Molecular signaling along the anterior-posterior axis of early palate development.

0.82 MB·English

by  Smith, Tara M.

REVIEWARTICLE
published:07January2013
doi:10.3389/fphys.2012.00488
Molecular signaling along the anterior–posterior axis
of early palate development
TaraM.Smith1,ScottLozanoff2,PaulP.Iyyanar1 andAdilJ.Nazarali1*
1LaboratoryofMolecularCellBiology,CollegeofPharmacyandNutrition,UniversityofSaskatchewan,Saskatoon,SK,Canada
2DepartmentofAnatomy,BiochemistryandPhysiology,UniversityofHawaiiSchoolofMedicine,Honolulu,HI,USA
Editedby: Cleft palate is a common congenital birth defect in humans. In mammals, the palatal
DanielGraf,UniversityofZurich, tissue can be distinguished into anterior bony hard palate and posterior muscular soft
Switzerland palate that have specialized functions in occlusion, speech or swallowing. Regulation of
Reviewedby: palate development appears to be the result of distinct signaling and genetic networks
DanielGraf,UniversityofZurich,
in the anterior and posterior regions of the palate. Development and maintenance of
Switzerland
ClaireA.Canning,A*STARAgency expression of these region-specific genes is crucial for normal palate development.
forScienceTechnologyand Numerous transcription factors and signaling pathways are now recognized as either
Research,Singapore anterior- (e.g., Msx1, Bmp4, Bmp2, Shh, Spry2, Fgf10, Fgf7, and Shox2) or
CarolinaParada,Universityof
posterior-specific (e.g., Meox2, Tbx22, and Barx1). Localized expression and function
SouthernCalifornia,USA
clearlyhighlighttheimportanceofregionalpatterninganddifferentiationwithinthepalate
*Correspondence:
AdilJ.Nazarali,Laboratoryof at the molecular level. Here, we review how these molecular pathways and networks
MolecularCellBiology,Collegeof regulate the anterior–posterior patterning and development of secondary palate. We
PharmacyandNutrition,University hypothesize that the anterior palate acts as a signalingcenter in setting up development
ofSaskatchewan,116Thorvaldson
ofthesecondarypalate.
Building,110SciencePlace,
Saskatoon,SKS7N5C9,Canada.
e-mail:[email protected] Keywords:anterior–posterioraxis,secondarypalate,development,signaling,migration,growthfactors
INTRODUCTION positionstarting attheanteriorendandproceedingposteriorly,
Cleftpalateisoneofthemostcommoncongenitalbirthdefectsin however, a clear understanding of how elevation occurs has yet
humans,occurringwithafrequencyof1:700to1:1000livebirths tobeachieved.Ultimately,theelevatedpalatalshelvesthengrow
(Gorlinetal.,2001).Acleftsecondarypalatecanoccurasaniso- towardoneanotheruntilthemedialedgeepitheliumfromeach
lated birth defect (non-syndromic), in conjunction with a cleft shelfcontactstoformthemidlineepithelialseam(MES)atE14.5.
lip,orasapartofanothersyndrome.Bothgeneticandenviron- Inadditiontogrowthofthepalateshelves,achangeintherelative
mentalfactorsplayrolesinthedevelopmentofcleftpalate(Dixon dimensionsofthehead(verticaldimensionsoftheheadincrease
etal.,2011). while the lateral maxillary width remains constant) allows the
During mammalian embryogenesis, the development of the palate shelves to contact one another at the midline (Diewert,
secondarypalateisregulatedbyanumberofcomplexnetworks 1978,1983).Epithelialcellsfromopposingpalateshelvesadhere
of growth factors and transcription factors. These molecular to one another through glycoproteins on their surface (Greene
networks and pathways work together to tightly regulate criti- and Kochhar, 1974; Pratt and Hassell, 1975; Souchon, 1975;
cal cellular processes in the palate including cell proliferation, Greene and Pratt, 1977) as well as through desmosomes (De
apoptosis,migration,andepithelial-mesenchymal transdifferen- AngelisandNalbandian,1968;MorganandPratt,1977).Contact
tiation.Thesecondarypalateoriginatesfromfirstbranchialarch andsubsequentfusionbeginsintheanteriormid-palateregions
neural-crestderivedmesenchymal cellscovered byamulti-layer andproceedsinboththeanteriorandposteriordirectionslikea
sheet of cells derived from the facial ectoderm (Noden, 1983). zipper(MorganandPratt,1977;Ferguson,1988).TheMESthen
Inthemouse,bilateralpalateshelvesfirstdevelopasoutgrowths undergoes a rapid degradationto forma secondary palatewith
fromthemaxillaryprocessesatembryonicday11.5(E11.5).The complete mesenchymal confluence (Ferguson, 1988; Berkovitz
shelvesthengrowverticallydowneithersideofthetongueuntil et al., 2009). Numerous mechanisms for the degradation of
E14.0(Ferguson,1988),afterwhichtheshelvesundergoarapid the MES have been proposed, including epithelial apoptosis
elevation to become horizontally oriented toward one another (Pourtois, 1966; Saunders, 1966; Farbman, 1968; Shuler, 1995;
abovethetongue.Growthofthestomodeumaswellasjawjoint Martínez-Álvarezetal.,2000a;Xuetal.,2006),migration(Carette
activity and neuromuscular function make it possible for the andFerguson,1992;Shuleret al.,1992;Martínez-Álvarez et al.,
embryotohavemouth-openingreflexes.Thesemovementsallow 2000a),andepithelial-mesenchymaltransformation(Fitchettand
thetonguetoflattenanddepress,andthedownwardpositioned Hay,1989;GriffithandHay,1992;Shuleretal.,1992;Kaartinen
palate shelves to reorient (Humphrey, 1969; Diewert, 1980). A etal.,1995;Proetzeletal.,1995;Sunetal.,1998;Cuietal.,2005).
number of changes occur within the palate shelves to facilitate Epithelial-mesenchymaltransformationhasbeenruledoutbased
therapidmovementoftheshelvesfromaverticaltoahorizontal on fate-maps (Vaziri Sani et al., 2005), but this theory is still
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Smithetal. A–Paxisinpalatedevelopment
unsettled. Mesenchymal confluencesignals the end ofpalatoge- rostral extension of the anterior palate from E11.5 to E14.5,
nesis at E15.5 (Ferguson, 1988). Finally, the anterior secondary the spatial relationship between R1 and the developing molar
palatefusestotheprimarypalateandthedorsalportionsofthe tooth bud remains unchanged (Welsh et al., 2007; Welsh and
secondarypalatefusewiththenasalseptummarkingthecomple- O’Brien,2009),makingthemolartoothbudanidealconvention
tionofproperpalataldevelopment.Distinct pathways/networks todelineatethetwostructurallydistinctregionsofpalate.
regulatedevelopmentateachstageofpalatogenesis,withdefects This review will provide an in depth look at the molecular
at any stage capable of resulting in cleft palate. In addition to processes involved in regulating the patterning and early devel-
problems with development of the palate proper, defects in the opmentofthesecondarypalate.Genesknowntobeinvolvedin
developmentofothercraniofacialelementsincludingthetongue thefusionofthepalateprocesseswillnotbediscussedindetail;
andmandiblecanresultinacleftpalate(Ferguson,1987). seeNawshad(2008)foracomprehensivereview.Themajorfocus
Analysisoftheliteratureonregionallyexpressedgenescanbe here will be to summarizeboth current information and devel-
difficultsinceastandardizedmethodofdeterminingtheanterior, oping new connections between the factors and genes involved
medial,andposteriorregionsofthepalateisnotinplace.Many inspecifyingandmaintainingtheA–Paxis.Wehypothesizethat
authorsfail to indicate how they define the region ofthe palate theanteriorpalateactsasasignalingcenterforsecondarypalate
that they are examining making comparison difficult between patterninganddevelopment.
articles. It is important for the field to adopt a standard con-
vention for defining the anterior, medial, and posterior palate ANTERIOR-SPECIFICGENEEXPRESSION
to ensure that these comparisons can be made. In the past, the A large number of anterior-specific genes specifically expressed
anteriorandposteriorhavebeendescribedinanumberofways. andactivewithintheanteriorpalate(Figure2)comparedtothe
We propose that the convention can be followed such that the posteriorpalatehighlightstheimportanceoftheanteriorregion
tissueanteriororposteriorto thefirstmolartooth budbecon- during secondary palate development (Zhang et al., 2002; Rice
sideredtheanteriororposteriorpalate,respectively. Themedial etal.,2004;Alappatetal.,2005;Yuetal.,2005;Levietal.,2006;
palatewouldbeconsideredpalatetissueintheplaneofthemolar Leeetal.,2007,2008;Welshetal.,2007;Heetal.,2008;Liuetal.,
toothbud(Figure1).Therationaleisthatthefirstformedpalatal 2008).
rugae(R1)demarcatestheexpressionboundaryofanterior(e.g.,
Msx1,Shox2,andFgf10)andposterior(e.g.,Meox2,Tbx22)spe- Msx1NETWORK
cific genes (Zhang et al., 2002; Yu et al., 2005; Li and Ding, The first network described to play an anterior-specific role in
2007;Pantalaccietal.,2008;WelshandO’Brien,2009;Bushand the developing palate involves the homeobox transcription fac-
Jiang, 2012). The first molar tooth bud lies immediately ante- torMsx1.MutationsinthehumanMSX1genehavebeenlinked
riorto theR1,whichformstheposteriorboundaryforanterior
Fgf10expression(Welshetal.,2007).Onthestructuralbasis,the
anteriortwo-thirdsofthepalateisthefuturehardpalate.During
FIGURE2|Schematicrepresentationofthekeyregulatorsinthe
anteriorpalate.Msx1andBmp4functioninanautoregulatoryloop
mechanisminthemesenchyme.Bmp4inducesShhexpressioninthe
epitheliumwhichsignalsbackstothemesenchymetopositivelyregulate
Bmp2toenhancecellproliferationinthemesenchyme.Msx1expressionis
controlledbyHoxa2inearlypalataldevelopment.Fgfsandtheirreceptors
FIGURE1|Schematicrepresentationfordefininganterior,medial,and areregulatedbySpry2forproperbalanceofproliferationandpreventionof
posteriorregionsofpalatalshelves.Tissueanteriororposteriortofirst prematureapoptosisintheepithelium.Fgf10inducesShhwhereasFgf7
molartoothisconsideredanteriororposteriorpalate,respectively.The actsasanantagonist.Msx1alsomaintainsproliferationbyinducingBmp7
palataltissueintheregionoffirstmolartoothbudisconsideredmedial. inthemesenchymealongthenasalepithelium.Genesrepresentedinred
Abbreviations:PP,primarypalate;PS,palatalshelves;M1,firstmolar arerestrictedtoeitheroralornasalsideofthepalate,whereasthose
toothbud. representedinbluearepresentacrosstheshelf.
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Smithetal. A–Paxisinpalatedevelopment
to isolated non-syndromic cleft palate (Vastardis et al., 1996; appearstoresultfromincreasedcell proliferationwhereexpres-
Lidral et al., 1998; Van den Boogaard et al., 2000; Suzukiet al., sionlevelsofbothMsx1anditsknowndown-streamtargetBmp4
2004; Tongkobpetch et al., 2006; Otero et al., 2007). Msx1- are up-regulated during the early stages of palate development
deficientmicedisplayneonatallethalityduetoawideopencleft (Smithetal.,2009).Geneticstudiesinhumanshavealsolinked
secondary palate (Satokata and Maas, 1994; Houzelstein et al., mutationsintheHOXA2genewithacleftsecondarypalate(Alasti
1997). Expression of Msx1 is localized exclusively to the ante- etal.,2008).Hoxa2actsupstreamofMsx1inthesecondbranchial
rior mesenchyme during the early stages of palate development archneuralcrestcells(Santagatietal.,2005).Thisnewgenetar-
from E12.5 to E13.5 (Zhang et al., 2002; Alappat et al., 2003) get providesadditional insight, asHoxa2is knownto beabsent
and functions through regulating the expression of Bmp4, Shh, from the migrating first branchial arch from which the palate
and Bmp2 at E12.5–E13.5 in the anterior palate (Zhang et al., shelves arise(Prince andLumsden, 1994). Clearly expression in
2002). Msx1 appears to regulate Bmp4 expression in the ante- the branchial arches prior to overt palate growth is not a pre-
rior mesenchyme, which subsequentlysignals to the epithelium requisiteofgenesthatareimportantinregulatingpalatogenesis.
andregulatesShhexpression;fromtheepitheliumShhthensig- WhetherHoxa2andFgfsrepresentdistinctregulatorynetworkof
nals back to the mesenchyme and regulates Bmp2 expression Msx1 or arepart of the same regulatory network remains to be
(Zhangetal.,2002).Inadditiontothislinearnetwork,Bmp4is determined.StrictregulationofMsx1expressioninthepalateis
involved in a reciprocal regulatory cycle controlling the expres- probablyduetoitsimportanceinregulatingproliferationinthe
sion of Msx1. The main function of Msx1 and its subsequent anteriorpalate.
networkappearstoberegulationofcellproliferationwithinthe The transcriptional activity of Msx1 can also be altered by
anterior mesenchyme (Zhang et al., 2002). It has been demon- other proteins in the palate. Msx1 undergoes post-translation
strated that although exogenous BMP is capable of inducing modification by sumoylation in vivo in a region of the protein
Msx1expressionandincreasingcellproliferationintheanterior that is responsible for regulating Msx1 interactions with other
palateithasnoeffectontheposteriorpalate(Zhangetal.,2002; proteins(GuptaandBei,2006).Thus,sumoylationofMsx1may
Hilliardetal.,2005).Sincefirstreported,numerousstudieshave help facilitateits abilityto interact withother transcriptionfac-
investigatedtheexpressionandfunctionofthegenesinthisnet- tors and therefore control its ability to regulate the expression
work.Allstudiesperformedtodate(seebelow)confirmthatthis ofothergenes.HaploinsufficiencyoftheSUMO1genehasbeen
networkisimportantinanteriormesenchymeproliferation;how- reported to lead to cleft palate through altering the sumoyla-
ever, the regulation of each of these genes is far more complex tion status of various proteins (Eya1, Pax9, and Msx1) in the
thansuggestedoriginally. palate (Alkuraya et al., 2006). However, it has also been sug-
The regulation of Msx1 expression has been linked to many gestedthatSUMO1expressionisnotnecessaryfornormalmouse
otherfactorsinthepalate.Msx1wasshowntobedownstreamof development(Zhangetal.,2008).Debatesalsoexistonwhether
theFoxe1genewithFoxe1nullmicehavingverylowexpressionof polymorphismsoftheSUMO1geneinhumansarelinkedtocleft
palatalMsx1andTgf-β3atE14(Venzaetal.,2011).LossoftheFgf palate(Songetal.,2008;AlmeidadeAssisetal.,2011).Whatrole
antagonistSpry2inthepiebalddeletionanimalmodel(discussed SUMO1playsinpalatedevelopmentistherefore unclearatthis
in more details in the section “Fgf Signaling Pathway”) results time.
inincreasedMsx1expressionaswellasaposteriorexpansionof InadditiontoregulatingBmp4andBmp2,Msx1regulatesthe
its expression border at E13.5 and E14.5; this increased expres- expression of Bmp7 and its antagonist Follistatin (Levi et al.,
sionleadstoanincreaseinproliferationwithinthepalate(Welsh 2006). Loss of Msx1 leads to a decrease in the anterior palatal
et al., 2007). Msx1 is Fgf-responsive in other regions of cranio- expressionofBmp7 butanincreaseinitsexpressioninthepos-
facial development (Bei and Maas, 1998; Alappat et al., 2003), terior palate (Levi et al., 2006). The Bmp antagonist Follistatin
although Fgf10 null palates do not exhibit altered Msx1 expres- is expressed throughout the palatal epithelium; in the anterior
sion (Alappat et al., 2005). These data suggest other Fgfs may palateitisprimarilyexpressedinarestricteddorsaldomainthat
be acting in the palate to regulate Msx1 expression (other pos- doesnotoverlaptheregionsofBmp4andBmp2expression(Levi
sible explanations are discussed in subsequent sections below). etal.,2006).Msx1nullmicealsoexhibitadecreaseinthelevelof
Fgf9mayplayanactiveroleinpalatedevelopment(Colvinetal., anteriorpalatalFollistatinexpression(Levietal.,2006).Together,
1999,2001)andlossofSpry2mayrelievetheantagonismofFgf9 these data highlight the important role of Msx1 in the regula-
resultingintheobservedupregulatedandexpandedMsx1expres- tion of the Bmp family and their antagonists in the palate, and
sion(Welshetal.,2007).Recently,Fgf9wasshowntoregulatecell provideanothermechanismbywhichitmayregulatethelevelof
proliferationinpalatalmesenchyme viaPitx2-dependentinduc- proliferationintheanteriorpalate.
tionofcyclinD1andcyclinD3intheTgfbr2fl/fl;Wnt1-Cremice Dlx5 is expressed in the anterior mesenchyme of the palate
(Iwataetal.,2012a),however,expressionofMsx1wasnotexam- andmutationsintheDlx5generesultinacleftsecondarypalate
inedinthisstudy.Fgf7isexpressedwithinthepalatemesenchyme (Levi et al., 2006; Han et al., 2009). Furthermore, loss of the
(Rice et al., 2004) and may also be involved in regulating Msx1 transcription factor MEF2C consequently leads to loss of Dlx5
expression and affected by loss of Spry2, although this has not expressioninthebranchialarchesresultinginacleftpalate(Verzi
beeninvestigated. et al., 2007). Although Dlx5 and Msx1 share similar expres-
Hoxa2, another homeobox gene, has recently been shown to sion domains it is unlikely that they are involved in regulating
regulatepalatalMsx1expression(Smithetal.,2009).Hoxa2null eachothersexpressionasMsx1expressionisnotalteredinDlx5
miceexhibitan81%penetranceofcleftpalate(Gendron-Maguire nullpalatesandviceversa(Hanetal.,2009).Dlx5/Msx1 double
etal.,1993;Rijlietal.,1993;BarrowandCapecchi,1999),which knockoutsshowarescueoftheMsx1nullcleftpalatephenotype
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Smithetal. A–Paxisinpalatedevelopment
(Levi et al., 2006; Han et al., 2009). Loss of Dlx5 in Msx1 null theanteriorpalateandposteriorpalatecorrespondinglydecreases
embryosalterstheexpressionofShh,Bmp7,andFollistatininthe and increases, respectively. Surprisingly, Bmp2 expression was
palate. Bmp7 expression in these doubleknockouts is increased unaltered in the Wnt5a null mutants (He et al., 2008), imply-
throughoutthepalate,whileexpressionofFollistatinisdecreased ing Bmp2 expression in the palate is regulated by an additional
(Levi et al.,2006; Hanet al.,2009). Shh expression isdecreased mechanism.DespiteadecreaseinBmp4andShhexpression,pro-
in Msx1 null palates but its domain is expanded in the double liferation was increased in the anterior mesenchyme, which is
knockouts suggesting that both Msx1 and Dlx5 are involved in contrarytowhatwouldnormallybeexpected(Heetal.,2008).
determining the areaofShh expression (Hanet al., 2009). Dlx5 Noggin is a polypeptide that binds to members of the Bmp
andFgf7 sharethesameexpressionregionintheanteriorpalate family preventing them from signaling. Noggin null mice show
mesenchyme onthenasalside.Fgf7 regionofexpressionislim- thatwithoutNoggin’srepressionofBmpsignaling,palatedevel-
ited in Dlx5 null mutants as well as in the Msx1/Dlx5 double opment does not proceed normally, with fusion between the
knockouts (Han et al., 2009). These data point toward a sys- palate and mandible ultimately leading to a cleft palate pheno-
tem where Dlx5 regulates the expression of Fgf7, which in turn type. Although Noggin null mice did not have changes in the
repressesShh.Ithasalsobeendemonstratedthatafeedbackloop expression of Msx1, Bmp4, orShh they did havereduced Shox2
andcrosstalkexistsbetweenBmp7andShh,whichplaysarolein andBmp2expressionintheanteriorpalateandanectopicexten-
refiningtheexpressiondomainofbothgenes(Hanetal.,2009). sionofBmp2expressionintotheposteriorregionofthepalate.In
Therefore,intheMsx1/Dlx5doubleknockoutsthelimitedBmp7 addition,decreasedproliferationrateswereseenexclusivelyinthe
expressionallowsanincreaseinShhexpression,whichlikelyleads anterior mesenchyme ofNoggin null palatewhich suggests that
to the observed increase in cell proliferation and rescues the lossofBmp2intheanteriorpalateeffectsproliferationandsup-
Msx1-inducedcleftpalate. ports the theory that posterior cells are not receptive to ectopic
Bmpexpression(Hilliardetal.,2005;Heetal.,2010).
BONEMORPHOGENICPROTEINSIGNALINGPATHWAYS The Bmp family plays an important role in maintaining the
Bmp4isknowntobedownstreamofMsx1inthepalate(Zhang A–P axisof the palateshelves aswell asregulating proliferation
etal.,2002).However,similartoMsx1,manyalternativeregula- (Nie, 2005). Bmp ligands regulate down-stream gene expres-
torypathwaysforBmp4havebeendescribedinrecentyears.The sion and cell processes through activation of cellular receptors.
transcription factor Tbx3 shows an overlapping expression pat- Bmpr1a and Bmpr1bareexpressed in anoverlappingpatternin
tern with Bmp4 in the developing anterior palate mesenchyme theanteriorpalate.TheBmpreceptorBmpr1aisessentialinthe
(Leeetal.,2007).Thesetwogenesregulateeachother’sexpression regulation of proliferation and patterning in the palate; a total
inthepalatewherebyTbx3inhibitstheexpressionofBmp4while lossoftheBmpr1ageneinallcraniofacialcellsleadstodecreased
Bmp4 induces Tbx3 expression (Lee et al., 2007). As expected proliferation as well as an anterior shift in the expression pat-
andbasedonthepreviouslyreportedroleofBmp4inthepalate, terns of the posterior-specific genes Pax9 and Barx1 (Liu et al.,
this regulatory loopacts byregulating the levelsofcell prolifer- 2005).ConditionallossofBmpr1aintheneuralcrestandderiva-
ation in the anterior palatal mesenchyme (Lee et al., 2007). In tives(Wnt1-Cre;Bmpr1af/−mice)leadstoananteriorcleftingof
thelimb,Tbx3expressionisdependentonBmp4(Tümpeletal., thesecondarypalateresultingfromdecreasedmesenchymalpro-
2002)andplaysanimportantroleinmaintainingnormalprolif- liferation(Lietal.,2011).Thesignificantlyreducedexpressionof
erationintheregion(Davenportetal.,2003).Tbx3nullembryos Msx1,Bmp4,Pax9,andShox2mayberesponsibleforthedefective
however, do not exhibit a cleft palate (Davenport et al., 2003) cellproliferation.TheseresultsindicatethatalthoughBmpr1bhas
and therefore the ability of Tbx3 to regulate Bmp4 expression acommonexpressionpatternitisnotabletocompensateforthe
and subsequently proliferation may be redundant with another lossofepithelialBmpr1aexpression(Lietal.,2011).Interestingly
regulatorymechanisminthepalate. when Bmpr1a is deleted from all craniofacial tissue the expres-
At the onset of palate development, the transcription factor sion of Msx1 is unaltered (Liu et al.,2005). Together these data
Tp63 regulates the expression of Bmp4 in the anterior palate. showthattheroleandnumberofBmpreceptorsinthepalateis
Loss of the Tp63 gene leads to cleft palate through altering the complexandyettobefullyunderstood.Thiscouldalsoimplya
expression of a variety of genes (including Bmp4) in the max- novel rolefor Bmp4and potentially other Bmps acting through
illary processes from which the palatal shelves emanate. This the Bmpr1a receptor in regulation of the spatial expression of
alteredgeneexpressionresultsindefectsoftheA–Paxisaswellas posterior-specificgenes.
theonsetofpalatedevelopment(Thomasonetal.,2008).These
observations indicate regulation of gene expression during and SONICHEDGEHOGSIGNALING
priortotheovertgrowthofthepalateshelvescaninfluencepalate Sonic hedgehog (Shh) is expressed in the epithelium through-
developmentandpatterning. out palatogenesis (Paiva et al., 2010) and proper regulation of
Bmp4actsupstreamofShhandBmp2withinthepalate(Zhang theShhsignaliscrucialfornormalpalatedevelopmenttooccur.
etal.,2002).NewstudiesdetailtheimportanceoftheWnt5asig- Expression is restricted to a striped pattern that corresponds to
nalingmoleculeinregulatingtheA–Paxisinthepalateincluding the rugae (Rice et al., 2006). Rugae develop through the thick-
theexpressionofBmp4(Heetal.,2008).IntheabsenceofWnt5a ening of the epithelium and condensation of the underlying
signaling, Bmp4 expression is down-regulated in the anterior mesenchyme. These rugae are suggested to act as centers that
palateatE13.5,whilebeingectopicallyup-regulated inthepos- coordinate patterning within the palate implying an important
terior palate (He et al., 2008). As predicted, Shh expression in roleforShhinthepatterningofthedevelopingpalate(Riceetal.,
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Smithetal. A–Paxisinpalatedevelopment
2004; Lin et al., 2011). The epithelial cells expressing Shh are at the MES (Rice et al., 2004; Alappat et al., 2005). In addi-
notactivelyproliferating,whereasthemesenchymalcellsunderly- tion, ectopic fusion of the palate shelves to the oral epithelium
ingtheseregionsaremorehighlyproliferativethanmesenchymal is observed in some animals, preventing normal shelf eleva-
cellsinotherareasofthepalate(Hanetal.,2009).Lossofrugae tion (Alappat et al., 2005). Similar to Msx1 expression, Fgf10
andrugae-specificmorphogenshasbeensuggestedtohamperthe isexpressed primarilyin the anteriorpalatemesenchyme atthe
molecularguidancenecessarytoregulatethegrowthofthepalate. early stages (E12–E13) of palatogenesis (Rice et al., 2004). The
Forexample,lossofWntsignalinginthepalateepitheliumblocks Fgf10ligandactsthroughthereceptorFgfr2b,whichalsoshows
the formation of the rugae and altered Shh expression which an anterior-specific expression pattern in areas of epithelium
in turn results in abnormalextension along the A–P axis and a adjacenttomesenchymeexpressingFgf10(Riceetal.,2004).Shh
uniqueanterioronlycleftpalatephenotype(Linetal.,2011).Shh expression is down-regulated in the epithelium of both Fgf10
isalsoadown-streamtargetoftheMsx1network thatregulates and Fgfr2b null embryos, leading directly to a severe reduction
cellproliferationintheanteriorpalate(Zhangetal.,2002).Loss inepithelial cell proliferationanda consequentlythin epithelial
of the Spry2 gene also leads to a disorganization in the expres- layer.Mesenchymal cell proliferationalsosignificantly decreases
sionpatternofShh,whichultimatelyleadstodeformitiesinthe due to a lack of reciprocal Shh signaling through its receptor
rugaeinthepalateoftheseknockoutanimals(Welshetal.,2007). Ptc1 (Rice et al., 2004). As discussed above, Msx1 expression
−/−
DoublenullmutantsofFgfintracellularantagonistsSpry2 act is not altered in Fgf10 null mutants (Alappat et al., 2005), nor
asFgfgain-of-function mutantwith highly disorganized palatal isBmp4 expression. However, the Bmp antagonist Sostdc1 does
rugae.Similarrugaedisorganizationwasalsoobservedinthecon- have reduced expression levels in Fgf10 null palates (Welsh and
ditionaldeletionofShh(K14-Cre;Shhfl/flmice)(Economouetal., O’Brien,2009).ThereforethealteredShhexpressionintheFgf10
2012).TheiranalysessuggeststhatFgfactsasanactivatingfactor orFgfr2bnullmicemaybeduetothedecreasedantagonismon
andShhactslikeSpry,functioningasaninhibitorofFgfsignaling Bmp signaling or directly due to loss of Fgf signaling and not
andofrugaedevelopment. throughalterationsintheMsx1network.Conditionallyknocking
Gli3,aproteinexpressed intheepithelium andmesenchyme outallFgfr2isoformsexclusivelyintheepitheliumalsoleadtoa
alongtheentireA–Paxisofthepalate,iscapableofactingasboth cleftpalate.OnceagainShhexpressionisdisorderedandthereis
anactivatorandrepressorofShhsignaling(Huangetal.,2008). alackofclearlydefinedrugaeduringthetimepalatogenesisnor-
IntheabsenceoftheShhsignal,theGli3proteinisprocessedby mally occurs. In this instance however, cell proliferation is only
proteinkinaseAallowingittoenterthenucleusandrepressthe decreasedwithintheepithelium, suggestingthatFgfr2receptors
expression of Shh target genes. Presence of the Shh signal pre- mustexistinmesenchymal cells,andberesponsibleforregulat-
vents the processing of the Gli3 protein, and therefore prevents ingcell proliferationinthesecells(Hosokawaetal.,2009).Loss
Gli3fromrepressingtheexpressionoftheShhtargetgenes(Wang of Fgf10 signaling alters cellular processes including apoptosis,
et al., 2000; Litingtung et al., 2002). In the limb, an antagonis- suggestingitplaysaroleincellsurvival.Fgf10nullmiceexhibit
ticrelationshipbetweenShhandGli3iscrucialinsettingupthe prematureandectopicfusionindicatingthatFgf10alsohasarole
A–P axis. Gli3 is expressed in the anterior region of the devel- inensuringproperfusion(Riceetal.,2004).
opinglimb,whereitrepressestheexpressionofShh.dHANDisa While Fgf10 regulates cell proliferation within the anterior
posterior-specificproteininthelimbthatisalsorepressedbyGli3 palate,ectopicexposuretoFgf10doesnotbringaboutanotice-
butisaknownactivatorofShhexpression.Together,thispathway able effect on the level of proliferation in the posterior palate
setsupanA–Paxisinthelimbthatensuresproperdevelopment (Yu et al., 2005), implying the down-stream effectors of Fgf10
(Niswander,2003).ThisimportantinteractionbetweenGli3and expression must not be present within the posterior region of
Shhinthelimbincombinationwiththeexpressionofbothgenes the palate.Thisprovidesfurther evidencethe anteriorandpos-
in the palate suggests a role for Gli3 in the palate. Not surpris- teriorregionsofthepalatearedistinctcellpopulationswithvery
ingly,Gli3nullmicedisplayacleftsecondarypalate;however,the differentregulatorymechanismsforthesamecellularprocesses.
cleftpalatephenotype wasnotdueto changeswithinthe palate Inadditiontoregulatingproliferation,apoptosis,andfusion,
itself, but rather due to defective growth of the tongue (Huang Fgf10 can also induce cell migration within the anterior palate.
etal.,2008). Theseresultsdemonstratethatregionaldifferences Fgf10expressionisnotonlylocalizedtotheanteriormesenchyme
and signaling pathways are not conserved between areas of the butisalsohigherintheoralregionoftheanteriormesenchyme
developing embryos. Hence, simply lining up the expression of (Rice et al., 2004). Fgf10 acts asa chemoattractant and induces
alloftheplayersinapathwaywithinthepalatedoesnotnecessar- the migration of anterior mesenchyme cells from the nasal to
ilyimplytheyfunctionbyasimilarmechanismasdescribedfor the oral side of the palate (He et al., 2008). The loss of Fgf10
otherareasofthedevelopingembryo. causespalateshelvestoassumeanabnormalshape(Alappatetal.,
2005), which could in part be explained by the loss of oral cell
FgfSIGNALINGPATHWAY migration.
Mutations in numerous members of the Fgf family have been TheFgfreceptorFgfr1bhasalsobeendescribedashavingan
linked to cleft palate in the human population (Riley et al., anterior and nasal specific expression pattern within the devel-
2007).ThebestunderstoodFgf-dependentpathwayinthepalate oping palate (Lee et al., 2008). As with other members of the
involves Fgf10 and its receptor Fgf2rb. Fgf10 null mice exhibit Fgf family, its expression is linked to the regulation of prolifer-
a wide open cleft palate that is due to abnormal palate shelf ationwithintheanteriorpalate.ExpressionofFgfr1bisnegatively
morphology and size, preventing the shelves from contacting regulated by the Wnt11 signaling molecule. In return, Fgfr1b
www.frontiersin.org January2013|Volume3|Article488|5
Smithetal. A–Paxisinpalatedevelopment
negatively regulates the expression of Wnt11 (Lee et al., 2008). exclusivelyintheanteriormesenchymeregionofthedeveloping
Atearlystages(E13.5)ofpalatedevelopment,thebalanceistilted secondarypalate,withitshighestexpressionoccurringduringthe
toward Fgfr1b allowing the palate to undergo cell proliferation. early stages of palate development. Mice deficient in the Shox2
However, as palate development proceeds (E14), the expression gene exhibit a rare form of cleft palate where the cleft only
balance is shifted away from Fgfr1b. At this stage proliferation occurs in the anterior part of the secondary palate. Expression
musttemporarilyhaltinorderfortheindividualpalateshelvesto of a number of genes critical for palatogenesis such as Jag2,
fuseandformacompletepalate(Leeetal.,2008).Thus,although Lhx8, Osr2, Pax9, Tgfb3, and Msx1 and its down-stream target
−/−
the most obvious role for the Fgf family is in regulating the Bmp4 do not change in the Shox2 palatal shelves (Yu et al.,
level ofproliferationwithin the palate, the Fgffamilyalso plays 2005).However,expressiondomainsofbothFgf10andFgfr2care
aroleinregulatingeventssuchasfusionbymaintainingminimal altered, which correspondswith altered proliferationand apop-
expressionofcertaingenesuntiltheappropriatetime. tosiswithintheanteriorpalateintheShox2nullmice(Yuetal.,
As discussed above, one specific animal model showed that 2005). These data indicate altering the expression ofgenes only
loss of the Fgf antagonist Spry2 leads to a cleft palate due to in one area of the palate can lead to clefting only in that spe-
alterations in the level of cell proliferation within the palate as cific area. However, Msx1 is also expressed in the very anterior
wellastheexpressionprofilesofnumerousgenesincludingMsx1 region of the palatal shelves and yet in Msx1 null mice a com-
(Welsh et al., 2007; Matsumuraet al., 2011). Spry2is expressed plete cleft of the secondary palate is observed (Zhang et al.,
−/−
intheepitheliumandmesenchymeatconsistentlevelsthrough- 2002) as it is in the Fgf10 mice (Rice et al., 2004; Alappat
out palatogenesis (Matsumura et al., 2011). The animal model et al., 2005). It is not known precisely why Shox2 exhibits this
discussed above has a piebald deletion, which is a collection of unusual anterior cleft. It may be that increased Fgf10 expres-
overlapping Mb-scale chromosomal deficiencies which includes sionissendingasignaltotheposteriorpalatetofuse(Yuetal.,
the Spry2 gene (Welsh et al., 2007), while another is a single 2005).
specific knockout of the Spry2 gene (Matsumura et al., 2011). While regulation of the Shox2 gene has been the subject of
Earlierreportsfromthegroupthatdevelopedtheanimalmodel recent investigations in palate development, a complete under-
lackingexclusivelySpry2indicatedthatanimalswerenotshown standing of Shox2 regulation remains elusive (Yu et al., 2005).
to have a cleft palate (Shim et al., 2005; Taketomi et al., 2005), BlockingofBmpsignalingwiththeantagonistNogginresultsina
however more recent reports have shown a prevalence for cleft down-regulationofShox2expressionwithintheexposedanterior
palate (Matsumura et al., 2011). The piebald deletion led to a mesenchymeatE12.5.ExposureofpalatalmesenchymetoBmp4,
highincidenceofcleftpalatewhilethetargeteddeletionofSpry2 Bmp2,andShhincultureisnotsufficienttoinduceShox2expres-
onlydisplayedthe cleftpalatephenotype inapproximately20% sion.ThesedatasuggestBmpsignalingisnotcapableofinducing
of animals. The differences in incidence rates are likely due to Shox2 expression on its own but is necessary for normal Shox2
otherdefectsresultingfromtheMb-scaleofthepiebalddeletion. geneexpression(Yuetal.,2005).
BothmutantsshowedthatalossofSpry2expressionleadstoan
increaseinthelevelofcellproliferationinthepalate(Welshetal., EPHRINSIGNALING
2007; Matsumura et al., 2011) which could be expected since Ephrin-B1 belongs to the transmembrane B-type subfamily of
its absence relieves inhibition on Fgf signaling which has been Eph/Ephrinsignalingmolecules(DavyandSoriano,2005).These
reportedtocontrolproliferationrates(Riceetal.,2004;Alappat signalingmoleculeshavetheabilitytocarryoutbidirectionalsig-
etal.,2005).Alsoseeninbothmutantanimalmodelswasaltered naling. Hence, cells expressing the Eph receptor tyrosine kinase
Msx1expression.Thetrueknockoutmodelshowedanincreasein can receive a forward signal whereas cells expressing ephrin
thelevelofMsx1expression,althoughregionspecificexpression (Efn) can be transduced to receive the reversesignal (Bush and
wasnotinvestigated(Matsumuraetal.,2011).Thepiebaldmuta- Jiang, 2012). The Efnb1 gene is expressed in the mesenchyme
tioninitiatesaposteriorexpansionofMsx1coincidingwithaloss of the anterior palate throughout the secondary palate devel-
of the anterior expansion of the posterior-specific transcription opment. Efnb1 forward signals to regulate anterior palatal shelf
factor Tbx22. While Tbx22 expression fails to reach its normal outgrowth by promoting cell proliferation through the activa-
anteriorexpressionboundary,Etv5andBarx1,whichareprimar- tionofERK/MAPpathway(BushandSoriano,2010).BothEfnb1
ilyexpressedintheposteriorpalate,expandtheirdomainstothe null miceandEfnb1+/− heterozygousfemalemicedevelopcleft
anterior(Welshetal.,2007).Theseresultssuggestantagonismof palatewithdecreasedcellproliferationintheanteriorpalatalmes-
Fgfs by Spry2 affects a number of networks in the palate lead- enchyme (BushandSoriano,2010). TheEfnb1 geneisX-linked
ingtogrosschangesintheirexpressionpatterns.Furtheranalysis andtheEfnb1+/− heterozygousembryosexhibitmosaicpattern
will be required to determine, which other factors are involved ofEfnb1expressioninthepalatethatcorrelateswithamosaicpat-
with the high rate of cleft palate in the piebald deletion mice. ternofproliferationandhenceamoreseveredysmorphogenesis
Although Fgf signaling is necessary for palate development, its ofthepalatalshelvescomparedtoEfnb−/−nullmice.Efnb1ispri-
action appears to require fine-tuning by repressors for normal marilyexpressedintheanteriormesenchyme,althoughthecleft
palatogenesistooccur. palate phenotype was along the entire axis (Bush and Soriano,
2010).Incontrast,lossofShox2(describedabove)whichisalso
Shox2NETWORK anteriorly restricted in the palate, induces a unique anterior-
Fgf10 expression is down-stream of the homeobox transcrip- delimited cleft palate (Yu et al., 2005). Hence, reduced Efnb1
−/−
tion factor Shox2 (Yu et al., 2005). The Shox2 gene is expressed signaling in the anterior palatal mesenchyme in Efnb null
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Smithetal. A–Paxisinpalatedevelopment
+/−
or Efnb1 heterozygous embryos may cross a threshold A–P
position at which initiation of fusion is required (Bush and
Soriano,2010).
Tgf-βPATHWAY
The role of Tgf-β family members in palatal shelf growth and
fusion is an area that has been well-studied. Loss ofxd func-
tion of Tgf-β2 (Sanford et al., 1997), Tgf-β3 (Kaartinen et al.,
1995; Proetzel et al., 1995; Martínez-Álvarez et al., 2000b), and
Tgf-β receptors Tgfbr1 (Dudas et al., 2006), Tgfbr2 (Ito et al.,
2003; Xu et al., 2006) are known to cause cleft palate. More
detailed information on this pathway was reviewed recently in
Bush and Jiang (2012). In the context of this review, we will
only highlight the anterior and posterior-specific roles of these
pathway members. The Tgf type I receptor Alk5 is expressed
exclusively in the anterior palatal epithelium and its activation
FIGURE3|Schematicrepresentationofthekeyregulatorsinthe
in Tgf-β3−/− palatalepithelium rescues palatalfusion, whereas posteriorpalate.Barx1andTbx22inducecellproliferationintheposterior
loss of Alk5 function in epithelium of wild-type palatal shelves palate.Hoxa2alsocontrolstheexpressionofBarx1intheearlystageof
palatogenesis.Mn1positivelyregulatesTbx22andrepresentsthefirst
preventspalatalfusion(Dudasetal.,2004).Interestingly, fusion
networkdeterminedtospecificallyregulatethelevelofproliferationinthe
of the posterior parts of palates is predominant following acti-
posteriorpalate.Meox2playsroleinfusionoftheposteriorpalate.
vation of Alk5 at E14 whereas its activation at E13.5 also facil-
itates fusion in the anterior region (Dudas et al., 2004). Thus,
there appearsto be an anterior to posterior direction of palatal Meox2NETWORK
fusion(Tayaetal.,1999)withTgf-β3signalingmediatedbyAlk5 Meox2 is a homeobox transcription factor with a posterior-
in the anterior epithelium (Dudas et al., 2004). The homozy- specificexpressionpatternthatbecomesincreasinglylocalizedto
gous knock-in of Tgf-β1 in the Tgf-β3 locus partially rescues the extreme posterior region of the palate as development pro-
the cleft palate phenotype of Tgf-β3−/− mice in the anterior ceeds(JinandDing,2006;LiandDing,2007).Micelackingthe
palate (Yang and Kaartinen, 2007). Since Tgf-β1 is expressed in Meox2 gene exhibit a low penetrance of cleft palate that results
the palatal epithelium along the A–P axis (Yang and Kaartinen, from a novel mechanism. Palate shelves grow, elevate and fuse;
2007), its partial rescue of anterior palatal fusion may also be however,fusionisweakandasthecraniofacialregionexpandsthe
mediated via Alk5 signaling in Tgf-β3−/− mice. Recent find- palateshelvespullapartfromoneanotherleadingtoacleftpalate
ings show that craniofacial abnormalities in Tgfbr2 −/− mice specifically in the posterior region. Histological analysis of the
is prevented following genetic manipulation of an alternative cleftpalatesclearlyshowthepalateshelveswerecompletelyabsent
non-canonicalTGF-βsignalingpathwaythroughAlk5/Tgf type ofthe medial epithelial edgeand were composed solelyof mes-
IIIreceptorcomplexandSMAD-independentTRAF6/TAK1/p38 enchyme. The mechanism responsible for the post-fusion cleft
signaling (Iwata et al., 2012b). The role of Tgf-β3 in apoptosis palate is not completely clear, but may involve a palatal growth
ofmedialedgeepithelium(MEE)iswell-established (Martínez- defectintheposteriorpalatethatpreventsthepalatesfrombeing
Álvarez et al., 2000a,b) and Tgf-β3 synthesized at the MEE able to keep up with the rest of craniofacial development (Jin
facilitates accumulation of chondroitin sulfate proteoglycans at and Ding, 2006). Alternatively, the loss of Meox2 may lead to
apical surface of MEE (Gato et al., 2002). Loss of Tgf-β3 dis- improperpalatalfusionandaweakseamthatdoesnotstandupto
rupts the normal distribution of E-cadherin, α-,and β-catenins themechanicalforcesofcraniofacialdevelopment(JinandDing,
in MEE and impairs cell-cell adhesion (Tudela et al., 2002). 2006). Meox2 has been reported to be Tgf-β responsive in the
Conditional deletion of β-catenin in the epithelium in K14- mammaryepithelialcellsbyinhibitingepithelialcellproliferation
Cre transgenic mice suppresses canonical Wnt signaling giving bybindingtothepromoterofp21throughTgf-β/Smadsignal-
rise to an abnormal and persistent MES. Loss of β-catenin also ingpathway(Valcourtetal.,2007).Interestingly,Xuetal.(2008)
induces down-regulation of Tgf-β3 and inhibition of apoptosis showedthatp21isrequiredforSmad4mediatedp38MAPKpath-
in MEE that subsequentlyleadsto a cleft palatephenotype (He way for apoptosis and MES degeneration. These works suggest
etal.,2011).Hence,regionspecificexpressionofTgf-β3isessen- thatMeox2couldplayaroleinTgf-β3mediatedfusion.However,
tial for proper palate development and these findings highlight thesemechanismsneedtobeinvestigatedfurther.
the interplay between the various pathways that govern palate
development. Tbx22NETWORK
Tbx22 alsohasaposterior-specific expression profilewithin the
POSTERIOR-SPECIFICGENEEXPRESSION developingpalate(Herretal.,2003).TheTbx22geneisaT-box
Although many genes important in palate development have protein that acts asa transcription factor regulating the expres-
regional specific expression and are expressed predominantly sion of down-stream genes. Alterations in the Tbx22 gene are a
in the anterior palate, several genes are also important in the commonsinglecauseofcleftpalateinhumans(Marçanoet al.,
posteriorregion(Figure3). 2004).AmissensemutationintheTbx22geneisresponsiblefor
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Smithetal. A–Paxisinpalatedevelopment
X-linked cleft palate (Marçano et al., 2004), whereby the mis- relative strength of Bmp and Fgf signaling (Welsh et al., 2007).
sense mutation affects the ability of Tbx22 to bind DNA and TheexpressionofBarx1isalteredinanumberofknockoutmouse
subsequently act as a transcriptional repressor (Andreou et al., lines.LossofSpry2viathepiebalddeletionnotonlyaffectsMsx1
2007).ThismutationisbelievedtopreventtheSUMO-1enzyme and Tbx22 expression, but also leads to an anterior expansion
fromsumoylatingtheTbx22protein.Intheabsenceofthispost- of Barx1 expression that may be involved in the increased cell
translationalmodification,Tbx22hasamuchloweraffinityforits proliferation seen in these palates (Welsh et al., 2007). The loss
DNAbindingsequence(Andreouetal.,2007),cannotrecognize of the Bmp receptor Bmpr1a also leads to an expansion of the
the DNA sequence and bind appropriately, and does not per- region in the palate expressing Barx1 (Liu et al., 2005). Hoxa2
formits normalrepressor functions. Notably,SUMO-1 isagain nullembryoshaveincreasedBarx1expressionattheearlystages
implicated in regulating palatogenesis. Based on the number of ofpalatedevelopment.Anincreaseinthelevelofcellproliferation
importantgenesknowntorequiresumoylationtofunctionprop- intheposteriorregionofthepalateisalsoobservedinHoxa2null
erly,haploinsufficiencyofSUMO-1isnotsurprisinglylinkedto mice (Smith et al., 2009). The alterations in both Fgf and Bmp
cleftpalatephenotype(Alkurayaetal.,2006). signalingcausingalteredBarx1expressionsupporttheviewthat
Tbx22expressioninSpry2piebaldmutantsisaffected asdis- regulationoftheregionalexpressionofBarx1involvesbothfami-
−/−
cussedabove.IntheabsenceofthisFgfantagonist,theexpression liesofsignalingmolecules.EvidenceforthiscomesfromTp63
ofTbx22failsto expand fromthe mostposterior regionsofthe mice where expression of Fgf8 at E11.5 in the anterior region
palate at E14.5. This altered expression profile coincides with a of maxillary processes is down-regulated, which coincides with
posterior shift in the expression of Msx1 as well as an increase areducedanteriorexpressionofitstargetgeneBarx1(Thomason
−/−
inproliferationthroughoutthepalateshelves(Welshetal.,2007). et al.,2008). Incontrast, the Tp63 mice(which exhibit cleft
(cid:2)
The5 regulatoryregionoftheTbx22genecontainsputativeMsx1 lip and palate phenotype) show increased expression of Bmp4
bindingsites(Herretal.,2003),however,Msx1nullmicedonot in the anterior region of the maxillary processes at E10.5 and
showanexpansionoftheTbx22expressiondomain(Fuchsetal., E11.5.Barx1isalsoregulatedbyrelativelevelsofFgf8andBmp4
2010), and Tbx22 null mice are not reported to have increased in developing chick facial primordia where BMPs reduce Barx1
Msx1expression(Pauwsetal.,2009).PalatalTbx22expressionhas expressionandantagonizeFgf-8signaling(Barlowetal.,1999).
been demonstrated to be independent ofFgf signaling, but was
reported to be repressed in culture by exogenous Bmp4 (Fuchs ATWHATSTAGEDOESTHEANTERO-POSTERIORMOLECULAR
etal.,2010).TakentogetherthissuggestsasystemwherebyMsx1 SIGNALINGGETESTABLISHED?
is involved in regulating Bmp4 expression which subsequently An intriguing question during palatal development is when
plays a role in the repression of Tbx22 expression, leading to a does the antero-posterior molecular signaling get established?
posterior-specificexpressionpattern. Althoughanswertothisremainselusive,availabledataindicates
Liu et al. describe a novel molecular network involving the a much earlier time in development and prior to palatal shelf
Tbx22 (Liu et al., 2008). The transcription factor Mn1 has a formation.TheanteriorlocalizationofMsx1andposteriorlocal-
medial-posterior-specific expression profile that generally over- izationofBarx1issettobedeterminedinthefirstbranchialarch
laps the Tbx22 expression profile. Loss of one or more copies whereMsx1islocalizedtodistalandBarx1totheproximalregion
of Mn1 leads to craniofacial abnormalities including a cleft (Barlow et al., 1999). In early mice palatal development, Barx1
secondary palate. In the Mn1 null embryos, Tbx22 expression expression is visible in the posterior region extending through
decreasesintheposteriorregionofthepalate,andMn1directly almost three quarters ofthe developing palatalshelves, whereas
regulatestheexpressionofTbx22inthepalate(Liuetal.,2008). Msx1 isrestricted to anarrowanteriorregionofthedeveloping
A marked decrease in proliferation in the medial and posterior palate (Welsh and O’Brien, 2009). Following rostral expansion,
palateshelvesalsooccurs,andisbelievedtobedueinparttothe the anteriorpalateextends withtheexpression ofMsx1 andthe
regulation ofa separategenetarget (Ccnd2) by Mn1 (Liu et al., first molar tooth bud serves as the posterior boundary of this
2008).Thisrepresentsthefirstnetworkdeterminedtospecifically extendedanteriorexpression(WelshandO’Brien,2009).Hence,
regulate the level of proliferationin the posterior palate. Tbx22 genes expressed in the presumptive hard and soft palateappear
expression appearsto beregulated byatleast two factors; Msx1 to be set up earlier along an A–P axis in the branchial arches.
acts asa repressor, whileMn1 acts asanactivator, and together ConsistentwiththisexpressionalonganA–Paxisinthefirstarch,
they determine the specific expression domain of Tbx22 in the Msx1 plays a role in incisor development and Barx1 in molar
posteriorregionofthepalate. tooth development (reviewed in Mitsiadis and Smith, 2006).
Interestingly,Bmp-Fgfsignalingalsogovernstoothdevelopment
Barx1NETWORK in a gradient manner along an A–P axis (Mitsiadis and Smith,
Barx1expressionhasapredominantlyposteriorexpressionpro- 2006). It is likely these genes playa similarrole in the orofacial
file that is complementary to the anterior expression of Msx1 structures.Indeedsimilartoitsroleinpalatedevelopmentwhere
(Barlow et al., 1999; Welsh and O’Brien, 2009). This region- Bmp4 is required to prevent the premature apoptosis of palatal
specific expression is initially set up in the branchial arches epithelium,Bmp4isessentialinblockingapoptosisinthedental
where Msx1 expression is localized to the distal regions of the epitheliuminaMsx1-dependentmannerregulatedbyTgf-βtype
first brachial arch and Barx1 expression is localized proximally IreceptorAlk-5(Zhaoetal.,2008).
(Barlow et al., 1999). The A–P axis derived from the regional The transcription factor Tp63 regulates the expression of
expression of Msx1 and Barx1 is believed to result from the Bmp4 in the anterior palate and loss of Tp63 in the maxillary
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Smithetal. A–Paxisinpalatedevelopment
processes in the medial region from which the palatal shelves mesenchyme. Bmp4 induces Shh expression in the epithelium
originate, results in improper Bmp signaling and a cleft palate whichsignalsbacksto mesenchyme to positively regulateBmp2
phenotype (Thomasonetal.,2008). However,conditional inac- to enhance cell proliferation in the mesenchyme (Zhang et al.,
tivationofBmp4intheearlymaxillarymesenchymeusingNestin 2002).CrosstalkalsoexistsbetweenBmp7andShh,whichplays
cre;Bmp4 null/flox(n/f) mice did not disrupt secondary palate aroleinrefiningtheexpressiondomainofbothgenes(Hanetal.,
developmentbutresulted instead inisolated cleftlip (Liu et al., 2009). Tbx3 and Bmp4 regulate each other’s expression in the
2005). Loss of Bmpr1a in facial primodia of Nestin cre;Bmpr1a palate.Tbx3inhibitstheexpressionofBmp4whileBmp4induces
n/f embryos, which did not impact Msx1 expression, resulted Tbx3 expression and regulates the levels of cell proliferation in
in reduced mesenchymal cell proliferation in maxillary process the anterior palatal mesenchyme (Lee et al., 2007). Regulatory
prior to the onset of secondary palate outgrowth resulting in feedbackloopexistsbetweenFgfr1bandWnt11.Fgfr1brepresses
smaller palatal shelves and subsequent cleft palate at birth (Liu expression of Wnt11 whereas Wnt11 signaling molecule nega-
etal.,2005).Incontrast,tissue-specificlossofBmpr1ainpalatal tively regulates Fgfr1b expression (Lee et al., 2008). Balance is
mesenchyme in Osr2-IresCre;Bmpr1af/f mutant mice results in titledtowardorawayfromFgfr1b,torespectivelyallowcellpro-
reducedexpressionofMsx1andanup-regulationinBmp4lead- liferationtoproceed(atE13.5)ortorecede(atE14)andfusion
ingtosubmucouscleftofthehardpalate(Baeketal.,2011).Thus, tooccur(Leeetal.,2008).Unliketheanteriorpalate,molecular
theBMPfamilyhighlightsthecomplexityofsignalinginvolvedin mechanismsofpalataloutgrowthintheposteriorpalatalregions
their early tissue specific role in orofacial development. Further are not yet well established. Mn1 directly regulates the expres-
earlyfatedeterminationstudiesareneededusinglineagespecific sion of Tbx22 in the palate (Liu et al., 2008) and Msx1 acts as
animalmodelstocharacterizethecomplexsignalingduringearly a repressor of Tbx22 in the palate (Welsh et al., 2007) which
palate development to clearly determine the origins of the A–P togetherdeterminestheposteriorexpressiondomainofTbx22in
molecularsignaling. thepalate.
CROSSTALKBETWEENNETWORKS/PATHWAYS ANTERIORPALATE-SIGNALINGCENTER
Palatal elevation and fusion is governed by transcription fac- Critical events such as elevation, maturation and fusion of sec-
tors, various growth factors and their receptors forming inter- ondary palatal shelves follow an anterior to posterior sequence
connected network of molecular pathways. Relative signals or (Tayaetal.,1999;Dudasetal.,2004)(Figure4).Duringmouse
gradients are strictly required to ensure proper development. palate development, at embryonic day E13.5–E14, the ante-
The anterior and posterior palatal tissues being the future hard riorpalateorientshorizontally abovethe tonguewhen the pos-
and soft palates differ in function and structure, show differ- terior palate is still lying vertically (Kaufman, 1992) providing
ence in the expression patterns along the A–P axis. Numerous a clear indication of the more dynamic growth in the anterior
pathways/networksinthepalateclearlydisplayreciprocalsignal- palatecomparedtotheposteriorpalate.Inaddition,theinitialsite
ing between the epithelium and mesenchyme. Genes expressed ofappositionandsubsequentfusionofthepalatalshelvesoccur
exclusivelyintheepitheliumhavebeenreported toregulatecel- firstintheanteriorhalfofthepalateandthesequenceofpalatal
lularprocessesandgeneexpressioninthemesenchyme,andvice closuremayberesultofsignalingactivityalongtheA–Paxis.
versa, such that reciprocal signaling occurs between the epithe- AlthoughShox2expressionremainsanterior-specificthrough-
lium and mesenchyme (Zhang et al., 2002; Yamamoto et al., out palatogenesis, it displays a dynamic pattern of expression.
2003; Rice et al., 2004; Nawshad et al., 2007). In recent years At the initial stages of palate growth, Shox2 expression is only
it has become increasingly evident that gene expression in the detected inthemostextreme anteriorregionsofthepalate(less
developingpalatenotonlydisplaysepithelial-mesenchymalspeci- than 25% of the length of the palate) (Yu et al., 2005; Li and
ficity,butalsoanterior–posterior(A–P)andoral-nasalspecificity Ding,2007).Asthepalateshelvescontinuetogrow,theexpression
(reviewedinMurrayandSchutte,2004;Hilliardetal.,2005;Bush of Shox2 expands until E14.5 when it covers the entire anterior
andJiang,2012).Interestingly,Tp63nullmutantshaveabnormal palate and most of the medial palate (60% of the length of the
outgrowth of the palate initially but by E12.5 the A–P specific palate shelf). Concurrent with the expansion of Shox2 expres-
expression patternswere normal despite abnormalshelf growth sion, the region of the palate expressing the posterior-specific
confirming the importance of setting up and maintaining the gene Meox2 shrinks (Li and Ding, 2007). This phenomenon
A–Paxis(Thomasonetal., 2008). Inadditionto their localized demonstratesnormaldevelopmentoftheanteriorpalaterequires
expression,geneshavebeenreportedtoelicitdifferentresponses recruitmentofcellsfromtheposterior,whichareconvertedinto
indifferent regionsofthepalate. Forexample, whileexogenous Shox2anterior-specificcells.Thishasbeensuggestedtobedueto
Fgf10 alters proliferation in the anterior palate, it has no effect a repression of Meox2 by Shox2 or a down-stream target of the
on proliferation in the posterior region of the palate (Yu et al., Shox2pathway(LiandDing,2007).
2005).Suchlocalizedexpressionandfunctionphenomenaclearly The rugae have been suggested to play an important role in
highlight the importance ofregional patterningand differentia- organizing and maintaining the A–P axis (Welsh et al., 2007;
tionwithinthepalateatthemolecularlevel.Overall,thenumber Pantalaccietal.,2008;WelshandO’Brien,2009).Rugaehavebeen
of genes involved in the development of the palate that dis- showntoformintheregionbetweenthelastformedandrugae8
playstrictlyregulateddomainsofexpressionisclearevidenceof (the first rugaeto form) in a sequential order(Pantalacci et al.,
regionaldifferentiationwithinthepalate.Msx1andBmp4func- 2008; Welsh and O’Brien, 2009). Rugae 8 has been denoted the
tioninanautoregulatoryloopmechanismintheanteriorpalatal “boundary rugae” as it appears to act to separate the anterior
www.frontiersin.org January2013|Volume3|Article488|9
Smithetal. A–Paxisinpalatedevelopment
FIGURE4|AtE13.5,theanteriorpalatalshelvesfirstflipuptoorient futurehardandsoftpalate,respectively.Fgf-Bmpgradients/thresholds
verticallywhentheposteriorpalatalshelvesarestilllyinghorizontaltoeach maintainproperpalatalgrowthandfusionthroughproliferation.Anterior
other(A).AtE14,theposteriorpalatalshelvesfollowtheanteriorpalatal Fgf10andBmpscontrolproliferationviaShhexpression.Thisdirectsanterior
shelvesinorientingvertically,whereastheanteriorpalatesbegintogrow palateflipupandverticalgrowthatE13.5–E14(A,B).AnteriorFgf-Bmp
verticallytowardeachothertomakecontact(B).AtE15,theanteriorpalatal gradientsalongwithposteriorFgf8regulateproliferationandgrowthvia
shelveshavemadecontactandfused,whereastheposteriorshelvesgrow Barx1intheposteriorpalateatE14–E15(B,C).Fusionisinitiatedatthe
vertically(C).AtE15.5,boththeanteriorandposteriorpalateshavefused(D). anteriorpalatebyTgf-β3throughitsreceptor(C).Thenthefusionextends
AtE16,thefusionbetweentheprimaryandsecondarypalateoccursatthe posteriorlythroughTgf-β-Meox2(D).Thefusionbetweentheprimaryand
futuresecondarychoana(E).Palatalshelvesaredividedintoanterior(pink) secondarypalatemarksthecompletionofpalatalfusionatE16(D,E),via
Msx1andposterior(aqua)Barx1expressiondomains(A–E)representing Bmpr1amediatedShox2andTgfβsignalingthroughitsreceptors.
and posterior domainsof the palate. Throughout palatal devel- higherintheanteriorpalatethantheposteriorpalate(Heetal.,
opment, expression of the anterior specific markers Shox2 and 2008),withWnt5adetectedexclusivelyinthemesenchyme(Paiva
Msx1remainanteriortorugae8andMeox2andTbx22stayposte- et al., 2010). The Wnt5a signal was consequently shown to act
riorofthisboundary(Pantalaccietal.,2008;WelshandO’Brien, asachemoattractantcausingcellstomigratefromtheposterior
2009). This rapid expansion of the palate anterior to rugae 8 region of the palate toward the anterior region. Evidence sug-
providesanalternateexplanationfortheanteriorgrowthofthe gests that Ror2 mediates the role of Wnt5a in the palatogenesis
palate to the one detailed above by Li and Ding (2007). The (He et al., 2008). As discussed above, Wnt5a also regulates the
majordifferenceisthatLiandDingdidnotdetectdifferencesin expressionofBmp4andShh,bothofwhichplayimportantroles
theproliferationratesoftheanteriorandposteriorpalate,while inthedevelopmentandgrowthoftheanteriorpalate(Heetal.,
Pantalacci et al. (2008) did detect a higher level in the anterior 2008).Hence,simpleupregulationofShox2anddownregulation
palate.Whichtheoryisdeemedtobecorrectwillrequirefurther of Meox2 may not result in the conversion of posterior cells to
investigations. anteriorcellsifthecellsaremigratingtowardthehigherWnt5a
Mice lacking expression of either Wnt5a or its noncanoni- signal. As cells enter the anterior region of the palate, Wnt5a
cal receptor Ror2 were found to exhibit a cleft palate (Schwabe andpotentiallyotherfactorsmayacttoaltertheexpressionpro-
etal.,2004;Heetal.,2008).Inaddition,bothgeneswereshown file of genes in the cells, causing them to transdifferentiate into
to exhibit an expression pattern whereby their expression was anterior-specificpalatecells.
FrontiersinPhysiology|CraniofacialBiology January2013|Volume3|Article488|10