1-s2.0-s0012160608010221-main

8132019 1-s20-S0012160608010221-main

httpslidepdfcomreaderfull1-s20-s0012160608010221-main 110

Facial clefting in Tp63 de1047297cient mice results from altered Bmp4 Fgf8 andShh signaling

Helen A Thomason a Michael J Dixon ab Jill Dixon a

a Faculty of Medical and Human Sciences Michael Smith Building University of Manchester Oxford Road Manchester M13 9PT England UK b Faculty of Life Sciences Michael Smith Building University of Manchester Oxford Road Manchester M13 9PT England UK

a b s t r a c ta r t i c l e i n f o

Article history

Received for publication 25 February 2008

Revised 19 June 2008

Accepted 19 June 2008

Available online 2 July 2008

Keywords

p63

Cleft palate

Cleft lip

Facial development

BMP

Shh

Fgf8

During embryogenesis the transcription factor Tp63 is expressed in the basal cells of multiple epithelial

tissues In humans mutations in TP63 have been identi1047297ed in 1047297ve distinct human developmental disorders

that are characterized by limb abnormalities ectodermal dysplasia and facial anomalies To dissect the

molecular pathogenesis of the bilateral cleft lip and cleft palate that results from mutation of Tp63 we

analysed Tp63 mutant mice At E105 Tp63-de1047297cient mice exhibited abnormal morphogenesis of the medial

nasal lateral nasal and maxillary processes Analysis of key signaling molecules revealed that these defects

result from increased Bmp4 signaling in the epithelia of the facial processes Acting antagonistically on Fgf8

and Shh this aberrant signaling led to a reduction in mesenchymal cell proliferation and increased cell death

in speci1047297c regions of the facial processes In addition a proliferative defect in the mesenchyme of the

maxillary processes at E115 resulted in absence of the anterior region of the palatal shelves and

subsequently cleft palate Our results are consistent with a role for Tp63 in the regulation of Bmp signaling

controlling the growth modelling and fusion events underlying facial development and shed new light on

the complex abnormality of facial clefting

copy 2008 Elsevier Inc All rights reserved

Introduction

Orofacial clefting (OFC) encompassing clefts of the lip and palate

is a common congenital disorder which occurs with an incidence that

has been estimated at 1 in 500ndash2500 live births (Murray 2002) OFC

may present as part of a syndrome where structures other than the lip

and palate areaffected or as an isolated entity Attemptsto dissect the

genetic basis of non-syndromic OFC have proven problematical as

both genetic and environmental factors are known to play a role in its

pathogenesis it has been estimated that as many as 14 interactingloci

maybe involved in non-syndromic clefting (Schliekelman and Slatkin

2002) Recent successes in delineating the molecular mechanisms

underlying OFC have been driven largely by the analysis of human

clefting syndromes In this regard mutations in the transcription

factor TP63 have been found to underlie 1047297ve distinct autosomal

dominant developmental disorders in man a subset of which exhibit

cleft lip and palate as a de1047297ning feature these disorders include

ectrodactyly ectodermal dysplasia clefting (EEC) syndrome (MIM

604292) and ankyloblepharon ectodermal dysplasia clefting (AEC)

syndrome (MIM 106260) (Celli et al 1999 McGrath et al 2001)

The TP63 gene is a member of the p53 family which encodes at

least six protein variants owing to two different transcription start

sites and alternative splicing Three isoforms contain N-terminal

transcriptional activation (TA) sequences while the other three (ΔN)

isoforms do not In addition alternative splicing towards the carboxy

terminus generates three subtypes α which encodes a sterile alpha

motif (SAM) domain and a transactivation inhibitory domain (TID)

and β and γ from which the SAM and TID domains are absent ( van

Bokhoven and Brunner 2002) The different protein isoforms all

display DNA-binding and tetramerisation domains but vary in their

ability to transactivate both p53-responsive genes and other Tp63

isoforms and in their ability to mediate cell cycle arrest and apoptosis

(Yang et al 1998 Ghioni et al 2002) The predominant isoform

expressed during embryogenesis is thought to be ΔN-Tp63α which is

found in the basal layer of many epithelial tissues (Yang et al 1999

Laurikkala et al 2006) Mice lacking all Tp63 isoforms die at birth and

exhibit severe developmental defects including limb truncations

epidermal defects and craniofacial anomalies (Mills et al 1999 Yang

et al 1999) Theepidermis is thin fails to stratify andlacksectodermal

appendages such as hairs whiskers teeth and several glands

including mammary salivary and lacrimal glands (Mills et al 1999

Yang et al 1999) The epidermal phenotype has been interpreted to

result from lack of proliferative potential of the Tp63-de1047297cient

epidermal stem cells (Yang et al 1999) or lack of commitment of

the immature ectoderm to epidermal lineages (Mills et al 1999)

Despite considerable research into the epidermal (Koster Huntzinger

and Roop 2002 Keyes et al 2006) and dental anomalies in Tp63

Developmental Biology 321 (2008) 273ndash282

Corresponding author Fax +44 161 275 5082

E-mail address jilldixonmanchesteracuk (J Dixon)

0012-1606$ ndash see front matter copy 2008 Elsevier Inc All rights reserved

doi101016jydbio200806030

Contents lists available at ScienceDirect

Developmental Biology

j o u r n a l h o m e p a g e w w w e l s ev i e r c o m d ev e l o p m e n t a l b i o l o g y

8132019 1-s20-S0012160608010221-main


knockout mice (Laurikkala et al 2006) the nature of the craniofacial

anomalies and their underlying developmental pathogenesis has not

been characterised

Development of the craniofacial complex involves a complex series

of events that requires the close co-ordination of programs for cell

migration growth differentiation and apoptosis Neural crest cells

which delaminate from the neural folds contribute to and migrate

through the mesenchymal tissue into the craniofacial region where by

thefourth week of embryonicdevelopment in man they participate inthe formation of the frontonasal prominence the paired maxillary

processes and the paired mandibular processes surrounding the

primitive oral cavity Theformation of the nasal placodes by the end of

the fourth week of embryogenesis divides the lower portion of the

frontonasal prominence into paired medial and lateral nasal pro-

cesses Towards the end of the sixth week of development merging of

the medial nasal processes with one another and with the maxillary

processes on each side leads to the formation of the upper lip and the

primary palate

The 1047297rst sign of overt development of the secondary palate occurs

during the sixth week of embryogenesis with outgrowth of paired

palatal shelves from the maxillary processes The palatal shelves

initially grow vertically down the sides of the tongue but during the

seventh week of development they elevate to a horizontal position

above the tongue contact and fuse to form a midline epithelial seam

which subsequently degenerates to allow mesenchymal continuity

across the palate These fusion processes are complete by the tenth

week of embryogenesis The development of the mammalian

secondary palate thereby divides the oronasal space into separate

oral and nasal cavities

To dissect the role of Tp63 during craniofacial development and to

determine why mutation of TP63 contributes to orofacial clefting in

humans we analysed the expression of Tp63 and characterised the

craniofacial defects in Tp63-null mice We demonstrate that loss of

Tp63 resulted in increased Bmp signaling in several areas of the facial

processes Consequently down-regulation of the key signaling

molecules Fgf8 and Shh led to reduced mesenchymal cell prolifera-

tion regional growth defects and altered morphogenesis of the nasal

processes resulting in a bilateral cleft of the upper lip We alsodemonstrate that a proliferative defect in the mesenchyme of the

maxillary processes at embryonic day 115 leads to absence of the

entire anterior region of the secondary palate during the initial stage

of palatogenesis Our results provide molecular evidence that Tp63-

mediated epithelial-derived signals are crucial during the outgrowth

and modeling of the facial processes and in the initial stages of palatal

growth

Materials and methods

Tissue preparation scanning electron microscopy

immunohistochemistry and in situ hybridisation

The generation and genotyping of the Tp63minus

minus

mice have beendescribed previously (Yang et al 1999) Mice were housed in

accredited animal facilities at the University of Manchester All

procedures were approved by local Ethical Review Committees and

are licensed under the Animal (Scienti1047297c Procedures) Act 1986 issued

by the Home Of 1047297ce Her Majestys Government London United

Kingdom BALBc-derived Tp63minusminus embryos were obtained by hetero-

zygous matings the morning of the vaginal plug being considered

E05 Tissues were 1047297xed in either 4 paraformaldehyde or Bouins

reagent and processed for histological examination scanning electron

microscopy or immunohistochemistry using standard protocols

(Dixon et al 2000 Knight et al 2006) Antibodies were obtained

from Santa Cruz (SC-8344 SC-8608 California USA) and R and D

systems (activated caspase 3 AF835 Minneapolis USA) The whole-

mount and section in situ hybridization protocols have been described

previously (Knight et al 2006 Wilkinson et al 1987) During all

assays wild-type and Tp63-null embryos were treated identically In

particular during whole-mount in situ hybridization assays to

compare gene expression levels embryos of both genotypes were

incubated in the detection reagent for identical periods of time

Organ culture and cell proliferation assays

E135 palatal shelves were dissected from Tp63 null mice and wild-type littermates in sterile Dulbeccos MENNUT Mix F-12 (Ham)

containing 005 mM L -glutamate 025 mM glycine and 15 mM Hepes

with 1 penicillinndashstreptomycin and freshly added L -ascorbic sodium

salt at 40 microgml 1047297nal concentration and cultured as described

previously (Ferguson et al 1984) Cell proliferation assays were

performed as described previously (Richardson et al 2006) Three

non-serial sections were counted from three Tp63minus minus and three wild-

type embryos from one or more litters All assays were analysed by

two individuals and the mean value recorded MannndashWhitney U

statistical tests were performed using SPSS 130

Results

ΔNp63α is widely expressed during facial development

To characterise the expression domains of Tp63 during facial

development we performed whole-mount in situ hybridization assays

on E95ndashE125 mouse embryos These studies indicated that Tp63

transcripts were widely expressed during facial development and

appeared at particularly high levels in the epithelia of the maxillary

and mandibular components of the 1047297rst branchial arch at E95 the

lateral and medial nasal processes as they fused at E105 the leading

edges of the upper lip and mandible at E1175 and the nasolacrimal

groove at E125 (Fig 1 AndashD) Immunostaining using a panel of

antibodies directed against all protein isoforms of Tp63 (pan-Tp63

4A4) ΔN-Tp63 and Tp63α isoforms were identical con1047297rming

previous observations that the main embryonic isoform is ΔN-

Tp63α (Yang et al 1999 Laurikkala et al 2006) Similar experiments

using a TA-Tp63-speci1047297c antibody failed to detect these isoforms atany of the embryonic stages examined These observations were

con1047297rmed by RT-PCR and real-time PCR (data not shown) We

observed strong staining for ΔN-Tp63α in all basal cells of the

ectoderm of the facial processes from E95 (data not shown) Initially

we detected intense Tp63 staining in basal cells of the ectoderm in the

most distal regions of the medial and lateral nasal processes at E105

(Fig 1E) Subsequently this expression domain was con1047297ned to only

the ectodermal component of the nasal processes at E115 ( Fig 1G)

Staining was present in all basal cells of the ectoderm covering the

maxillary processes during E10ndashE12 (Fig 1 FH) and at all stages of

development of the secondary palate from E115 to E145 (Fig 1 IndashL)

and at E155 (data not shown)

Tp63minus

minus

embryos display bilateral cleft lip and cleft palate

Tp63minus minus embryos exhibited a fully penetrant bilateral cleft lip and a

complete cleft of the secondary palate (n =137) (Fig 2 AndashD) This

phenotype was 1047297rst apparent at E115 when the maxillary processes

were observed to be smaller in Tp63minus minus mice than in Tp63+minus or Tp63++

littermates (Fig 2 E F) In addition the medial nasal processes had a

more rounded appearance and failed to meet the lateral nasal

processes in the most distal regions (Fig 2F) Histological analysis of

a developmental series of Tp63minusminus embryos determined that the facial

processes were abnormally shaped andthe lateral nasal processes and

maxillary processes appeared smaller than those of their wild-type

littermates (Fig 2 Gndash J) The medial and lateral nasal processes

remained apart in all Tp63minusminus embryos examined although serial

sections through Tp63-null embryos revealed that the posterior

274 HA Thomason et al Developmental Biology 321 (2008) 273ndash 282

8132019 1-s20-S0012160608010221-main


region of the medial nasal processes fused with the anterior region of

the maxillary processes via an ldquoepithelial bridgerdquo (Fig 2 J) In addition

the defects of the secondary palate were particularly severe in theanterior region which was completely absent in Tp63minus minus embryos at

E125 and markedly smaller at E135 (Fig 2 KndashN) Skeletal analysis of

E185 Tp63minus minus embryos revealed abnormalities of the nasal capsule

absence of the premaxilla and palatal processes of the maxilla and

palatine bone (Fig 2 O P) Although the overall morphology of the

Tp63minus minus mice suggested that the defect in palatogenesis occurred as a

result of abnormal growth and patterning during early embryogen-

esis we con1047297rmed that the palatal shelves were competent to fuse

with one another using in vitro organ culture (Ferguson et al 1984)

Palates from E135 wild-type and Tp63minus minus mice were dissected placed

in close proximity and cultured for 24 or 48 h Histological analysis

con1047297rmed that after 24 h all Tp63minus minus explants had fused in the midline

and in most cases the epithelial seam had started to degenerate (Fig

2 Q R) After 48 h of culture all Tp63minus

minus

explants were fully fused withno evidence of a midline epithelial seam (data not shown) These data

indicate that the palates of Tp63minus minus mice are capable of fusion and that

the cleft palate occurs as a result of failure of growth of the individual

palatal shelves

Tp63-mediated signaling is essential for cell survival

Scanning electron microscopy revealed that the facial processes of

wild-type mice were covered by 1047298attened regular periderm cells

whereas the tips of the lateral nasal processes and the maxillary

processes of Tp63minus minus embryos were decorated with rounded irregular

cells that appeared to be sloughing away from the underlying

epithelium (Fig 3 AndashD) These cells were not periderm in nature

indeed Tp63-null mice do not form a periderm layer as judged by

keratin 17 immuno1047298uorescence assays (data not shown) These

abnormal cells were evident in histological sections (Fig 3 EndashH) and

stained positively for activated caspase-3 con1047297rming that the cellswere undergoing inappropriate cell death (Fig 3 I J) We were unable

to 1047297nd any areas of cell death within the mesenchyme of the facial

processes or secondary palate in Tp63minus minus embryos (Fig 3 I J and data

not shown) In light of these observations we hypothesized that while

the epithelial cell death might be a contributory factor to the cleft lip

it could not account for the smaller maxilla observed in Tp63minusminus

embryos We therefore analysed neural crest cell migration by

examining the expression patterns of the neural crest marker Sox10

(Southard-Smith et al 1998) These experiments determined that

Sox10 expression patterns were comparable in the Tp63minus minus embryos

and their wild-type littermates indicating that the migration of

neural crest cells into the developing branchial arches was unaffected

in Tp63minus minus mice (Fig S1)

Tp63minus minus embryos display mesenchymal proliferation defects

To further investigate the pathogenesis of the cleft lip and palate in

the Tp63minus minus mice we analysed cell proliferation in E105ndashE125 mutant

embryos and their wild-type littermates using BrdU incorporation

assays We hypothesised that for the facial processes to form the

correct shape to allow contact and fusion there were likely to be

different proliferation rates in different regions of the facial processes

Thus the medial nasal lateral nasal and maxillary processes were

divided into sub-regions which were analysed individually It proved

impossible to assess proliferation within the epithelia of the facial

processes as it appeared unhealthy and in many areas was detached

from the mesenchyme (Fig 2L) These assays determined that the

mesenchyme within the facial processes of E105 wild-type embryos

Fig1 Expression of Tp63 during facial morphogenesis (AndashD) Whole-mount in situ hybridization (A) At E95 Tp63 is expressed throughout the ectoderm of the facial processes and

the maxillary and mandibular components of the 1047297rst branchial arch (B) At E105 Tp63 is detected in the ectoderm surrounding the nasal pit and in the maxillary processes (C) At

E1175 Tp63 transcripts are identi1047297ed in the leading edges of the mandible and the fused regions of the medial nasal processes (D) Tp63 is expressed intensely in the nasolacrimal

groove at E125 (arrowed) (EndashL)Immunohistochemistryusingthe p63antibody4A4 on coronal sections of wild-typeembryos(E andF) At E105 Tp63 expressionis detectedin basal

cells of the ectoderm in the most distal portions of the medial and lateral nasal processes (E) and throughout the ectoderm of the mandibular and maxillary process (F) (G and H) At

E115 Tp63 expression is con1047297ned to the ectodermal component of the nasal pit (G) and is expressed highly in the fusion region between the medial nasal process and maxillary

process (arrowed) (H) (IndashL) During development of the secondary palate Tp63 staining is detected in the ectoderm throughout the oral cavity from E115 (I) the developing tooth

germs from E125(J)and in thepalatal shelves from E125to E145(JndashL) fnpfrontonasal process mndmandiblemx maxillary process mnpmedial nasalprocesslnp lateral nasal

process p palate t tongue tg tooth germ Scale bars EndashK 200 microm L 100 microm

275HA Thomason et al Developmental Biology 321 (2008) 273ndash 282

8132019 1-s20-S0012160608010221-main


was highly proliferative with the percentage of labelled cells rangingfrom 42 to 58 Although there was no signi1047297cant difference in

proliferation within any region of the facial processes of Tp63minus minus mice

compared to control littermates at this age the lateral nasal processes

and the maxillary processes had signi1047297cantly lower total cell counts

than those of the wild-type littermates (P values=005 Fig S2) The

total cell count in these processes was also signi1047297cantly lower at E115

(P =005 Fig 4) Whenproliferation rates were assessed at E115 three

regions of the facial processes in Tp63minus minus embryos were found to

contain signi1047297cantly fewer BrdU-labelled cells region 4 of the lateral

nasal process (P =0014) region 2 of the medial nasal processes

(P =0014) and region 4 of the maxillary processes (P =0027 Fig 4 L ndash

N) Intriguingly region 4 of the maxillary process is the medial aspect

from which thepalatal shelves emerge late on E11 These data ledus to

extend our studies to the next stage of palate development After

comparisons of the morphology of the wild-type and Tp63minus

minus

embryosat E125 we were unable to 1047297nd any evidence of palatal shelf

outgrowth from the anterior maxillary processes of Tp63minus minus embryos

We therefore divided the mid and posterior palate into medial and

lateral regions At E125 we demonstrated that the posterior palate

contained signi1047297cantly fewer cells predominantly due to a signi1047297-

cantly lower total cell count in the medial aspect (P =005 Fig S3 G)

although both the medial and lateral aspects of the posterior palate

were signi1047297cantly more proliferative (P = 005 Fig S3 H)

Altered signaling in the facial processes of p63minus minus embryos

To analyse the molecular events underlying the facial clefting

observed in Tp63minus minus mice we performed whole-mount in situ

hybridization on E105 to E125 mouse embryos focussing on key

Fig 2 Phenotype of Tp63minusminus mice (AndashD) Tp63minusminus mice exhibit a fully penetrant bilateral cleft lip (B) and complete cleft of the secondary palate (D) (E and F) The phenotype is 1047297rst

apparent at E115 whenin comparisonto wild-type littermates the maxillaryprocesses of Tp63minusminus embryos appearsmaller andthe medialnasalprocesses appearrounded atthe tips

and bulge medially (F) (Gndash J) Coronal sections of E115 embryos reveal small unusually shaped facial processes in Tp63minusminus embryos An epithelial bridge between the medial nasal

processesand themaxillary processesis evident (arrowedin panel J)(KndashN) Developmentof the anterior secondary palate Coronal sections indicate thatin comparisonto wild-type

littermates palatal outgrowth in Tp63minusminus embryos is absent at E125 (L) and rudimentary at E135 (N) (O and P) Ventral views of the craniofacial skeleton of E185 Tp63minusminus embryos

reveal abnormalities of the nasal capsule absence of the premaxilla and palatal processes of the maxilla and palatine bone (asterisk in panel P) (Q and R) In vitro palate culture

Histological analysis of palates dissected from E135 wild-type and Tp63minus minus embryos after culture for 24 h indicated that Tp63minus minus explants fused in the midline with no evidence of an

epithelial seam (arrowed in the wild-type palate Q) mnd mandible mx maxillary process mnp medial nasal process lnp lateral nasal process p palate t tongue ns nasal

septum pmx pre-maxilla Scale bars A and B 1 mm CndashF 500 microm GndashL 300 microm M and N 200 microm


8132019 1-s20-S0012160608010221-main


signaling molecules known to be important during facial morphogen-

esis including members of the Bmp Shh and Fgf families ( Wilkie and

Morriss-Kay 2001 Richman and Lee 2003) At E105 and E115 the

secreted signaling molecule Bmp4 is expressed at low levels in the

epithelia covering the medial and lateral nasal processes and

maxillary and mandibular processes in wild-type embryos (Francis-

West et al 1994) In contrast in Tp63minus minus embryos Bmp4 transcripts

appeared markedly elevated in the epithelium of the caudal region of

the lateral nasal processes at E105 and in the anterior region of the

maxillary processes at E105 and E115 (Fig 5 AndashD) Alterations in Bmp

signaling have previously been shown to affect the expression of Fgf8

(Barlow et al 1999 Shigetani et al 2000) which is expressed in both

complementary and overlapping domains with Bmp4 (Crossley and

Martin 1995) At E105 and E115 Fgf8 expression was detected in the

epithelia of the medial and lateral nasal processes and the maxillary

and mandibular processes of wild-type embryos In Tp63minusminus embryos

however we observed marked changes in Fgf8 expression At E105

Fgf8 transcripts were absent from the epithelia covering the distal

tips of the medial nasal processes the entire lateral nasal processes

and the anterior region of the maxillary processes (Fig 5 E F) AtE115 Fgf8 expression was absent from the epithelia of the anterior

maxillary processes and was down-regulated in the rostral region of

the lateral nasal processes (Fig 5 G H) All of these epithelial regions

overlie areas of the mesenchymein which we identi1047297ed a proliferative

defect (Fig 4 L ndashN) To investigate the downstream consequences of

the loss of Fgf8 expression we examined the expression of the Fgf8

target gene Barx1 (Barlow et al 1999) These assays determined that

the medially located expression domains of Barx1 were reduced in the

mesenchyme of the Tp63minusminus maxillary processes although a more

lateral expression domain was still present in posterior regions (Fig 5

I J) Subsequently we examined the expression pattern of Shh which

is expressed in the ectoderm of the facial primordia (Echelard et al

1993 Hu and Helms 1999) and has been shown to interact with BMP

and Msx1 in regulating mammalian palatogenesis (Zhang et al 2002)

In Tp63minus minus embryos we found that expression of Shh was down-

regulated in the medial nasal processes and roof of the oral cavity and

was absent from the entire maxillary process (Fig 5 K L)

As the major site of altered gene expression appeared to be the

anterior region of the maxillary processes we performed section

in situ hybridization to identify the precise regions affected in

Tp63minus minus embryos These studies con1047297rmed that Bmp4 was present

at much higher levels in the epithelia of Tp63minus minus embryos

particularly in the medial region of the anterior maxillary

processes (Fig 6 A B) corresponding to region 4 in which we

identi1047297ed a proliferative defect in the mesenchyme (Fig 4 N)

Conversely Fgf8 expression was lost from this region (Fig 6 E F)

In the posterior region of the maxillary processes Bmp4 was not

expressed in wild-type embryos nor Tp63minus minus embryos (Fig 6 C D)

Fgf8 expression in the posterior region of the maxillary processesof Tp63minus minus embryos was comparable to that observed in wild-type

littermates (Fig 6 G H) Expression of Barx1 appeared to be

reduced in the mesenchyme of the anterior region of the maxillary

processes of Tp63minusminus embryos but was unaffected in the posterior

region (Fig 6 IndashL)

Expression of Msx1 which is localised in the nasal processes and

anterior maxillary process in wild-type embryos appeared slightly

up-regulated in the maxillary and medial nasal processes of Tp63minusminus

embryos (Fig S4 A B) Expression domains of additional markers

including the Fgf8 target genes Pyst1 (Kawakami et al 2003) Pea3

Erm and Pax3 (Firnberg and Neubuser 2002) which are expressed in

the nasal processes as well as Alx4 Dlx5 and Pax9 were unaltered in

Tp63minusminus embryos in comparison to wild-type controls (Fig S4 and data

not shown) Examination of markers expressed during later stages of palatal development including Satb2 Osr2 Pax9 and Shox2 (FitzPatrick

et al 2003 Lan et al 2004 Yu et al 2005) indicated that although

the anterior region of the Tp63minus minus palate at E125 and E135 exhibited

abnormal growth the anterior to posterior patterning of the palatal

shelves was unaffected for example Shox2 which is expressed

strongly in the mesenchyme of the anterior palate in wild-type

embryos was expressed in the Tp63minus minus rudimentary palatal shelves at

E135 (Fig S4 O P) These data con1047297rm that the altered signaling

events during outgrowth of the facial processes at E105 and E115

result in the absence of the entire anterior region of the secondary

palate during the initial stage of palatogenesis At E135 although the

palatal shelves exhibit rudimentary growth this is insuf 1047297cient for the

palatal shelves to make contact and fuse ultimately resulting in a cleft

of the secondary palate Nevertheless the intrinsic molecular

Fig 3 Cell death in Tp63minusminus embryos (AndashD) Scanning electron microscopy of the facial

processes at E115 (A and C) The epithelia of wild-type embryos appear smooth and are

covered in 1047298attened periderm cells with distinct cellndashcell junctions and central nuclei

(B) In contrast the lateral nasal processes and maxillary processes of Tp63minusminus embryos

exhibit an area of rounded cells covered in 1047297lamentous debris (D) At higher

magni1047297cation the cells appear to be sloughing away from the underlying epithelium

(EndashH) Histological analysis of coronal sections indicatesthat the epitheliumcoveringthe

maxillary processes of E115 Tp63minusminus embryos appears irregular with dark condensed

nuclei (arrowed in panel H) (Panels G and H are higher magni1047297cations views of the

boxed regions in panels E and F) (I and J) Anti-activated caspase 3 immunohistochem-

istry (I) No apoptotic cells are detected in the wild-type embryo whereas numerous

cells in the Tp63minusminus embryos exhibit immunoreactivity (arrowed) con1047297rming that the

cellsare undergoing inappropriate cell death(J) mndmandiblemx maxillary processmnp medial nasal process lnp lateral nasal process n nuclei Scale bars A and B

200 microm C and D 20 microm E and F 200 microm G and H 50 microm I and J 100 microm


8132019 1-s20-S0012160608010221-main


mechanisms which underlie anterior-posterior patterning are unaf-

fected in Tp63minusminus embryos

Discussion

Tp63minus minus mice exhibit abnormal facial morphogenesis

In humans mutations in TP63 have been identi1047297ed in 1047297ve distinct

developmental disorders including EEC and AEC syndromes which

are characterised by varying degrees of limb abnormalities ectoder-

mal dysplasias and facial clefting To dissect the molecular pathogen-

esis of the facial anomalies resulting from mutation of Tp63 we have

analysed Tp63 mutant mice Loss of all Tp63 isoforms in mice results in

severe developmental defects including limb truncations epidermal

defects and facial anomalies (Mills et al 1999 Yang et al 1999)

However with the exception of the epidermal (Koster Huntzinger and

Roop 2002 Keyes et al 2006) and dental anomalies(Laurikkala et al

2006) the phenotype of the Tp63-null mice has not been analysed in

detail In this study we have characterised the fully penetrant bilateral

cleft lip and cleft palate observed in Tp63minusminus embryos and have

determined the underlying mechanisms Development of the upper

lip involves a series of highly co-ordinated genetically controlled

morphogenetic events including outgrowth and expansion of the

facial processes programmed cell death and fusion and subsequent

breakdown of the epithelial seam Perturbation of any of these tightly

controlled steps may result in cleft lip Central to all of these steps is

the establishment of correct facial morphogenesis indeed embryos

derived from the AJ and CLFr strains of mice which have a high

Fig 4 Cell proliferation in the facial processes of Tp63minusminus embryos at E115 (Andash J) Immunohistochemistry of coronal sections using an anti-BrdU antibody The regions of the medial

nasal lateral nasal and maxillary processes were de1047297ned by the dashed lines as illustrated in panels AndashD and divided into regions as illustrated in panels panels Endash J (K) Total

mesenchymal cell counts of the entire facial processes When compared to wild-type littermates (blue columns) Tp63minusminus embryos (yellow columns) have signi1047297cantly fewer cells in

the lateral nasal processes and maxillary processes Proliferation assays indicate that three regions of the facial processes of Tp63minus minus embryos have signi1047297cantly fewer BrdU-labelled

cells region 4 of the lateral nasal process (P =0014) (L) region 2 of the medial nasal process (P =0014) (M) and region 4 of the maxillary process (P =0027) (N) These regionally-

speci1047297c differences are masked when the processes are analysed as a whole Error bars are illustrated an asterisk denotes a signi 1047297cant 1047297nding using a MannndashWhitney U statistical

test mx maxillary process mnp medial nasal process lnp lateral nasal process Scale bars A and B 300 microm C and D 200 microm E ndash J 100 microm


8132019 1-s20-S0012160608010221-main


frequency of spontaneous cleft lip have more prominent and medially

convergent medial nasal processes (Millicovsky et al 1982 Traslerand Ohannessian1983) We observed that the medial nasal processes

of Tp63minusminus embryos at E115 were extremely rounded at their leading

edges and were distinctly more prominent Total cell counts revealed

that the lateral nasal processes and the developing maxillary

processes of Tp63minusminus embryos were signi1047297cantly smaller than those

of the wild-type littermates at E105 and E115 We excluded the

possibility that these defects arise from altered neural crest cell

migrationor from increased cell death in themesenchymeof thefacial

processes and identi1047297ed three areas of signi1047297cantly reduced mesench-

ymal cell proliferation at E115 As Tp63 is expressed in the basal cells

of the epithelia covering the facial processes these data indicate that

the loss of Tp63 signi1047297cantly disrupts epithelial signaling during

outgrowth of the facial processes resulting in two distinct but related

facial defects cleft lip and cleft secondary palate

The origin of the cleft lip in Tp63minus minus mice

During the critical stages of outgrowth of the facial processes in

Tp63minus minus embryos we observed marked changes in the expression levels

of key signaling molecules including Bmp4 and Fgf8 The BMPs are a

group of secreted signaling molecules of the TGFβ superfamily which

regulate diverse developmental processes including cell proliferation

apoptosis differentiation and tissue morphogenesis (Wozney 1998)

Regulation of BMP signaling is complex forexample BMPs frequently

stimulate transcription of their own antagonists (Merino et al 1998

Stottmann et al 2001 Laurikkala et al 2003) In the chick Ashique

and co-workers observed that BMP signaling is required to stimulate

proliferation and outgrowth of branchial arch mesenchyme but also

observed that the chick globular process and anterior maxillary

prominence respond to increased BMP levels by programmed cell

death (Ashique et al 2002) This is particularly intriguing as we found

Fig 5 Altered signalling in thefacial processesof Tp63minusminus embryos detected by whole-mount insitu hybridization Allimages areventral viewsexcept E andF which arelateralviews

(AndashD) Bmp4 transcripts are up-regulated in the caudal region of the lateral nasal processes of Tp63minusminus embryos at E105 (B red arrow) and in the anterior region of the maxillary

processes at E105 and E115 (black arrows) (B and D) (E and F) At E105 Fgf8 transcripts are absent from the lateral nasal processes the tips of the medial nasal processes (arrowed)

andthe anteriorregionof themaxillary processes(asterisk)(G andH) AtE115whileexpressionof Fgf8 is clearly visible in theposterior regionof themaxillary processesexpression

in the anterior regionremains down-regulatedIn addition the lateral nasalprocesses exhibit patchy expressionof Fgf8 in theepithelium surrounding thenasalpits (asteriskin panel

H) (I and J) The medially located expression domain of the Fgf8-target gene Barx1 is down-regulated in the anterior region of the maxillary processes of Tp63minusminus embryos (arrowed)

although transcripts are present posteriorly (K and L) Shh transcripts are absent from the anterior region of the maxillary processes of E115 Tp63minus minus embryos (arrowed) mnd

mandible mx maxillary process mnp medial nasal process lnp lateral nasal process


8132019 1-s20-S0012160608010221-main


wide-spread apoptotic cell death in the epithelia covering the caudal

region of the lateral nasal processes and most rostral region of themaxillary processes in Tp63minusminus embryos at E115 These areas of cell

death correlated precisely with regions of increased Bmp4 expression

(compare Fig 3 D and Fig 5 D) During development of the upper lip

apoptosis is tightly regulated with dying cells only being observed in

the epithelia of the fusing medial and lateral nasal processes and

maxillary processes at E115 ( Jiang et al 2006) This tight regulation

ensures that apoptosis does not occur before the facial processes have

made contact Examination of chick facial primordia using scanning

electron microscopy has indicated that the lateral nasal processes

exhibit a region of apoptosis on the medial surface that likely

correlates with elevated BMP4 expression (Cox 2004) As several

studies have linkedBMP signaling with cell death (Barlow and Francis-

West 1997 Graham et al 1994 Zhou and Niswander 1996) we

conclude that theincreased cell death observed in Tp63minus

minus

mutants is adirect consequence of elevated Bmp signaling (Fig 5 AndashD) We also

observed loss of Fgf8 signaling in the epithelia of the nasal and

maxillary processes and identi1047297ed proliferative defects in the

mesenchyme underlying these regions in Tp63-null embryos This is

extremely signi1047297cant as Fgf8 expression within the facial processes

has been correlated with high levels of cell proliferation and

expansion of the frontonasal mass mesenchyme (McGonnell et al

1998 Bachler and Neubuumlser 2001) In addition ectopic application of

Fgf8 has been shown to be capable of substituting for the facial

ectoderm in order to stimulate proliferation promote cell survival and

regulate gene expression in the facial mesenchyme (Firnberg and

Neubuser 2002) Conversely stripping of the epithelium decreases

proliferation (Hu and Helms 1999 Schneider et al 2001) Our results

therefore provide evidence that the cleft lip observed in Tp63minusminus

mice

results from a combination of inappropriate cell death in the lateral

nasal process and maxillary processes together with reducedproliferation in the medial and lateral nasal processes and maxillary

processes Interestingly there are few genetic models of cleft lip with

or without cleft palate those that have been reported exhibit a wide

variety in the penetrance of clefting ( Juriloff and Harris 2008) The

fullypenetrant cleft lip phenotype characterised hereis reminiscent of

that observed in mice with conditional ablation of Bmpr1a signaling

(Liu et al 2005) Nestin creBmpr1a mice displayed elevated apoptosis

not only in the epithelium of the medial nasal process but also

extending into the underlying mesenchyme (Liu et al 2005) Whether

the cleft lip arises from increased Bmp signaling as we have identi1047297ed

in our study or loss of Bmp signaling it is clear that the emerging

network linking Tp63 and BMP signaling which has also been

documented in other model organisms (Bakkers et al 2002) and

developmental systems (Laurikkala et al 2006) is essential in theregulation of proliferation and apoptosis in the developing facial

processes

The altered signaling events in the maxillary process underlie the cleft

secondary palate in Tp63minus minus mice

Our section in situ hybridization data allowed us to demonstrate

that the altered signaling events in the developing facial processes

also had a deleterious affect upon the secondary palate At E115

Bmp4 which is expressed in the anterior palate only (Zhang et al

2002) was markedly up-regulated in the epithelia of the maxillary

processes of Tp63minusminus embryos Fgf8 expression by comparison was

lost from this region in Tp63minusminus embryos We also observed a

signi1047297cant mesenchymal proliferative defect in the anterior medial

Fig 6 Altered gene expression in the anterior maxillary process of Tp63minusminus embryos (AndashD) At E115 section in situ hybridization on coronal sections indicates that Bmp4 transcripts

are up-regulated in the anterior region of the maxillary processes speci1047297cally in the medial region from which the palatal shelves originate (arrowed in panel B) (C and D) Bmp4

transcripts are not present in the posterior region of the maxillary processes of wild-type or Tp63minusminus embryos (EndashH) Fgf8 transcripts are detected in the anterior region of the

maxillary processes of wild-type embryos speci1047297cally in the medial region (E) but are absent from this region in Tp63minusminus embryos (arrowed in panel F) (G and H) Fgf8 expression

appears to be unalteredin theposterior regionof Tp63minusminus embryos (IndashL) Expressionof the Fgf8 target gene Barx1 is reduced in theanteriorregion of themaxillary processesof Tp63minusminus

embryos (J) but unaltered posteriorly (L) mnd mandible mx maxillary process mnp medial nasal process lnp lateral nasal process Scale bars 200 microm


8132019 1-s20-S0012160608010221-main


region of the developing maxillary processes which subsequently

affected the initial stages of palatogenesis In the posterior region of

the maxillary processes where Bmp4 is not expressed Fgf8 signaling

and consequently growth and patterning in the posterior region of

the palate in Tp63minus minus embryos appeared to be unaffected While

expression of Fgf8 has not been reported for any of the later stages of

palatal growth we postulate that expression of Fgf8 in the maxillary

processes at E115 acts to initiate palatal shelf outgrowth This signal is

de1047297

cient in Tp63-null mice leading to a lack of the anterior palate atE125 and ultimately results in a cleft secondary palate Tp63-null mice

also exhibited loss of Barx1 expression which has previously been

shown to result from perturbation of BMPFgf8 signaling (Barlow et

al 1999) and down-regulation of Shh which is essential for the

survival and proliferation of facial mesenchyme cells (Hu and Helms

1999 Ahlgren and Bronner-Fraser 1999 Jeong et al 2004) and has

important roles in both tooth germ and palate development (Zhang et

al 2000 2002) Taken together our data uncover a crucial role for

Tp63 in the regulation of Bmp Shh and Fgf8 signaling during facial

development In this regard it is interesting to note that a recentstudy

of Tp63-bound genes included those involved in cell adhesion

proliferation and cell death and included members of the Notch

Wnt and Tgf β signaling pathways including SMO BMP1 BMP 7

TGFβ1 and TGFβR3 (Yang et al 2006) Exactly how Tp63 regulates the

expression of Bmp4 Fgf8 or Shh during facial development is currently

unknown In this regard however chromatin immunoprecipitation

studies have indicated that Tp63 binds directly to Shh whose

expression is induced in response to overexpression of Tp63 in vitro

and is down-regulated in 1047297broblasts extracted from Tp63minusminus mice

(Caserta et al 2006) In addition previous reports suggested that Fgf8

transcripts were severely down-regulatedabsent in Tp63 mutant limb

buds (Yang et al 1999 Mills et al 1999) while other studies

demonstrated both Fgf8 and Shh expression in the Tp63 mutant

dental lamina (Laurikkala et al 2006) To understand fully the role of

Tp63 it will be important to determine how Tp63 regulates its target

genes during facial dental and limb development versus its role in

maintaining tissue-speci1047297c functions in the ldquostem cell nicherdquo or in

preventing differentiation during epidermal development (Senoo

et al 2007)Recently analysis of the mechanisms underlying cleft lip and

palate in knockout mice has helped to de1047297ne the genetic pathways

governing fusion of the lip and palate Simultaneously analysisof DNA

samples from syndromic cases of cleft lip and palate has identi1047297ed

previously uncharacterised genes such as IRF6 in which mutations

cause orofacial clefting (Kondo et al 2002) In addition analysis of

DNA samples from non-syndromic cleft lip and palate patients has

indicated minor roles for mutations in MSX1 FOXE1 GLI2 MSX2 SKI

and SPRY2 in the susceptibility for orofacial clefting (Riley et al 2007)

Furthermore polymorphisms in IRF6 have been found to be strongly

associated with non-syndromic cleft lip and palate and account for

approximately 12 of clefting (Zucchero et al 2004) Identi1047297cation of

further Tp63 targets involved in controlling the balance between

apoptosis and proliferation during outgrowth of the facial processesmay reveal important information that furthers our knowledge of

facial morphogenesis and how this is perturbed in orofacial clefting

Acknowledgments

We thank Frank McKeon for the Tp63 knockout mice and the 4A4

antibody Karin Nylander for the ΔNp63 antibody Paul Sharpe Paul

Trainor Rulang Jiang and Denis Headon for generously providing

cDNA probes We thank Helen Worthington for statistical advice and

Les Lockey of the University of Manchester Electron Microscopy Unit

for invaluable advice We con1047297rm that there is no con1047298ict of interest

associated with this work We thank the Medical Research Council

(G0400264 to JD G0400955 to MJD) and the National Institutes of

Health (P50-DE016215 to MJD) for funding this work

Appendix A Supplementary data

Supplementary data associated with this article can be found in

the online version at doi101016jydbio200806030

References

Ahlgren SC Bronner-Fraser M 1999 Inhibition of Sonic hedgehog signaling in vivoresults in craniofacial neural crest cell death Curr Biol 18 1304ndash1314

Ashique AM Fu K Richman JM 2002 Endogenous bone morphogenetic proteinsregulate outgrowth and epithelial survival during avian lip fusion Development129 4647ndash4760

Bachler M Neubuumlser A 2001 Expression of members of the Fgf family and theirreceptors during midfacial development Mech Dev 100 313ndash316

Bakkers J Hild M Kramer C Furutani-Seiki M Hammerschmidt M 2002 Zebra1047297shDeltaNp63 is a direct target of Bmp signaling and encodes a transcriptionalrepressor blocking neural speci1047297cation in the ventral ectoderm Dev Cell 2617ndash627

Barlow AJ Francis-West PH 1997 Ectopic application of recombinant BMP-2 andBMP-4 can change patterning of developing chick facial primordia Development124 391ndash398

Barlow AJ Bogardi JP Ladher R Francis-West PH 1999 Expression of chick Barx-1and its differential regulation by FGF-8 and BMP signaling in the maxillaryprimordia Dev Dyn 214 291ndash302

Caserta TM Kommagani R Yuan Z Robbins DJ Mercer CA Kadakia MP 2006p63 overexpression induces the expression of sonic hedgehog Mol Cancer Res 4759ndash768

Celli J Duijf P Hamel BC Bamshad M Kramer B Smits AP Newbury-Ecob RHennekam RC Van Buggenhout G van Haeringen A et al 1999 Heterozygousgermline mutationsin thep53 homolog p63are thecauseof EECsyndrome Cell 99143ndash153

Cox TC 2004 Taking it to the max the genetic and developmental mechanismscoordinating midfacial morphogenesis and dysmorphology Clin Genet 65163ndash176

Crossley PH Martin GR 1995 The mouse Fgf8 gene encodes a family of polypeptidesand is expressed in regions that direct outgrowth and patterning in the developingembryo Development 121 439ndash451

DixonJ Brakebusch CFaumlssler RDixon MJ2000 Increasedlevels ofapoptosis in theprefusion neural folds underlie the craniofacial disorder Treacher Collinssyndrome Hum Mol Genet 9 1473ndash1480

Echelard Y Epstein DJ St-Jacques B Shen L Mohler J McMahon JA McMahonAP1993 Sonic hedgehog a member of a family of putative signaling molecules isimplicated in the regulation of CNS polarity Cell 75 1417ndash1430

Ferguson MW Honig LS Slavkin HC 1984 Differentiation of cultured palatalshelves from alligator chick and mouse embryos Anat Rec 209 231ndash249

Firnberg N Neubuser A 2002 FGF signaling regulates expression of Tbx2 Erm Pea3

and Pax3 in the early nasal region Dev Biol 247 237ndash250FitzPatrick DR Carr IM McLaren L Leek JP Wightman P Williamson K

Gautier P McGill N Hayward C Firth H et al 2003 Identi1047297cation of SATB2 asthe cleft palate gene on 2q32-q33 Hum Mol Genet 12 2491ndash2501

Francis-West PH Tatla T Brickell PM 1994 Expression patterns of the bonemorphogenetic protein genes Bmp-4 and Bmp-2 in the developing chick facesuggest a role in outgrowth of the primordia Dev Dyn 201 168ndash178

Ghioni P Bolognese F Duijf PH Van Bokhoven H Mantovani R Guerrini L 2002Complex transcriptional effects of p63 isoforms identi1047297cation of novel activationand repression domains Mol Cell Biol 22 8659ndash8668

Graham A Francis-West P Brickell P Lumsden A 1994 The signalling moleculeBMP4 mediates apoptosis in the rhombencephalic neural crest Nature 372684ndash686

Hu D Helms JA 1999 The role of sonic hedgehog in normal and abnormalcraniofacial morphogenesis Development 126 4873ndash4884

Jeong J Mao J Tenzen T Kottmann AH McMahon AP 2004 Hedgehog signaling inthe neural crest cells regulates the patterning and growth of facial primordiaGenes Dev 18 937ndash951

Jiang R Bush JO Lidral AC 2006 Development of the upper lip Morphogenetic andmolecular mechanisms Dev Dyn 235 1152ndash1166

Juriloff DM Harris MJ 2008 Mouse genetic models of cleft lip with o r without cleftpalate Birth Defects Res A Clin Mol Teratol (Epub ahead of print)

Kawakami Y Rodriguez-Leon J Koth CM Buscher D Itoh T Raya A Ng JKEsteban CR Takahashi S Henrique D et al 2003 MKP3 mediates the cellularresponse to FGF8 signalling in the vertebrate limb Nat Cell Biol 5 513ndash519

KeyesWM VogelH Koster MI Guo XQi Y Petherbridge KM RoopDR BradleyAMills AA 2006 p63heterozygous mutantmiceare notproneto spontaneous orchemically induced tumors Proc Natl Acad Sci USA 103 8435ndash8440

Knight AS Schutte BCJiang R Dixon MJ2006 Developmental expressionanalysisof the mouse and chick orthologues of IRF6 The gene mutated in Van der Woudesyndrome Dev Dyn 235 1441ndash1447

Kondo S Schutte BC Richardson RJ Bjork BC Knight AS Watanabe YHoward E de Lima RL Daack-Hirsch S Sander A et al 2002 Mutations inIRF6 cause Van der Woude and popliteal pterygium syndromes Nat Genet 32285ndash289

Koster MI Huntzinger KA Roop DR 2002 Epidermal differentiation transgenicknockout mouse models reveal genes involved in stem cell fate decisions and

commitment to differentiation J Investig Dermatol Symp Proc 7 41ndash

45


8132019 1-s20-S0012160608010221-main


Lan Y Ovitt CE Cho ES Maltby KM Wang Q Jiang R 2004 Odd-skipped related2 (Osr2) encodes a key intrinsic regulator of secondary palate growth andmorphogenesis Development 131 3207ndash3216

Laurikkala J Kassai Y Pakkasjarvi L Thesleff I Itoh N 2003 Identi 1047297cation of asecreted BMP antagonist ectodin integrating BMP FGF and SHH signals from thetooth enamel knot Dev Biol 264 91ndash105

Laurikkala J Mikkola ML James M Tummers M Mills AA Thesleff I 2006 p63regulates multiple signalling pathways required for ectodermal organogenesis anddifferentiation Development 133 1553ndash1563

Liu W Sun XBraut AMishina Y Behringer RR Mina MMartin JF 2005 Distinctfunctions for Bmp signaling in lip and palate fusion in mice Development 132

1453ndash

1461McGonnell IM Clarke JD Tickle C 1998 Fate map of the developing chick faceanalysis of expansion of facial primordia and establishment of the primary palateDev Dyn 212 102ndash118

McGrath JA Duijf PH Doetsch V Irvine AD de Waal R Vanmolkot KRWessagowit V Kelly A Atherton DJ Grif 1047297ths WA et al 2001 HayndashWellssyndrome is caused by heterozygous missense mutations in the SAM domain of p63 Hum Mol Genet 10 221ndash229

Merino R Ganan Y Macias D Economides AN Sampath KT Hurle JM 1998Morphogenesis of digits in the avian limb is controlled by FGFs TGFbetas andnoggin through BMP signaling Dev Biol 200 35ndash45

Millicovsky G Ambrose LJ Johnston MC 1982 Developmental alterationsassociated with spontaneous cleft lip and palate in CLFr mice Am J Anat 16429ndash44

Mills AA Zheng B Wang XJ Vogel H Roop DR Bradley A 1999 p63 is a p53homologue required for limb and epidermal morphogenesis Nature 398 708ndash713

Murray JC 2002 Geneenvironment causes of cleft lip andor palate Clin Genet 61248ndash256

Richardson RJ Dixon J Malhotra S Hardman MJ Knowles L Boot-Handford RP

Shore P Whitmarsh A Dixon MJ 2006 IRF6 is a key determinant of thekeratinocyte proliferationdifferentiation switch Nat Genet 38 1329ndash1334

Richman JM Lee S 2003 About face signals and genes controlling jaw patterningand identity in vertebrates BioEssays 25 554ndash568

Riley BM Mansilla MA Ma J Daack-Hirsch S Maher BS Raffensperger LMRusso ET Vieira AR Dodeacute C Mohammadi M et al 2007 Impaired FGFsignaling contributes to cleft lip and palate Proc Natl Acad Sci USA 1044512ndash4517

Schliekelman P Slatkin M 2002 Multiplex relative risk and estimation of the numberof loci underlying an inherited disease Am J Hum Genet 71 1369ndash1385

Schneider RA Hu D Rubenstein JL Maden M Helms JA 2001 Local retinoidsignaling coordinates forebrain and facial morphogenesis by maintaining FGF8 andSHH Development 128 2755ndash2767

Senoo M Pinto F Crum CP McKeon F 2007 p63 is essential for the proliferativepotential of stem cells in strati1047297ed epithelia Cell 129 523ndash536

Shigetani Y Nobusada Y Kuratani S 2000 Ectodermally derived FGF8 de1047297nes themaxillomandibular region in the early chick embryo epithelialndashmesenchymal

interactions in the speci1047297cation of the craniofacial ectomesenchyme Dev Biol 22873ndash85

Southard-Smith EM Kos L Pavan WJ 1998 Sox10 mutation disrupts neural crestdevelopment in Dom Hirschsprung mouse model Nat Genet 18 60ndash64

Stottmann RW Anderson RM Klingensmith J 2001 The BMP antagonists Chordinand Noggin have essential but redundant roles in mouse mandibular outgrowthDev Biol 240 457ndash473

Trasler DG Ohannessian L 1983 Ultrastructure of initial nasal process cell fusionin spontaneous and 6-aminonicotinamide-induced mouse embryo cleft lipTeratology 28 91ndash101

van Bokhoven H Brunner HG 2002 Splitting p63 Am J Hum Genet 71 1ndash13

Wilkie AO Morriss-Kay GM 2001 Genetics of craniofacial development andmalformation Nat Rev Genet 2 458ndash468Wilkinson DG Bailes JA McMahon AP 1987 Expression of the proto-oncogene int-

1 is restricted to speci1047297c neural cells in the developing mouse embryo Cell 5079ndash88

Wozney JM 1998 The bone morphogenetic protein family multifunctionalcellular regulators in the embryo and adult Eur J Oral Sci 106 (Suppl 1)160ndash166

Yang A Kaghad M Wang Y Gillett E Fleming MD Dotsch V Andrews NCCaput D McKeon F 1998 p63 a p53 homolog at 3q27ndash29 encodes multipleproducts with transactivating death-inducing and dominant-negative activitiesMol Cell 2 305ndash316

Yang A Schweitzer R Sun D Kaghad M Walker N Bronson RT Tabin CSharpe A Caput D Crum C McKeon F 1999 p63 is essential for regenerativeproliferation in limb craniofacial and epithelial development Nature 398714ndash718

Yang A Zhu Z Kapranov P McKeon F Church GM Gingeras TR Struhl K 2006Relationships between p63 binding DNA sequence transcription activity andbiological function in human cells Mol Cell Biol 24 593ndash602

Yu L Gu S Alappat S Song Y Yan M Zhang X Zhang G Jiang Y Zhang ZZhang Y Chen Y 2005 Shox2-de1047297cient mice exhibit a rare type of incompleteclefting of the secondary palate Development 132 4397ndash4406

Zhang Y Zhang Z Zhao X Yu X Hu Y Geronimo B Fromm SH ChenYP 2000 A new function of BMP4 dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ Development 1271431ndash1443

Zhang ZSong Y Zhao XZhangX Fermin CChen Y 2002 Rescueof cleft palateinMsx1-de1047297cient mice by transgenic Bmp4 reveals a network of BMP and Shhsignaling in the regulation of mammalian palatogenesis Development 1294135ndash4146

Zhou H Niswander L 1996 Requirement for BMP signaling in interdigital apotosisand scale formation Science 272 738ndash741

Zucchero TM Cooper ME Maher BS Daack-Hirsch S Nepomuceno B Ribeiro LCaprau D Christensen K Suzuki Y Machida J et al 2004 Interferon regulatoryfactor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate N Engl JMed 351 769ndash780


8132019 1-s20-S0012160608010221-main


knockout mice (Laurikkala et al 2006) the nature of the craniofacial

anomalies and their underlying developmental pathogenesis has not

been characterised

Development of the craniofacial complex involves a complex series

of events that requires the close co-ordination of programs for cell

migration growth differentiation and apoptosis Neural crest cells

which delaminate from the neural folds contribute to and migrate

through the mesenchymal tissue into the craniofacial region where by

thefourth week of embryonicdevelopment in man they participate inthe formation of the frontonasal prominence the paired maxillary

processes and the paired mandibular processes surrounding the

primitive oral cavity Theformation of the nasal placodes by the end of

the fourth week of embryogenesis divides the lower portion of the

frontonasal prominence into paired medial and lateral nasal pro-

cesses Towards the end of the sixth week of development merging of

the medial nasal processes with one another and with the maxillary

processes on each side leads to the formation of the upper lip and the

primary palate

The 1047297rst sign of overt development of the secondary palate occurs

during the sixth week of embryogenesis with outgrowth of paired

palatal shelves from the maxillary processes The palatal shelves

initially grow vertically down the sides of the tongue but during the

seventh week of development they elevate to a horizontal position

above the tongue contact and fuse to form a midline epithelial seam

which subsequently degenerates to allow mesenchymal continuity

across the palate These fusion processes are complete by the tenth

week of embryogenesis The development of the mammalian

secondary palate thereby divides the oronasal space into separate

oral and nasal cavities

To dissect the role of Tp63 during craniofacial development and to

determine why mutation of TP63 contributes to orofacial clefting in

humans we analysed the expression of Tp63 and characterised the

craniofacial defects in Tp63-null mice We demonstrate that loss of

Tp63 resulted in increased Bmp signaling in several areas of the facial

processes Consequently down-regulation of the key signaling

molecules Fgf8 and Shh led to reduced mesenchymal cell prolifera-

tion regional growth defects and altered morphogenesis of the nasal

processes resulting in a bilateral cleft of the upper lip We alsodemonstrate that a proliferative defect in the mesenchyme of the

maxillary processes at embryonic day 115 leads to absence of the

entire anterior region of the secondary palate during the initial stage

of palatogenesis Our results provide molecular evidence that Tp63-

mediated epithelial-derived signals are crucial during the outgrowth

and modeling of the facial processes and in the initial stages of palatal

growth

Materials and methods

Tissue preparation scanning electron microscopy

immunohistochemistry and in situ hybridisation

The generation and genotyping of the Tp63minus

minus

mice have beendescribed previously (Yang et al 1999) Mice were housed in

accredited animal facilities at the University of Manchester All

procedures were approved by local Ethical Review Committees and

are licensed under the Animal (Scienti1047297c Procedures) Act 1986 issued

by the Home Of 1047297ce Her Majestys Government London United

Kingdom BALBc-derived Tp63minusminus embryos were obtained by hetero-

zygous matings the morning of the vaginal plug being considered

E05 Tissues were 1047297xed in either 4 paraformaldehyde or Bouins

reagent and processed for histological examination scanning electron

microscopy or immunohistochemistry using standard protocols

(Dixon et al 2000 Knight et al 2006) Antibodies were obtained

from Santa Cruz (SC-8344 SC-8608 California USA) and R and D

systems (activated caspase 3 AF835 Minneapolis USA) The whole-

mount and section in situ hybridization protocols have been described

previously (Knight et al 2006 Wilkinson et al 1987) During all

assays wild-type and Tp63-null embryos were treated identically In

particular during whole-mount in situ hybridization assays to

compare gene expression levels embryos of both genotypes were

incubated in the detection reagent for identical periods of time

Organ culture and cell proliferation assays

E135 palatal shelves were dissected from Tp63 null mice and wild-type littermates in sterile Dulbeccos MENNUT Mix F-12 (Ham)

containing 005 mM L -glutamate 025 mM glycine and 15 mM Hepes

with 1 penicillinndashstreptomycin and freshly added L -ascorbic sodium

salt at 40 microgml 1047297nal concentration and cultured as described

previously (Ferguson et al 1984) Cell proliferation assays were

performed as described previously (Richardson et al 2006) Three

non-serial sections were counted from three Tp63minus minus and three wild-

type embryos from one or more litters All assays were analysed by

two individuals and the mean value recorded MannndashWhitney U

statistical tests were performed using SPSS 130

Results

ΔNp63α is widely expressed during facial development

To characterise the expression domains of Tp63 during facial

development we performed whole-mount in situ hybridization assays

on E95ndashE125 mouse embryos These studies indicated that Tp63

transcripts were widely expressed during facial development and

appeared at particularly high levels in the epithelia of the maxillary

and mandibular components of the 1047297rst branchial arch at E95 the

lateral and medial nasal processes as they fused at E105 the leading

edges of the upper lip and mandible at E1175 and the nasolacrimal

groove at E125 (Fig 1 AndashD) Immunostaining using a panel of

antibodies directed against all protein isoforms of Tp63 (pan-Tp63

4A4) ΔN-Tp63 and Tp63α isoforms were identical con1047297rming

previous observations that the main embryonic isoform is ΔN-

Tp63α (Yang et al 1999 Laurikkala et al 2006) Similar experiments

using a TA-Tp63-speci1047297c antibody failed to detect these isoforms atany of the embryonic stages examined These observations were

con1047297rmed by RT-PCR and real-time PCR (data not shown) We

observed strong staining for ΔN-Tp63α in all basal cells of the

ectoderm of the facial processes from E95 (data not shown) Initially

we detected intense Tp63 staining in basal cells of the ectoderm in the

most distal regions of the medial and lateral nasal processes at E105

(Fig 1E) Subsequently this expression domain was con1047297ned to only

the ectodermal component of the nasal processes at E115 ( Fig 1G)

Staining was present in all basal cells of the ectoderm covering the

maxillary processes during E10ndashE12 (Fig 1 FH) and at all stages of

development of the secondary palate from E115 to E145 (Fig 1 IndashL)

and at E155 (data not shown)

Tp63minus

minus

embryos display bilateral cleft lip and cleft palate

Tp63minus minus embryos exhibited a fully penetrant bilateral cleft lip and a

complete cleft of the secondary palate (n =137) (Fig 2 AndashD) This

phenotype was 1047297rst apparent at E115 when the maxillary processes

were observed to be smaller in Tp63minus minus mice than in Tp63+minus or Tp63++

littermates (Fig 2 E F) In addition the medial nasal processes had a

more rounded appearance and failed to meet the lateral nasal

processes in the most distal regions (Fig 2F) Histological analysis of

a developmental series of Tp63minusminus embryos determined that the facial

processes were abnormally shaped andthe lateral nasal processes and

maxillary processes appeared smaller than those of their wild-type

littermates (Fig 2 Gndash J) The medial and lateral nasal processes

remained apart in all Tp63minusminus embryos examined although serial

sections through Tp63-null embryos revealed that the posterior


8132019 1-s20-S0012160608010221-main


















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minus















mice
















processes


















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de1047297












































Acknowledgments













References






























45


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1453ndash
































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minus




palatal shelves















































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minus























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Discussion































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minus















mice
















processes


















8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main

















minus























8132019 1-s20-S0012160608010221-main



























































































8132019 1-s20-S0012160608010221-main




Discussion































8132019 1-s20-S0012160608010221-main









































8132019 1-s20-S0012160608010221-main

















minus















mice
















processes


















8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main



























































































8132019 1-s20-S0012160608010221-main




Discussion































8132019 1-s20-S0012160608010221-main









































8132019 1-s20-S0012160608010221-main

















minus















mice
















processes


















8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main




Discussion































8132019 1-s20-S0012160608010221-main









































8132019 1-s20-S0012160608010221-main

















minus















mice
















processes


















8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main









































8132019 1-s20-S0012160608010221-main

















minus















mice
















processes


















8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main

















minus















mice
















processes


















8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main










de1047297












































Acknowledgments













References






























45


8132019 1-s20-S0012160608010221-main






1453ndash
































8132019 1-s20-S0012160608010221-main






1453ndash
































1-s2.0-s0012160608010221-main

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