family (which we also encounteled in
Ohapter 3) are good candidates for the lateral and dorsal signals. Tendons arise
fton cells that come from the dorso-lateraldomain of the sclerotomeand specifilotry express the transcription factor Scleraxis. This tendon-Progenitor region is
ftirtr6sd at the boundary of the sclerotome and myotome.
tegulation of the Pax homeobox genes in the somite by signals from the notodord and neural tube seems to be important in determining cell fate. Pax3 is
iiinfti:lly expressedin all cells that will form somites.Its expressionis then moduruted by signals from BMP-4 and Wnt proteins so that it becomes confined to
mscle precursors. It is then further downregulated in cells that differentiate as
nhe muscles of the back, but remains switched on in the migrating presumptive
mscle cells that populate the limbs. Mice that lack a functional Pax3gene-Splotch
nnrrenB-lack limb muscles. In the chick, Paxl has been implicated in the
tfirrrnation of the scapula, a key element in the shoulder girdle, part of which is
mtributed by somites. Unlike the Paxl-expressingcells of the vertebrae, which
ue of sclerotomal origin, the blade of the scapula is formed from dermomyotome
mlls of chick somites 77-24, whereas the head of the scapula is derived from
Meral plate mesoderm.All the scapula-formingcells expressPax1.
Dorsal
Fig. 4.16 A signalfrom the notochord
inducessclerotomeformation. A graft of
to the dorsalregion
notochord
anadditional
embryosuppresses
of a somitein a 10-somite
the formationof the dermomyotomefrom the
the
dorsaloortionof the somite,andinduces
whichdevelopsinto
formationof sclerotome,
Thegraftalsoaffectsthe shapeof
cartilage.
the neuraltube.
Dorsal
ll€ntnl
!
lateralizingsignal
ventralizingsignal
!
I
donalizingsiqnal
Fig.4.17 A model for patterningof somite
differentiation.Thesclerotomeisthoughtto
the
signal,probably
bya diffusible
bespecified
protein,
from
the notochord
hedgehog
Sonic
andthefloorplateof the neuraltube(blue
fromthe dorsalneuraltube
arrows).
Signals
wouldspecifythe
andectoderm(redarrows)
togetherwith lateralsignals
dermomyotome,
(greenarrow)from the lateralplatemesoderm.
R.L.:1994.
Afterlohnson,
166
.
4 : p A T T E R N T NTGHE vERT EBRATBo
E Dy p L AN n :T HE soMrrE S A N D E A R LvN E R V oussysrE r\i l
Summary
Somitesareblocksof mesodermal
tissuethat areformedaftergastrulation.
Theyfosequentially
in pairson eithersideof the notochord,startingat the anteriorendof tit
embryo.Thesomitesgiveriseto the vertebrae,
to the muscles
of the trunk andllmb:
andto the dermisof the skin.Thepre-somitic
mesodermispatternedalongitsanterc*
posteriordimensionwhile cellsare in the node,and the first manifestation
of tiir:
patternisthe expression
genes
pre-somitic
the
Hox
in
the
mesoderm
of
beforesom
formation.Thesomitesarealsopatternedby signalsfromthe notochord,neuraltum.
particular
give
andectoderm,whichinduce
regionsof eachsomiteto
riseto musce
cartilage,
or dermis.
The regionalcharacterof the mesodermthat givesriseto somitesis specifie:
evenbeforethe somitesform.Thepositionalidentityof the somitesisspecified
byrt
combinatorial
expression
alongthe antero-posterfcr
of genesof the Hoxcomplexes
axis,from the hindbrainto the posteriorend,with the orderof expression
of thesr
genesalongtheaxiscorresponding
to theirorderalongthechromosome.
Mutatior:r
overexpression
of a Hoxgeneresults,in general,in localizeddefectsin the anter::i
partsof the regionsin whichthe geneis expressed,
andcancausehomeotictransiomations.We canthinkof Hoxgenesasprovidingpositionalinformationthat speciiir:
the identityof a regionand its laterdevelopment.
Theyact on downstreamtarge=
aboutwhichwe knowrelatively
little.
it
t-
rL
-: l
L. . -: --
' l----
The role of the organizerand neural induction
We shall now look at the role of the crucially important organizer region bo--: -:
neural induction and in organizing the antero-posterioraxis in vertebrateemb:--:.
The Spemannorganizer of amphibians, the shield in zebrafish, Hensen'snodt ,r
the chick, and the equivalent node region in the mouse all have a similar gl- - .L
organizing function in vertebrate development.They can induce a complete !-,: '
axis if transplanted to another embryo at an appropriate stage,and so are ab.. .
organize and coordinate both dorso-ventral and antero-posterior aspectsof --::
body plan, as well as induce neural tissue from ectoderm. In the mouse,the a:,:
rior visceral endoderm is required as well as the node for the induction of s=-.
tures anterior to the end of the notochord. such as the head and forebrair
'..'
Section3.9).
During gastrulation, the ectoderm lying along the dorsal midline of the emr- .
becomesspecifledas neurectoderm,the neural plate. During the subsequeDtS=i:
of neurulation, this forms the neural tube, which eventually differentiatesinrc --:.
central nervous system-the brain and the spinal cord-and the nervesthat in:.:
vate the skeletal muscles.The neural tube also throws offneural crest cells,rr';:--:
migrate throughout the body to give rise to the sympathetic and paraslnnpatic: .
neryous systemsand other structures. The nervous system must develop il --::
correct relationship with other body structures, particularly the mesodern:.
derived structures that give rise to the skeleto-muscularsystem.Thus, patter::---:
of the nervous systemmust be linked to that of the mesoderm,and this is coc- nated through the organizer,which is involved in both. In this part ofthe chac:=:
we consider the patterning of the neural tube up to shortly after its closure --,:
also considerthe specificationand formation of the neural crest cells.We wili -r::.:
look at the hindbrain at a later stage,when that region becomessegmented-- neural crest cells have migrated.
I
)
RN D N E U R A TIN DU C TION
TH E R OLEOF TH E OR GA N IZE A
167
The function of the organizer has been best studied in amphibians, and we have
already described its role in the dorso-ventral patterning of the mesoderm of
XEnoW (see Section 3.19).We now discuss its profound effects on the antero'
posterior axis. To do this we must first return to the late blastula and early gastrula,
before the stage of somite fortration discussedin the previous Part of the chapter.
{-6 The inductive capacity of the organizer changesduring gastrulation
htu
F"
hnD
ru
:tr
bm
tft
rn-
h amphibians, the action of the organizer is dramatically dernonstrated in what is
classicallyknown as primary embryonic induction. In the early amphibian gastrtrla'
the Spemann organizer is located in the dorsal lip of the blastopore. If this is
grafted to the ventral side of the marginal zone of another gastnrla' it can induce
e complete secondembryo (Fig.4.1s).This secondembryo can have a well defined
head and trunk region, and even a tail, but is joined to the main embryo along the
rris (seeFig. 1.10).A variety of other treatments, such as grafting dorsal vegetal
bl,astomerescontaining the Nieuwkoop center to the venffal side, produce a similm result (see Fig. 3.32), but what all these treatments have in common is that'
directly or indirectly, they result in the formation of a new Spemann-organizer
region. One important question, which is still umesolved, is whether or not the
Eganizer contains functionally sepalate organizers for the head, trunk, and tail
rcgions. This question arises from classic experiments that showed that a graft of
tte blastopore dorsal lip (which contains the organizer region) from an early
gestrula induces a complete body axis and centlal nervous system, whereas a
lbrsal lip from a mid-gastrula induces a trunk and tail but no head, while a dorsal
lip from a late gastrula induces only a tail (seeFig. 4.18).This is interpreted to mean
that as gastrulation proceeds, the antero-Posterior axis becomes specified,
mrl so the cells that make up the organizer at later stagesof gastrulation induce
mly posterior structures. Is this due to a change in the quantity of inducing
rLmals produced by the organizel as gastrulation proceeds,or are different signals
fuolved in speci$ring different regions of the anteroposterior axis?
aniuerfantplantfromeailygastnda
IC-
:F
m
}lDE
r
re
ro
r&
s
&
dtF
rr4
adi
ttu
ld
Fig. 4.18 Theinductivepropertiesofthe
organizerchangeduring gastrulation.
A graft of the organizerregion,from the dorsal
lip of the blastoporeof an earlyfrog gastrulato
the ventralsideof anotherearlygastrula,results
of an additionalanterioraxis
in the development
at the siteof the graft (left panels).A graft from
the dorsallip regionof a lategastrulato anearly
gastrulaonlyinducesformationoftail structures
(rightpanels).
168
.
4 : p A T T E R N T NG
T HE vERT EBRATBo
E Dy p L AN u : T H E soMtrE s A N D E A R LyN E R vous sysrE M
Flg. 4.19 Different parts of the Xenopus
organizerregion give riseto different tissues.
(leftpanel)the organizer
In the earlygastrula
is
locatedin the dorsallipofthe blastopore.
Thecellsofthe leadingedge(orange)
arethe
firstto internalize
andgiveriseto anterior
endoderm
ofthe neurulastage(rightpanel).
Thedeepcells(palebrown)arenextto
internalize
andgiveriseto the pre-chordal
plate,
the mesoderm
anteriorto the notochord,
which
formsmuchofthe head.Theremainder
ofthe
givesriseto notochord
(red).
organizer
Adopted
C.ondNiehrs,
C.:2001.
fromKiecker,
The cells present at the dorsal lip ofthe blastoporc in axenop)s early gastrula gh
rise during gastrulation to anterior endodem, prospective head mesoderm (ft
pre-chordal plate), and the chordamesodem that forms the notochord (Fig. af$"
As well as providing cells for these axial structures, the organizer region h
patteming and inductive properties: it helps to pattern the adjacent more vegeidl
mesoderm, as we saw in Chapter 3, and it induces the neural plate in the adjacrr*
dorsal ectoderm. The early gastrula organizer is a complex signaling center \,rifr
the different parts expressing different genes,having different inductive capacitk*
and giving rise to different structures. Becauseof these complex properties, unfustanding how the organizer region organizes the overall pattern of the antero"
posterior axis is not straightforward. As gastrulation proceeds and cells mm
inwards, the cellular composition of the dorsal lip changes, and at one level ttiir
explains the different inductive proper[ies displayed by the organizer over time in
experiments such as those describedabove.For example,in the early gastrula-rh
vegetal portion of the organizer that is fated to produce pre-chordal mesod€m
expressesproteins, such as the transcription factor XOtx2, that are characterift
of anterior structures. Experiments investigating the inductive capacity of differtil
parts ofthe organizer show that the ability to induce headsis also restricted to ' -vegetal region. The more dorsal part ofthe organizer is characterized by expresslu
of the transcription factor Xnot, and can induce trunk and tail structures but d
heads.
The avian equivalent to the Spemannorganizer is Hensen'snode, the region d
the anterior end of the primitive streak in the chick blastoderm (seeFig. 3.1f,r
In normal development the node contributes cells to head mesoderm, notochcnd_
somites, and gut endoderm, as well as producing inducing signals. The properticr
of the avian node have been investigated by transplanting quail nodes to chirft
embryos. If for example, a quail node from a head-process-stageembryo is grafud
beneath a chick epiblast at the same stageof development,the transplanted nodp
can induce the formation of an additional axis with somites, but no anterior neurdil
tissue (Fig.4.20).Induction occursif the graft is placed quite closeto the embryotr
own primitive streak; the $aft then induces the non-axial mesoderm to forro
somites and other axial structures. AsinXenopus,grafting an earlier-stagenode im
the area opaca of an earlier stage embryo can induce the formation of a new bodfu
axis, complete with neural tissue.
In the mouse, the node precursors can induce a similar axis duplication m
transplantation to the lateral epiblast of an early embryo, with the exception d
A N D N E U R A LIN D U C TION
TH E R OLEOF TH E OR GA N IZE R
q
Fig.4.2O Hensen'snodecan inducea new
node
axisin avianembryos.WhenHensen's
stage
froma quailembryoat the head-process
isgraftedto a positionlateraltothe primitive
streakof a chickembryoat the samestageof
a newaxisformsat the siteof
development,
(Atthe head-process
stage,
transplantation.
of the primitivestreakiscomplete,
elongation
the notochord-the headprocess-hasstarted
to formanteriorto the node,butthe nodehas
not yet startedto regress.)Histological
showsthatalthoughsomeof the
examination
somitesof this newaxisareformedfrom the
graftitself(quailtissuecaneasilybe
seeFig.4.14),
fromchicktissue,
distinguished
othershavebeeninducedfrom hosttissuethat
Graftinga
formsomites.
doesnot normally
a newaxislacking
nodeat thisstageproduces
neuraltissue.Graftingat anearlierstageinto
the areaopacawillinducea completenewbody
axis,completewith neuraltissue.
i.-l
i[1
_l
[gi,ft'
r {tlttP
+rgil[,
rhu
Ttr'r[[li
Fad
rmfrffih
rfriq
rfuF'
ffitf,n"
l5m
il ttuih
*tu
h.tu
tu
rirniir
M
btfuls,
iltmr
lffi
tstrd
. E_IffiX
Ml.
:irrrfine
forebrain, which requires additional signals from the anteriorvisceral endoderm
il{p Fig. 3.39).
in the organizer and are known
-f ngmber of proteins are specifically expressed
M be required for its function (Fig. 4-271.Goosecoid,for example, is an early and
reIlent marker of the organizer that is expressedin the cells that will give rise
ffi furcgut, pre-chordal plate, and notochord, and which have internalized by the
mftIaastrula stage.Goosecoidcan mimic almost all the properties of the organizer,
iM although it is required for head formation in the normal course of developrlrisrl goosecoid
mRNA injected into venffal blastomeres induces a secondary axis
lb*ing a head. This indicates that additional signals are involved in the induction
osthe head and tfie central nervous system. Signaling Proteins secreted by cells of
ft organizer are probable candidates. Head formation in Xenopttsapparently
rmFrires the inhibition of both BMP and Wnt, and even Nodal signals, which are
' ' g produced in the gastrula at this time: an extra head can be induced by
tdh simultaneous ectopic inhibition of BMP and Wnt signaling in early gastrulas.
rtn *e saw in Chapter 3, antagonists of BMPs and Wnts are secreted by the
rymizer: the proteins Chordin, Noggin, and Follistatin can antagonize BMP, and
lllffikopf antagonizeswnt signaling. The protein cerberus can inhibit wnt, Nodal,
BMP signaling. It is important to bear in mind, however, that although
d
:lnqnyof the same proteins are produced in and around the organizer in different
mebrates, they do not all have precisely the same functions in all our model
@
rffi
;mrimels.
fffi
lffi
Mll
c; The neural plate is induced in the ectoderm
mrm
rf u
hM
rh@
bnm
tud
169
region
Gmerin organizer
Genes
enroding
tlanscfption
fadors
Genes
encoding
secreted
proteins
shh
Cerfurus
'&
induction of neural tissue from ectodem was first indicated by the organizerummsplantexperiment in frogs illustrated in Fig' 4.18; in the secondary embryo
M forms at the site of transplantation, a nervous system develops from the host
@derm that would normally have formed ventral epidermis. This suggestedthat
rsrral tissue could be induced from asyet unspecified ectoderm by signals emanatmgfrom the organizer mesoderm. The requirement for induction was confirmed
huperiments that exchangedprospectiveneural plate ectoderm for prospective
u4*lermis before gastrulation; the transplanted prospective epidermis developed
Fig.4.21 Genesexpressedin the Spemann
organizerregion of the Xenopusgastrula,
and in Hensen'snode in the mousegastrula.
Thereisa similarpatternof geneactivityin the
genesbeing
with homologous
two animals,
of someofthese
Theexpression
expressed.
is not confinedto the
genes,suchasBrochyury,
Shh= Sonichedgehog.
organizer.
H E sOMITE SA N D E A R LYN E R V OU S
5Y S TE M
y. O.2 The nervous system of Xenopusis
inducedduring gastrulation.The
left panels
showthe normaldevelopmental
fateof
ectodermat two differentpositions
in the early
gastrula.Theright panelsshow
the
transplantation
of a pieceof ventralectoderm,
whosenormalfateisto form epidermis,
from
the ventralsideof an earlygastrula
to the dorsal
sideof another,whereit replaces piece
a
of
dorsalectodermwhosenormalfate
isto form
neuraltissue.In its newlocation,the
transplantedprospective
epidermisdevelops
not asepidermisbut asneuraltissue,
andforms
part ofa normalnervoussystem.
Thisshows
that the ventraltissuehasnot yet
been
determinedat the time of transplantation,
and
that neuraltissueis inducedduringgastrulation.
rnto ne'ral tissue, and, the-tralsplanted
prospective neurar tissue into
epideruri
{Fig.4.22).This shows
that the
u^ rv'uduuu
fo.*"tion of
oftfrl
ule nervous system is dependent
on u
inductive signal.
An enormous amount of effort
was devoted in the 1g30sand 1g40s
to tryitrgb
identify the signars involved
in neurar ioarrJor in amphibians.
Researchers
encouraged by the finding that
a dead organizer region could :ll
'ren
s
induce rcrd1
tissue' It seemed to be merery
a matter or rr"ra work to isolate
the chemfot
responsibre.
the search was fruitless, for it
appeared that an enormous vrr
ety of substances
^'as, were capable
of varying degreesof neural induction.
fu it hrrnr*l
out' this was becausenewt ectoderm,
the main experimental material
use*
sem
to have a high propensity to
develop into oeu.ar tissue on its
own. This is not &
casewith xenoQrus
ectoderm, although proronged
culture of disaggregatedectfu
mal cells can result in their Oif"ru"t"tioo
,
experiments
inxenopus,it
was
round
th",*" "i#;:H ;H"ffi;";ffi::1[ffi
a nucleopore filter (which prevents
.uu .ora.t but allows the passage
of qdh
large molecules, such as proteins),
and that contact with organizer
mesoderm.#
ing about 2 hours is required
for induction,o o..*. The morecules
responsibrefu
neural induction have still not
been definitivelyiaentified, although
there are ,*
some strong candidates.
Neural tissue can be induced
in the chick epiblast-in both
the area perlucrh
and the :*ea opaca-otfrom the primitive*treak mesoderm.
*1r
Indnin
activity is initiany located_in
the anterior piJtive
streak and Hensen,snode,
later' during regression of the
d
node, r".oi",
.onnned to the region ofpre.somrfo
mesoderm just anterior to the
node. By the four-somite stage
inJuctive i-"o*
disappeared;the competence
ofthe ectoderm to respond disappears
at aboutrftr
sametime.
A key point in the study of neural
induction was the finding that
the disagrqp,
tron of xenopts gastrura-stage
animal caps removed an inhibitor
oro"*r
derehilD
ment; this was subsequently
identifieJ as BMp<. In the late
blastula, BM,o am
expressedthroughout the
ectoderm (seeFig. 3.62) but expression
is ;*q.dm
lost in the neural plate. Neurar
induction.ota
trr"rerore be due to tie prodrcim
of proteins by the organizer that
bound to nup,
hfted their inhibitory artur
"oa
TH E R OIE OF TH E OR GA N IZE A
RN D N E U R A LIN OU C TION
'fi$ BMP' induce expression of their own genes, this would also suppressBMp gene
crylression. BMP inhibitors such as Noggin and chordin
are p.oauc"a in the organm (seesection 4.6), and their inhibition of BMp proteins produces
the gradient of
hlP sipaling that helps pattern the early mesoderm
(see Section 3.19).These
deerrrations led to the so<aled defaurt moder for
neural induction in xenop,s.
ftis proposes that the default state of the dorsal ectoderm
is to develop as neural
'but that this pathway is blocked by the presence of BMps,
which promote
''e,
ft epidermal fate. The role of the organizer is to lift
this inhibition by brocking
DIP activity; tfre affected region of ectoderm will then
develop as neural ectoderm.
&fieins produced by the organizer can act on ectoderm
urat hes adjacent to it at the
hginning of gastrulation (seeFig.4.19), and as gastmlation proceeds,
internalized
c[s derived from the organizer influence the fate of the
ectoderm overrying t]rem.
slimination of organizer signals individually
in amphibians has rather modest
rfrc* on neural induction. But when
the BMp antagonists chordin, Noggin, and
Histatin
were simurtaneousry depleted in the organizer of the
frog xenow
rli'nhs using antisense morpholino origonucleotides (see Box 5A, p. 189),
there
ms a dramatic failure of neural and other dorsal development
and L expansion
drcntral and posterior fates.
lhere are problems with the default model, however,
as neurar induction in
wx'ry$
and the chick has been shown also to require the growth
factor FGF,
arnwhen BMp inhibition is lifted by the presence of
Noggin and chordin. In the
fi.*'
FGFis secreted by the hypobrast, which as gastrulation proceeds
becomes
rgressively dispraced towards the anterior of the embryo
uy trre endoblast
Fc section 3.3).Arso, some genes that appear to be required for neural
induction
ft 6ick are not activated by BMps in ectoderm, but
are activated by FGF. one
gene is churchitr:when its expression is downregrrated
G
using an antisense
rpholino
oligonucreotide (see Box sA, p. 189) the neural plate
ioes not form.
ifuidl
can be activated by FGF, and this in turn leads to repression
of genes
ft*erted5lis
of mesoderm and activation of the gene for the neurar-specific
ItEscdption factor Soxz.
KF is not thought to be the sole inducer of the neurar
tube in vertebrates,
h*,
and inhibition of BMp signaling is likely to have a part
to play. FGFactiffis tle mitogen-activated protein (MAp) kinase intracenuiar
rtg""ri"g pathway
ffiEEg. 8.4) and there is evidence that MAp kinase can interfere with BMp
signal_
an inhibitory phosphorylation of smad-l, which is part
of
the
BMp
ir
-TTt
signaling pathway (seeFig. 3.5S).
(raer experiments in chick embryos
suggest that BMps are invorved in setting
:boundary ofthe neural plate. BMps are expressedat
the border ofthe neurar
e,and the application of BMp to the border region causes
inward displacement
narrowing of the plate, while the apprication of BMp antagonists
causeswiden-a seemsthat, in the chick embryo at least, two separatedecisions are required
rurd-plate formation: one is between mesendoderm or
neural ectodem fate
+rblast cells that sets the medial edge of the prospective neurar plate (the
edge
r will be adjacent to the streak when gastrulation stops),
and involves FGFand
; the other is made at the lateral edgesofthe neurar prate, where
neurar and
ral ectoderm meet, and is likely to involve inhibition of BMp
signaling.
h zebrafish, there is a clear difference in the mechanism
of induction of
:nor neural tissue close to the shield in the dorsal region,
and the induction of
lnsterior neural ectoderm that will give rise to the spinal cord. whereas
the
in zebrafish contributes to the induction of anterior
neurar tissue by
BMP signaling, the ectoderm that develops into the spinal
cord is some
away from the organizer on the ventral_vegetal side
of the embryo,
171
t: TH E S OMITE S
A N D E A R LYN E R V OU S Y S TE M
Flg.4.23 Prospectivespinalcord in the
zebrafishembryo is distant from the
organizer.Theectodermthat will form the
spinalcordissituatedon the othersideofthe
gastrulafrom the organizer,
in the
ventral-vegetal
ectoderm,andistoo far awavto
beinfluenced
bysignals
fromthe organizer.
FCF
signals
in the ventral-vegetal
regioninducethis
ectodermasneurectoderm
and BMpspromote
its developmentasposteriorneuraltissue.
Adopted
fromKudoh,T.,et ot.:2004.
where organizer signarsdo not reach (Fig.
a.23).The initiator of neural deveropmerrfr
in this ventral-vegetal ectoderm
is FGi and BMp signals, which are
high in th
ventral region, in this caseact to push
the neurectoderm towards a posterior fuI
Neurar induction is a_comprexmultistep
processand the very fi,,t stages
@E
likely to occur in the brastula,
even before a ostio.t organizer region
becornq
detectable' The individual signaling proteins
have murtiple roles in deveropme4
and their rores change over time, which
makes it difficurt to disentangle their
mcil
contributions to any particular process.
Their developmental roles can arso diffi*
between different veftebrates. An
essential similarity in the mechanism
of ne.,dil
induction among vertebrates is rikery,
however, as Hensen,s node from a chirh
embryo can induce neural gene express
ion in xenopusectoderm(Fig. 4.24),
wtfofr
suggests that there has been an
evolutionary conseryation of inducing
signah
Moreover, early nodes induce gene
expression characteristic of anterior amphrihdm
neural structures,whereasolder
nodesinduce expressiontypical ofposterior
strre
tures' These resurts are in line with
the theory that the vertebratJnode
specifcr
different antero-posterior positionar
varuesat djfferent times, ana ttr"y-.orrt
_ ru
essentialsimilarity of Hensen,snode
and the Spemannorganizer.
In chick embryos, the spinal cord
is ,ro, ,irrrpty an extension of the posterir
most neural plate. The complete spinal
cord is generated from a small region
d
Ftg.4.24 Hensen,snodefrom a chick
embryo
can inducegeneexpressioncharacteristic
of
neuraltissuein Xenopusectoderm.Tissues
from differentpartsof the primitive_streak
stageof a chickepiblastareplacedbetween
two fragmentsof animalcaptissue(prospective
ectoderm)from a Xenopus
blastula.The
inductionin the Xenopus
ectodermof genesthat
characterize
the nervoussystemisdetectedbv
lookingfor expression
of mRNAsfor-nurr.l ."il
adhesion
molecule(N-CAM)
andneurooenic
factor-3(NF-3),whichareexpressed
spJcifically
in neuraltissuein stage30Xenopus
embryos.
Onlytransplants
from Hensen's
nodeinduce
the expression
oftheseneuralmarkers
in the
Xenopus
ectoderm.(EF-1oisa common
transcriptionfactorexpressed
in all cells.)
AfterKntner,C.R.,
et o!.:1991.
3o
s5
s <p
:E
FE
i-e
l<rttrmr
3an"
A N D N E U R A LIN D U C TION
TH E R OLEOF TH E OR GA N IZE R
I nelral
I tube
|- (spinal
I ord)
I transition
I zone
I rtt
f zone
[Isal
173
Fi1.4.25 Thespinalcord is formed from a
zoneof stem cellsthat arisesin the posterior
neuralplate. In the chickembryo,a restricted
regionof stemcellsformsin the posteriorneural
platealongside
the nodeandanteriorprimitive
streakjustbeforethe onsetof somitogenesis
of
(yellow).Theregionis markedby expression
5ox7.Thisstem-cellregionmovesposteriorly
leavingbehind
with the nodeasit regresses,
progenitor
neuralcellsthatwillformthe spinal
cord.Thecompletespinalcordisformedfrom
this stem-cellzone.
M.,etol.:2005.
Adopted
fromDelfino-Mochin,
pdiferating stem cells that develops in the ectoderrr at the posterior end of the
nraf phte on both sides of the node and primitive streak (Fig. 4.25). This zone
lbmes
distinct just before somite formation begins. The stem-cell zone moves
along with the node, leaving behind progenitor cells that produce the
Lrciorly
qfual cord.
Tlrenervoussystemcanbe patternedby signalsfrom the mesoderm
the mechanisms of neural induction eventuallv turn out to be, it is clear
the neural plate can be patterned by signals from the mesoderm. Pieces of
taken from different positions along the antero-posterior axis of a newt
and placed in the blastocoel of an early newt embryo induce neural strucat the site of transplantation. The structures that are formed correspond
or less to the original position of the transplanted mesoderm: pieces of
irr mesoderm induce a head with a brain, whereas posterior pieces induce a
witlr a spinal cord (Fig. a.26). Another indication of positional specificity in
W
:re*w
lig. 4.26 lnduction of the nervoussystem
by the mesodermis region specific.
Mesodermfrom differentpositionsalong
axisof earlynewt
the dorsalantero-posterior
neurulasinducesstructuresspecificto its region
to ventralregions
of originwhentransplanted
Anteriormesoderminduces
of earlygastrulas.
a headwith a brain(top panels),whereas
posteriormesoderminducesa posterior
trunkwith a spinalcordendingin a tail
(bottom panels).
O.:1933.
AfterMongold,
174
.
4 : P A T T E R N T NG
r HE vERT EBRATBo
E Dy p L AN r t: T H E soMrrE s A N D E A R ty N E R V oussysrE M
induction comes from the observation that pieces of the neural plate themsehu
induce similar regional neural structures in adjacent ectoderm when transplantd
beneath the ectoderm ofanother gastrula. Indications that gene expression in th
mesoderm may be influencing gene expression in the ectoderm come from fre
observation of coincident expression of several Hox genes in the notochord and il
the pre-somitic mesoderm and ectoderm at the same position along the anteto
posterior axts:.Nhboxl in xenopts and HoxbTin the mouse are examples of gw
that have coincident expressionofthis type.
Hox genesare involved in patterning the hindbrain, as we shall seelater, but th
gene exPression cannot be detected in the anterior-most neural tissue of fu
mouse-the midbrain and forebrain. Lrstead, homeodomain transcription facfmi
such as otx and Emc are expressedanterior to the hindbrain and speciff patten
in the anterior brain in a manner similar to the Hox genes more posteriorly. Th
Drosopthila
orthodmticlegene and the mouse otx genes are homologous and provlh
a good example of the conservation of gene function during evolution. orthoderffi
is expressed in the posterior region of the future Drosoprhila
brain and mutatim
in the gene result in a greatly reduced brain. In mice, otxT andotx2 arc expressedil
overlapping dornains in the developing forebrain and hindbrain, and mutatior
in otxT leads to brain abnormatties and epilepsy. Mice with a defective otx gm
can be partly rescued by replacing it with orthodenticle,even though the sequeur
similarity of the two proteins is confined to the homeodomain region. Human (rr
can even resste orthndmticlemtrtants inDrosopthiln.
one model for neural ectoderm patterning inxenoyntssuggeststhat qualitatiwfir
different inducers are present in the mesoderm at different positions along th
antero-posterior axis. Many experiments fit quite well with a simpler fry6ggnd
model of neural patterning, however, in which differences are due to quantitatirc
rather than qualitative differencesin the inducing signals(Fig.a.27l.In this modef
the first, activating, signal is produced by the whole mesoderm and indus
the ectoderm to become anterior neural tissue. A second signal transforms part d
this tissue so that it acquires a more posterior identity. This latter transforming
signal would be graded in the mesoderm with the highest concentration at rt.
posterior end. In Xeropra,chick, and zebrafish, wnts are the posteriorizing factm*
W
|T--'.iii-@,
s ess sttt
ffi
ry
Flg. 4.27 Modelsof neuralpafterning by
induction.Toppanel:in thetwo-signal
model,
onesignalfromthe mesoderm
firstinduces
anteriortissuethroughoutthe corresponding
ectoderm.A second,graded,signalfrom the
mesodermthen specifiesmoreoosterior
regions.
Bottompanel:in analternative
model,
qualitativelydifferentinducersarelocalizedin
the mesoderm.
AfterKelly,O.G.,et ol.: 199 .
$€ti
ffiffin
TH E R OLEOF TH E OR GA N IZE R .A NN
DE U R A TIND U C TION
175
rs inceased concentrations of these proteins cause cells in the neural plate to
dqrt a more posterior fate. Another component of the posteriorizing signal is
Etinoic acid. In the mouse, the anterior visceral endoderm is the source of
tohibitory signals that protect anterior tissue from these posteriorizing signals.
r.r Thereis an organizerat the midbrain-hindbrain boundary
Ih developing brain is divided into three main regions-forebrain, midbrain, and
rindhrqin (Fig. 4.28). The hindbrain has a segmented organization, which will be
tr*russed in the next section, whereas the midbrain and forebrain are unsegmtred- An organizing region located at the midbrain-hindbrain boundary, also
hown as the isthmus, regulates patterning of the midbrain. If this region is
sEfted into the anterior midbrain it respecifies the tissue as posterior midbrain,
frl tansplanting it into the forebrain converts that tissue into midbrain. FGF-8is
:ryressed at the isthmus and is a good candidate for signaling posterior midbrain
ilroctures; induced expression of FGF-8in the chick anterior midbrain results in
ft expression of genes characteristic of the posterior midbrain. Zebrafish that
lfre the mutation acerebe\ar,which is due to loss of FGF-8function, lack the posterirmidbrain regions associatedwith the organizer. The development of the organfu region is linked to the expression of Otx2 in the midbrain and Gbox2in the
hrfhrqiri; the border between the expression of these two genes is important for
rrt*nizs1 development. FGF-8is subsequently expressed on the midbrain side of
thborder and Wnt on the hindbrain side.
Thehindbrainis segmented
into rhombomeres
by boundaries
restriction
F19.4.28 The nervoussystemin a 3-day
chick embryo.Thehindbrainisdividedinto
(r1to r8).Thepositions
eightrhombomeres
of
the cranialnerveslll to Xllareshownin green.
bl to b4arethe fourbranchial
arches.
b1 gives
riseto thejaws.s = somite.
Adopted
A.: 1991
.
fromLumsden,
of the posterior region of the head and the hindbrain involves segmenofthe neural tube along the antero-posterior axis. This type ofpatterning
not occur along the spinal cord, where the patteur ofdorsal root ganglia and
motor neryes at regular intervals-one pair per somite-is imposed by the
In the chick embryo, three segmentedsystemscan be seen in the postehead region by 3 days of development: the mesoderm on either side of the
is subdivided into somites, the hindbrain (the rhombencephalon) is
into eight rhombomeres,and the lateral mesodermhas formed a seriesof
archesthat are populated by neural crest cells (seeFig. 4.28).
lDwelopment of the hindbrain region of the head involves several interacting
ts. The neural tube produces the segmentally arranged cranial neryes
innervate the face and neck and neural crest cells, which in turn give rise both
Xnripheral nerves and most of the facial skeleton. In addition, the otic vesicle
rise to the ear. The main skeletal elements of the head in this region develop
the first three branchial arches, into which neural crest cells migrate. For
, the first arch gives rise to the jaws, while the second arch develops into
bony parts ofthe ear. This region ofthe head is a particularly valuable model
studying patterning along the anterlposterior axis becauseofthe presence of
nrrmerous different stnrchrres ordered along it.
hmediately after the neural tube of the chick embryo closesin this region, the
hindbrain becomesconstricted at evenly spacedpositions to define the eight
(seeFig. 4.28).The cellular basisfor these constrictions is not underbut may involve differential cell division or changes in cell shape.Whatever
underlying cause, it seems that the boundaries between rhombomeres are
iers of cell-lineage restriction; that is, once the boundaries form, cells and their
176
.
4 : P A T T E R N T NTG
HE vERT EBRATBo
E Dy p tAN r : T HE soMrrE S A N D E A R LvN E R V oussysrE M
Fig.4.29 Lineagerestriction in
rhombomeresof the embryonicchick
hindbrain.Singlecellsareinjectedwith a label
(rhodamine-labeled
dextran)at anearlystage
(leftpanel)or a laterstage(rightpanel)of
neurulation,
andtheirdescendants
aremapped
2 dayslater.Cellsinjectedbeforerhombomere
boundaries
form giveriseto someclonesthat
(darkred)aswellas
spantwo rhombomeres
thosethatdo not crossboundaries
(red).
Clonesmarkedafterrhombomereformation
nevercrossthe boundaryof the rhombomere
thattheyoriginatein (blue).
Adopted
A.: 1991.
from Lumsden,
F19.4.30Signalingby ephrinsandtheir
receptors,Ephrinsbindingto their receptors
(Eph)cangenerate
a bidirectional
signal,in
whichthe ephrinitselfalsogenerates
a signalin
the cellthat carriesit.
descendantsare confined within a rhombomere and do not cross from one sidc ltr
a boundary to the other. Marking of individual cells shows that before the consrrb
tions become visible, the descendants of a given labeled cell can populate tm
adjacent rhombomeres. ffier the constrictions appear, however, descendann d
cells then within a rhombomere never cross the boundaries and are thu5 66nfinsfl
to a single rhombomere (Fig.4.z9l.It seemsthat the cells of a rhombomere shm
some adhesive property that prevents them mixing with those of adjacent rhm:
bomeres.This property involves ephrins, membrane-boundproteins that interd
with Eph receptors on adjacent cells and which can generate bidirectional siglrh
(Fig. 4.30). Eph receptors and ephrins are separately expressedin altern*iry
rhombomeres, thus preventing cell mixing at the boundaries. This implies th*
cells in each rhombomere may be under the control of the same genes, and ttru
the rhombomere is a developmental unit. A rhombomere is thus behaving lih
a compartment, which is an important feature of insect development, as we sawin
Chapter 2, but seemsto be rare in vertebrates.
The idea that each rhombomere is a developmental unit is supported by tfu
observation that when an odd-numbered and an even-numbslsd lrrsulborm
from different positions along the antero-posterior axis are placed next to er.Nft
other after a boundary between them has been surgically removed, a new bord
ary forms. No boundaries form when different odd-numbered rhombomercr
are placed next to each other, however, suggesting that their cells have sirnilu.
surfaceproperties.Rhombomeres13 and 15 expressEph4,wtrict,is activatedby '
transcription factor Krox-20, which is expressedin two stripes in the neural plr
that become13 and 15.
The division of the hindbrain into rhombomeres has functional significauc
in that each rhombomere has a unique identity and this determines how it rryim
develop. As we will see later, their development is under the control of Hox gnp
expression. we will first consider the behavior of the neural crest cells rhrr.
originate from the neural tube in the hindbrain and initially migrate over fu
rhombomeres. They populate the branchial arches, subsequently giving dse tu
structures such as the lower jaw
e!$
TH E R OIE OF TH E OR GA N IZE R
A N D N E U R A LIN D U C TION
177
4.11 Neuralcrest cellsarisefrom the borders of the neural plate
Neural crest cells are induced at the borders of the neural plate, which corne
together to form the dorsal portion ofthe neural tube. From there, they migrate to
give rise to a wide variety ofcell types, as describedin Chapter 8. The induction of
tle neural crest is a multistep process that starts in the early gastrula stage and
continues until neural-tube closure. A current model for neural-crest induction is
ttrat crest cells form in two bands in the ectoderm along each lateral border ofthe
neural plate, where the level of BMP signaling is just above the level that would
block neural-plate formation. In addition, Wnt, FGF,and retinoic acid signals appear
to be involved. Induction leads to activation ofthe transcription factors Sox9 and
Sox10,which in turn activate the gene snail, an early marker of neural crest.
The cranial neural crest that migrates from the rhombomeres of the dorsal
region of the hindbrain has a segmental arrangement, correlating closely with the
rhombomere from which the crest cells come. This has been revealedby labeling
chick neural crest cells in vivo and following their migration pathways. Crest cells
frrom rhombomeres 2, 4, and 6 populate the first, second, and third branchial
mches,respectively(Fig.4.31).
The crest cells have already acquired a positional value before they begin to
nigrate. When crest cells of rhombomere 4 are replaced by cells from rhombomere 2 taken from another embryo, these cells enter the secondbranchial arch
tut develop into structures characteristic of the first arch, to which they would
normally have migrated. This can result in the development of an additional lower
tsw in the chick embryo. However, neural crest cells have some developmental
Filesticity and their ultimate differentiation depends on signals from the tissues
imo which they migrate.
Paralogs
Hoxa
Hoxb
Hoxd
Rffi
Fig. 4.31 Expressionof Hoxgenesin the
branchialregion of the head.Theexpression
of genesofthreeparalogous
Hoxcomplexes
in
(rhombomeres
11to r8),neural
the hindbrain
crest,branchial
arches(b1to b4),andsurface
ectodermisshown.Hoxol andHoxdTarenot
expressed
at this stage.Thearrowsindicatethe
migrationof neuralcrestcellsintothe branchial
arches.
Notethe absence
of neuralcrest
migrationfrom 13and15.
AfterKrumlouf,
R.:1993.
178
.
4 : p A T T E R N T NG
T HE vERT EBRATBo
E Dy p L AN u : T HE soMl rE S A N D E A R LyN E R V oussysrE M
4.I2 Hox genesprovide positional information in the developinghindbrain
Fig.4.32 Geneexpressionin the hindbrain,
Thephotograph
showsa coronalsection
throughthe hindbrainof a 9.5-daypost-coitum
mouseembryo,whichistransgenic
for two
reporterconstructs.Thefirst constructcontains
the /ocZgeneunderthe controlof an enhancer
from Hoxb2,whichdirectsexDression
in
rhombomeres
3 and5 (revealed
asblue
staining).
Thesecondconstructcontains
an
phosphatase
geneunderthecontrolof
alkaline
anenhancer
fromHoxb7,
whichdirects
expression
in rhombomere4 (revealed
as
greenish-brown
staining).
A similarenhancer
directingexpression
in rhombomere
4 existsfor
Hoxb2.Anterioris uppermost,andthe positions
of fiveof the rhombomeres
areindicated(r2 to
16).Scalebar= 0.1mm.
Photogroph
courtesy
ofl. Shorpe,
A.
from Lumsden,
ondKrumlouf,
R.:1996.
Hox gene expressionprovides a possiblemolecular basisfor the identities of bd
the rhombomeres and the neural crest at different positions in the hindbmin;
No Hox genesare expressedin the most anterior part of the head but Hox gers
are expressed in the mouse embryo hindbrain in a well defined pattern, whi&
closely correlates with the segmental pattern (seeFig. 4.31). It is clear that fre
three paralogous groups involved have different anterior margins of expressi@,
paralog 7 (i.e.Hoxb7,Hoxc1,etc.)being most anterior, followed by paralogs2 and 3For example, Hoxb3 has its most anterior region of expression at the border d
rhombomeres 4 and 5, while Hoxb2 has its anterior border at the border d
rhombomeres 2 and 3 (Fig.4.32).In general, the paralogousgenesof the diffeftd
Hox complexeshave similar patterns of expression.
The pattern ofHox gene expressionin the ectoderm and branchial archesat e
particular position along the antero-posterior axis is similar to that in the neurafl
tube and neural crest, and it may be that the crest cells induce their positionail
values in the overlying ectoderm during their migration.
Tlansplantation of rhombomeres from an anterior to a more posterior positim
alters the pattern of Hox gene expression so that it becomesthe same as tla(
normally expressedat the new location. The signals responsible for this reprograrlming originate from the neural tube itself and not the surrounding tiszuer
Studies ofthe control ofHox gene expression at the molecular level have providerl
some indication as to how their pattern of expression is controlled. For examph,
although t};leHoxb2gene is expressedin the three contiguous rhombomeres 3, 4
and 5, its expression in rhombomeres 3 and 5 is controlled quite independently
from its expression in rhombomere 4. The regulatory regions of the Hoxb2ger
carry two separatecis-regulatoryregions that regulate its expression in these three
rhombomeres. Expressionin rhombomeres 3 and 5 is controlled through one d
these regions, while expression in rhombomere 4 is controlled through the other
(seeFig. 4.321.ln rhombomeres 3 and 5, Hoxb2is activatedin part by the transcrip
tion factor IGox-2O,which is expressedin these rhombomeres but not in rho*
bomere 4. There are binding sites for IGox-20 in the regulatory DNA element tbat
activates expression of Hoxh2in rhombomeres 3 and 5. How the spatially organized
expression of transcription factors such as Krox-20 is achieved is not yet knownIn the caseof Hoxb4,whose anterior boundaly of expressionis at rhombomerc G
there is evidence that it is induced and localized by signaling both within tbe
neural tube and from adjacent somites. There is also a role for retinoic acid in
patterning the hindbrain, as its absence leads to loss of hindbrain rhombomeres"
whereas an excesscausesa transformation of cell fate from anterior to posteriorAn experiment showing that Hox genes determine cell behavior in the rhon
bomeres comes from the misexpression of HoxbTin an anterior rhombomereMotor axons from a pair of rhombomeres project to a single branchial arch,
rhombomere 12 projects to the first branchial arch while 14 projects to the second
arc}r. HoxbT is expressed in 14 but not in r2. lf Hoxbl is misexpressed in an 12
rhombomere, this then sendsaxons to the secondarch.
Gene knock-outs in mice have also shown that the Hox genes are involved
in patterning the hindbrain region, though the results are not always easy to
interpret; knock-out of a particular Hox gene can affect different populations of
neural crest cells in the same animal, such as those that will form neurons and
those that will form skeletal structures. Knock-out of tlneHoxa2gene, for example,
results in skeletal defectsin that region ofthe head correspondingto the normal
domain of expression of the gene, which extends from rhombomere 3 backr,vards.
TH E R OTEOF TH E OR GA N IZE R
A N D N E U R A LIN D U C TION
Segmentation itself is not affected, but the skeletal elements in the second
branchial arch, all of which come from neural crest cells derived from rhombomere 4, are abnormal. The usual elements, such as the stapes of the inner ear,
ere absent, but instead some of the skeletal elements normally formed by the first
branchial arch develop, such as Meckel's cartilage, which is a precursor element in
the lower jaw. Thus, suppression of Hoxa2causesa partial homeotic transformation
cf one segment into another. The converseeffect is seenwhen Hoxa2is misexpressed
in a more anterior position (seealso Section 4.41.lf Hoxa2is misexpressedin all
tissues of the first branchial arch, in which no Hox genes are normally expressed,
ttere is a homeotic transformation into secondbranchial arch. ln addition, if Hox2a
fo transfected into prospective anterior neural crest, the cells do not develop in
their normal way and the skeletal structures they give rise to are abolished.
Theseobservations,together with those describedearlier in this chapter, show
tftat during gastrulation the cells of vertebrates acquire positional values along the
ryrrFnoFposterioraxis, and that this positional identity is encoded by the genes of
&e Hox complexes. Many of the anatomical differences between vertebrates are
grobably simply due to differences in the subsequent targets of Hox gene actions,
mhich result in the emergence of different but homologous skeletal structures&e mammalian jaw or the bird's beak, for example.
r"rs The embryo is patterned by the neurula stage into organ-forming regions
l*can still regulate
dt the neumla stage, the body plan has been established and the regions of
rffip embryo that will form limbs, eyes, heart, and other organs have become
&rmined
(Fig.4.33).This contrasts sharplywith the blastula stage,at which time
m mch determination has occurred. The basic vertebrate phylotypic body plan is
esablished during gastnrlation. But although the positions of various organs
''lfrhnc
,m fixed, there is no overt sign yet of differentiation. Numerous grafting experinnms have shown that the potential to form a given organ is now confined to
rynr;fic regions. Each of these regions has, however, considerable capacity for
nrr$nhtisn, so that if part of the region is removed a normal structure can still
[im- For example, the region of the neurula that will form a forelimb will, when
iMplanted to a different region, still develop into a limb. If part of a future limb
nGi.n is removed, the remaining part can still regrrlate to develop a normal limb.
'llhdrvelopment of limbs and other organsis discussedin Chapter 8.
alongboth the antero-posterior
and dorso-ventralaxesis closelyrelatedto
duringgastrulation.
of the Spemann
organizer
anditsmorphogenesis
When
to the ventralsideof an earlygastrula,the Spemannorganizerinducesboth
dorso-ventralaxisand a new antiro-posterioi axis,with the developmentof
twinned embryo.In the zebrafishthe embryonicshieldis the organizer,
h chickdwelopment,Hensen'snode servesa functionsimilarto that of the
organizeland it too can specifya new antero-posterioraxis.In the mouse,
can specifya new axisapartfrom the most anteriorforebrain,for which
by the anteriorvisceral
endodermisalsonecessary.
wtebrate nervoussystem,which forms from the neural plate, is induced
ty earlysignalswithin the ectodermand by signalsfrom the mesodermthat
= b lie beneath prospectiveneural plate ectoderm during gastrulation.
179
Post€dor
Fig. 4.33 TheXenopus
embryo hasbecome
regionalizedby the neurulastage,Various
organssuchaslimbs,heart,andeyeswill
develop
fromspecific
regions(red)ofthe
neurulaaftergastrulationiscomplete.
Someof theseregions,
likethe limbbuds,
arealready
determined
at thisstageand
will not form anyotherstructure.The
boundaries
of the regionsarenot sharply
defined,however,andwithineachregionor
'field'considerable
regulation
isstillpossible.
r80
4 : P A T T E RNINC THE VERTEBRATE BODY PLAN II: T H E
SOM IT ES AN D EAR LY N ER VO U S SYST EM
Forsomeproteinsthat caninduceneuraltissueinXenopus,
suchasNoggin,thisabilir,
is due to theirinhibitionof BMpsignaling.
patterning
of the neuralplatecanpan:.
be accountedfor by a two-signalmodel:the ectodermis first specified
as anterioneuraltissueandthen a secondsetof signals,possiblygraded,specifies
moreDoste_
riorstructures.
The hindbrainis segmented
into rhombomeres,
with the cellsof eachrhombomer:
respecting
theirboundaries.
Neuralcrestcellsfromthe hindbrain
populate
specific
mesc*
dermalregionssuchasthe branchial
archesin a position-dependent
fashion.
A Hoxgenc
codeprovides
positional
values
fortherhombomeres
andneuralcrestcellsofthehindbrairegion,whileothergenesspecifymoreanteriorregions.Bythe neurula
stage,aftergasr.lation,the bodyplanhasbeenestablished.
gastrulationand
activity
norganizer
v
J}
the Hoxgenecomplexesareexprexed alongtne antero_posterior
axis
Hoxgeneexpression
establishes
positionalide'ntityfor mesoderm,endoderm,andectode-
I
mesodermdevelopsinto notochord,
somites,and lateralplatemesoderm
Jt
somitesreceivesignalsfrom notochord,
neuraltube,andectoderm
V
somite
develops
intosclerorome
and dermomyotome
\ <.\
earl ysi gnal sand mesoderm i nduc e
neural plate from ectoderm
n
JL
V
mesodermsignalsgiveregionai
identityto neuraltuoe
-n
v
rhombomeres
andneuralcrestin t-=
hindbrain
arecharacterized
by regic-.
patternsof Hoxgeneexpression
The germ layersspecifiedduring blastulaformationbecomepatterned
alongthe
antero-posterior
anddorso-ventral
axesduringgastrulation.
Theprimaryembryonic
organizeris involvedin the initialpatterningthat underlies
the regionalization
of the
antero-posterior
axis.positionalidentity of cellsalong the antero_posterior
axisls
encodedby the combinatorial
expression
of genesof the four Hoxcomplexes,
which
providea codefor regionalidentity.Thereis both spatial
and temporalco-linearir,
betweenthe orderof Hoxgeneson the chromosomes
andthe orderin whichtheyare
expressed
alongthe antero-posterior
axisof the embryofrom the hindbrainback_
wards.Inactivation
or overexpression
of Hoxgenescanleadbothto localized
abnor_
malitiesandto homeotictransformations
of one ,segment'of the axisinto anotnei-_
indicatingthat thesegenesare crucialin specifyingregional
identity.At the end or,
gastrulation,the basicbody plan has been laid down
and the nervoussysterr
induced.specificregionsof eachsomitegive riseto cartilage,
muscle,and dermis.
and these regionsare specifiedby signalsfrom the notochord,
neurartube, anc
epidermis.Inductionand patterningof the nervoussysteminvolves
both signalsir
the earlyembryoand from the underrying
mesoderm.
In the hindbrain,
Hoxgene
expression
providespositionalvaluesfor both neuraltissueandneural
crestcells.
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