Molecular basis of cleft palates in mice

Abstract

Cleft palate, including complete or incomplete cleft palates, soft palate clefts, and submucosal cleft palates, is the most frequent congenital craniofacial anomaly in humans. Multifactorial conditions, including genetic and environmental factors, induce the formation of cleft palates. The process of palatogenesis is temporospatially regulated by transcription factors, growth factors, extracellular matrix proteins, and membranous molecules; a single ablation of these molecules can result in a cleft palate in vivo. Studies on knockout mice were reviewed in order to identify genetic errors that lead to cleft palates. In this review, we systematically describe these mutant mice and discuss the molecular mechanisms of palatogenesis.

Core tip: Cleft lip and/or palate is one of the most frequent congenital craniofacial anomalies observed. Multifactorial conditions, including genetic and environmental factors, induce the formation of cleft palates. We screened knockout mice with cleft palate phenotypes and observed approximately 180 mice with the anomaly. In order to understand the molecular regulatory mechanisms of palatogenesis and to identify genetic errors that lead to cleft palates, we aimed to review studies performed using knockout mice with cleft palates.

INTRODUCTION

Cleft lip and/or palate (CL/P) is the most frequent congenital craniofacial anomaly observed in humans, with an incidence of 1 per 700 births worldwide[1]. Furthermore, 55% of the patients with CL/P are reported to have a multiple malformation syndrome[2]. CL/P involves a multifactorial etiology, both genetic and environmental. Teratogens that cause CL/P in humans include common environmental exposures, such as alcohol, smoking, infections, dioxin, estrogen, retinoic acid, and altitude (reviewed by Murray[1]). The offspring of parents with CL/P present a higher incidence of CL/P than those without a family history[1]. Gene-environment interactions for non-syndromic CL/P have also been reported[1]. Cleft palate (CP) cases include complete CP, incomplete CP, and soft palate clefts. The mildest form of cleft palates is the soft palate cleft or bifid uvula because the initial palatal fusion occurs in the anterior region of secondary palatal shelves. Incomplete CP and soft palate clefts can manifest together with submucosal CP. This review focuses on studies performed using knockout mice with CP, aiming to clarify the molecular regulatory mechanisms of palatogenesis and to identify genetic errors underlying mammalian cleft palates.

MAMMALIAN PALATOGENESIS

The palate is formed with the primary and secondary palate. The primary palate is derived from the frontonasal prominence and becomes a small anterior part of the adult hard palate. The secondary palatal shelves extend bilaterally from the internal aspects of the maxillary prominences and will become the adult hard and soft palates. The process of palatogenesis consists of several stages: palatal shelf formation, elevation, and midline fusion of the palatal shelves (Figure 1). The secondary palatal shelves develop between embryonic day (E) 11.5 and 12.5 in the mouse embryo (Figure 1A). At E13.5, the palatal shelves grow downward on each side of the tongue (Figure 1B). As the jaws develop, the tongue descends and the palatal shelves elevate to a horizontal position above the dorsum of the tongue (E14). Continuing their growth, the bilateral palatal shelves meet at the midline and fuse between E14.5 and E15.5 (Figure 1C).

Figure 1.

Palatogenesis in mice. Hematoxylin and eosin staining of coronal sections of the head of a wild-type mouse at embryonic day (E) 12.5 (A), E13.5 (B), and E14.5 (C, D). A: Mouse palatal shelves (p) develop from the maxillary prominences; B: By E13.5, the palatal shelves grow downward on each side of the tongue (t); C and D: At E14.5, the palatal shelves face each other along the midline above the tongue and fuse, separating the oral cavity (oc) from the nasal cavity (nc). The arrow in (D) indicates the medial edge epithelial (MEE) cells that constitute the midline epithelial seam. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Texas Southwestern Medical Center and Tokyo Medical and Dental University. mes: Mesenchyme; epi: Epithelium.

The palatal shelves are composed of the neural crest-derived mesenchyme and ectoderm-derived epithelia, which cover the palatal mesenchyme (Figure 1D). Both elevation and fusion of the secondary palatal shelves occur in the midline from anterior to posterior. The secondary palatal shelves also fuse with the primary palate, separating the oral and nasal cavities. The anterior two-thirds of the palate forms the hard palate with neural crest-derived palatal bones (Figure 2A). The posterior one-third of the palate forms the bone-free soft palate and is involved in the palatopharyngeal sealing. Disruption at any stage of the formation, elevation, growth, or fusion of the secondary palatal shelves results in CP[3].

Figure 2.

View of the palate from wild-type and Tbx1^-/- mice with cleft palates. A-D: Ventral view of the maxilla of newborn wild-type (A) and Tbx1^-/- mice with cleft palates (B-D). The palate consists of the primary palate (pp) and the secondary palate (sp), which consists of a hard palate (hp) and a soft palate (sp) (A). Tbx1^-/- mice show complete cleft palate (CP) (arrowheads in B), incomplete CP (dashed line in C), and soft palate clefts associated with anterior CP (dashed line in D). An anterior CP (an arrow in D) is present at the junction between the primary palate and secondary palate, while the posterior palate remains fused; E-G: Ventral view of the cranial base of newborn wild-type (E) and Tbx1^-/- mice (F, G) stained with alizarin red for mineralized bone and alcian blue for cartilage. Fusion of the bilateral palatal bones (pa) observed in the wild-type (dashed line in E) is absent in Tbx1^-/- mice (dashed lines in F, G). The palatal shelves in the maxilla (mx) of Tbx1^-/- mice with complete CP (oval dashed line in F) failed to grow toward the midline. Note the visible presphenoid bone (ps) associated with CP (F, G). Modified and used with permission from Funato et al[4]. ns: Nasal septum; pt: Pterygoid bone.

MOUSE MODELS FOR STUDYING THE MOLECULAR MECHANISMS OF PALATAL DEVELOPMENT

Major advances have been achieved regarding the molecular mechanisms that regulate palatal development using genetically engineered mice. Deletions in many genes of mice result in CP and the most frequent phenotype seen is complete CP (Figure 2B). Uniquely, Tbx1^-/- mice present various phenotypes of CP[4], including complete CP (Figure 2B), incomplete CP (Figure 2C), and anterior CP (Figure 2D). Bone staining showed that some mice potentially had a submucosal CP (Figure 2G). These observations are in agreement with various CP phenotypes in humans.

In order to elucidate the molecular pathogenesis of CL/P, we conducted a literature search on PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and the Mouse Genome Informatics (MGI) from the Jackson Laboratory (http://www.informatics.jax.org). The search was limited to knockout mice with CP and excluded the teratogen-induced CP (Table 1). We also investigated diseases/syndromes using the Online Mendelian Inheritance in Man (OMIM) (http://omim.org). Not all the molecules involved in cleft palates in mice are correlated to CL/P in human (Table 1). When genes in Table 1 were analyzed by biological function using BioCarta (http://www.biocarta.com) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database (http://www.genome.jp/kegg/pathway.html), the transforming growth factor (TGF), hedgehog, Wnt, fibroblast growth factor (FGF), and mitogen-activated protein kinase (MAPK) signaling pathways were found to be critical in palatogenesis (Table 2). When genes were analyzed by molecular function using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) database (http://pantherdb.org)[5], the most significantly enriched molecular function was the “transcription factor”, especially the “homeobox transcription factors” (Table 2 and Figure 3).

Table 1.

Molecules involved in cleft palate in mice

Knockout mice with cleft palates			Humans
Gene/category	Protein	Ref.	OMIM	Syndrome	CL/P
Growth factors, antagonist, and receptors
Acvr1/Alk2	Activin A receptor, type I	[33]	1102576	Fibrodysplasia ossificans progressiva	nr
(Wnt1-Cre-mediated ablation)
Acvr2a	Activin A receptor, type IIA	[34]	1102581	nr	nr
Bmp4	Bone morphogenetic protein 4	[35]	1112262	Microphthalmia, syndromic 6	r
				Orofacial cleft 11
Bmp7	Bone morphogenetic protein 7	[36]	1112267	nr	nr
Bmpr1a/Alk3	Bone morphogenetic protein receptor, type IA	[35]	1601299	Juvenile polyposis syndrome,	nr
(Nestin-Cre-mediated ablation)				Polyposis syndrome
Chrd	Chordin	[37]	1603475	nr	nr
Ctgf	Connective tissue growth factor	[38]	1121009	nr	nr
Edn1	Endothelin 1	[39]	2131240	Auriculocondylar syndrome 3	r
Egfr	Epidermal growth factor receptor	[17]	1131550	nr	nr
Fgf9	Fibroblast growth factor 9	[40]	1600921	uc	nr
Fgf10	Fibroblast growth factor 10	[13,41]	1602115	Aplasia of lacrimal and salivary glands	nr
				LADD syndrome
Fgf18	Fibroblast growth factor 18	[42,43]	1603726	nr	nr
Fgfr1	Fibroblast growth factor receptor 1	[44]	1136350	Nonsyndromic cleft lip/palate	r
				Hartsfield syndrome
				Hypogonadotropic hypogonadism 2
				Pfeiffer syndrome
Fgfr2	Fibroblast growth factor receptor 2	[13,45]	1176943	Apert syndrome	r
(knockout) (Krt14-Cre-mediated ablation)				Crouzon syndrome
				Pfeiffer syndrome
				Saethre-Chotzen syndrome
Fst	Follistatin	[46]	1136470	nr	nr
Gabrb3	Gamma-aminobutyric acid A receptor, beta 3	[47]	1137192	Epilepsy, childhood absence, susceptibility to, 5	r
Gdf11/Bmp11	Growth differentiation factor 11	[48]	1603936	nr	nr
Gpr124	G protein-coupled receptor 124	[49]	1606823	nr	nr
Inhba	Inhibin, beta A/activin A	[50]	1147290	nr	nr
Pdgfc	Platelet-derived growth factor C	[51]	1608452	nr	r [52]
Pdgfra	Platelet-derived growth factor receptor, alpha polypeptide	[53,54]	1173490	Gastrointestinal stromal tumor, somatic	r
(knockout) (Wnt1-Cre-mediated ablation)				Hypereosinophilic syndrome, idiopathic, resistant to imatinib
Tgfb2	Transforming growth factor, beta 2	[55]	1190220	Loeys-Dietz syndrome, type 4	r
Tgfb3	Transforming growth factor, beta 3	[15,16,18]	1190230	Arrhythmogenic right ventricular dysplasia 1	r
Tgfbr1/Alk5	Transforming growth factor, beta receptor I	[56,57]	1190181	Loeys-Dietz syndrome, type 1	r
(Wnt1-Cre-, and Nestin-Cre-mediated ablation)
Tgfbr2	Transforming growth factor, beta receptor II	[12,58]	1190182	Loeys-Dietz syndrome, type 2	r
(Wnt1-Cre-, and KRT14-Cre-mediated ablation)
Vegfa	Vascular endothelial growth factor A	[59]	2192240	nr	nr
Membrane proteins
Ceacam1	Carcinoembryonic antigen-related cell adhesion molecule 1	[60]	1109770	nr	nr
Efna5	Ephrin A5	[61]	1601535	nr	nr
Efnb1	Ephrin B1	[62]	1300035	Craniofrontonasal dysplasia	r
Efnb2	Ephrin B2	[63]	1600527	nr	nr
Fzd2	Frizzled class receptor 2	[64]	1600667	nr	nr
Itga5	Integrin alpha 5	[65,66]	1135620	nr	nr
(knockout) (Mesp1-Cre-mediated ablation)
Itgb1	Integrin beta 1	[67]	1135630	nr	nr
(Col2a1-Cre-mediated ablation)
Itgb8	Integrin beta 8	[68]	1604160	nr	nr
Jag1	Jagged1	[69]	2601920	Alagille syndrome	nr
(Wnt1-Cre-mediated ablation)
Jag2	Jagged2	[70]	1602570	nr	nr
Kcnj2	Potassium inwardly-rectifying channel, subfamily J, member 2	[71]	1600681	Andersen syndrome	r
				Atrial fibrillation, familial, 9
				Short QT syndrome 3
Lrp6	Low density lipoprotein receptor-related protein 6	[72]	1603507	nr	nr
Ror2	Receptor tyrosine kinase-like orphan receptor 2	[73]	1602337	Robinow syndrome, autosomal recessive	r
				Brachydactyly, type B1
Ryk	Receptor-like tyrosine kinase	[74]	1600524	nr	nr
Ryr1	Ryanodine receptor 1, skeletal muscle	[75]	1180901	Central core disease	nr
				King-Denborough syndrome
				Minicore myopathy with external ophthalmoplegia
Sc5d/Sc5dl	Sterol-C5-desaturase (fungal ERG3, delta-5-desaturase) homolog (S. cerevisae)	[76]	1602286	Lathosterolosis	nr
Shh	Sonic hedgehog	[13,77]	1600725	Holoprosencephaly-3	r
(KRT14-Cre-, and Sox2-Cre-mediated ablation)				Microphthalmia with coloboma 5
				Single median maxillary central incisor
Smo/Smoh	Smoothened, frizzled class receptor	[78]	1601500	Basal cell carcinoma, somatic	nr
(Wnt1-Cre-mediated ablation)
Tctn2	Tectonic family member 2	[79]	1613846	Meckel syndrome 8	r
Wls/Gpr177	Wntless homolog (Drosophila)	[80]	1611514	nr	nr
(Wnt1-Cre-mediated ablation)
Wnt5a	Wingless-type MMTV integration site family, member 5A	[81]	1164975	Robinow syndrome, autosomal dominant	r
Wnt9b	Wingless-type MMTV integration site family, member 9B	[82,83]	1602864	nr	nr
(knockout) (Foxg1-Cre-mediated ablation)
Transcription and nucleolar factors
Alx1	Aristaless-like homeobox 1	[84]	1601527	Frontonasal dysplasia 3	r
Alx3	Aristaless-like homeobox 3	[85]	1606014	Frontonasal dysplasia 1	r
Alx4	Aristaless-like homeobox 4	[85]	1605420	Frontonasal dysplasia 2	Cleft alae nasi
				Parietal foramina 2
				Craniosynostosis 5
Anp32b	Acidic (leucine-rich) nuclear phosphoprotein 32 family, member B	[86]	nr	nr	nr
Arid5	AT-rich interaction domain-containing protein 5A	[87]	1611583	nr	nr
Asxl1	Additional sex combs like 1	[88]	1612990	Bohring-Opitz syndrome	r
				Myelodysplastic syndrome, somatic
Barx1	BarH-like homeobox 1	[89]	1603260	nr	nr
Cdc42	Cell division cycle 42	[90]	1116952	nr	nr
(Prrx1-Cre-mediated ablation)
Chd7	Chromodomain helicase DNA binding protein 7	[91,92]	1608892	CHARGE syndrome	r
(heterozygotes) (Wnt1-Cre-mediated ablation)				Hypogonadotropic hypogonadism 5 with or without anosmia
Cited2	CBP/p300-interacting transactivator, with Glu/Asp-rich C-terminal domain, 2	[93]	1602937	Atrial septal defect 8	nr
				Ventricular septal defect 2
Crebbp/Cbp	CREB binding protein	[94]	1600140	Rubinstein-Taybi syndrome	nr
Dlx1	Distal-less homeobox 1	[95]	1600029	nr	nr
Dlx2	Distal-less homeobox 2	[95]	1126255	nr	nr
Dlx5	Distal-less homeobox 5	[96,97]	1600028	Split-hand/foot malformation 1 with sensorineural hearing loss	r
Dph1/Ovca1	DPH1 homolog (S. cerevisiae)	[98]	1603527	nr	nr
Eya1	Eyes absent 1 homolog (Drosophila)	[99]	1601653	Branchiootic syndrome 1	r
				Branchiootorenal syndrome 1, with or without cataracts
				Anterior segment anomalies with or without cataract
Foxc2/Mfh1	Forkhead box C2	[100]	1602402	Lymphedema-distichiasis syndrome	r
Foxd3	Forkhead box D3	[101]	1611539	uc	nr
(Wnt1-Cre-mediated ablation)
Foxe1/Titf2/Fkhl15	Forkhead box E1	[102]	1602617	Bamforth-Lazarus syndrome	r
				Nonsyndromic orofacial clefting
Foxf2	Forkhead box F2	[103]	1603250	nr	nr
Gbx2	Gastrulation brain homeobox 2	[104]	1601135	nr	nr
Gli2	GLI family zinc finger 2	[8]	1165230	Culler-Jones syndrome	r
				Holoprosencephaly-9
Gli3	GLI family zinc finger 3	[105]	1165240	Greig cephalopolysyndactyly syndrome	r
				Pallister-Hall syndrome
Gsc	Goosecoid homeobox	[106]	1138890	Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities	nr
Hand2/dHand	Heart and neural crest derivatives expressed 2	[107]	1602407	nr	nr
Hic1	Hypermethylated in cancer 1	[108]	1603825	nr	nr
Hoxa2	Homeobox A2	[19]	1604685	Microtia with or without hearing impairment (AD)	r
				Microtia, hearing impairment, and cleft palate (AR)
Irf6	Interferon regulatory factor 6	[109,110]	1607199	van der Woude syndrome	r
				Orofacial cleft 6
				Popliteal pterygium syndrome 1
Jmjd6/Ptdsr	Jumonji domain containing 6	[111]	1604914	nr	nr
Kat6a/Moz/Myst3	K (lysine) Acetyltransferase 6A	[112]	1601408	nr	nr
Lhx7	LIM homeobox gene 7	[113]	nr	nr	nr
Lhx8	LIM homeobox gene 8	[11]	1604425	nr	r
Luzp1	Leucine zipper protein 1	[114]	1601422	nr	nr
Mef2c	MADS box transcription enhancer factor 2	[115]	1600662	Chromosome 5q14.3 deletion syndrome	nr
(Wnt1-Cre-mediated ablation)				Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations
Meox2	Mesenchyme homeobox 2	[116]	1600535	nr	nr
Mn1	Meningioma 1	[117]	1156100	Meningioma	nr
Mnt	Max binding protein	[118]	1603039	nr	nr
Msx1	Msh homeobox 1	[10,23]	1142983	Ectodermal dysplasia 3, Witkop type	r
				Orofacial cleft 5
				Tooth agenesis, selective, 1, with or without orofacial cleft
Msx2	Msh homeobox 2	[119]	1123101	Craniosynostosis, type 2	r
(missense mutation)				Parietal foramina 1
				Parietal foramina with cleidocranial dysplasia
Nabp2/Obfc2b/hSSB1	Nucleic acid binding protein 2	[120,121]	1612104	nr	nr
Osr2	Odd-skipped related transcription factor 2	[9]	1611297	nr	r
Pak1ip1	PAK1 interacting protein 1	[122]	1607811	nr	nr
Pax9	Paired box gene 9	[6]	1167416	Tooth agenesis, selective, 3	nr
Pbx1	Pre B cell leukemia homeobox 1	[83]	1176310	Leukemia, acute pre-B-cell	nr
Pds5a	PDS5, regulator of cohesion maintenance, homolog A (S. cerevisiae)	[123]	1613200	nr	nr
Phc1/Rae28	Polyhomeotic homolog 1	[124]	1602978	uc	nr
Pitx1	Paired-like homeodomain 1	[7,125]	1602149	Clubfoot, congenital, with or without deficiency of long bones and/or mirror-image polydactyly	r
				Liebenberg syndrome
Pitx2	Paired-like homeodomain 2	[126]	1601542	Axenfeld-Rieger syndrome, type 1	nr
				Iridogoniodysgenesis, type 2
				Peters anomaly
Pnn	Pinin	[127]	1603154	nr	nr
Prdm16	PR domain containing 16	[128]	1605557	Cardiomyopathy, dilated, 1LL	nr
				Left ventricular noncompaction 8
Prrx1/Prx1/Mhox	Paired related homeobox 1	[129]	1167420	Agnathia-otocephaly complex	r
Ptch1/Ptc1	Patched 1	[130]	1601309	Basal cell nevus syndrome	r
(Wnt1-Cre-mediated ablation)				(Gorlin syndrome)
				Holoprosencephaly type 7
Pygo2	Pygopus 2	[131]	1606903	nr	nr
(CMV-Cre-mediated ablation)
Rax	Retina and anterior neural fold homeobox	[132]	1601881	Microphthalmia, isolated 3	nr
Recql4	RecQ protein-like 4	[133]	1603780	Baller-Gerold syndrome	r
				RAPADILINO syndrome
				Rothmund-Thomson syndrome
Runx2	Runt-related transcription factor 2	[134]	1600211	Cleidocranial dysplasia	r
Sall3	Spalt-like transcription factor 3	[24]	1605079	nr	nr
Satb2	SATB homeobox 2	[135,136]	1608148	Glass syndrome	r
Shox2	Short stature homeobox 2	[22]	1602504	nr	nr
Sim2	Single-minded family bHLH transcription factor 2	[137]	1600892	nr	nr
Smad4 (Osr2-Cre-mediated ablation)	SMAD family member 4	[138]	1600993	Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome	nr
				Myhre syndrome
Smad7	SMAD family member 7	[139]	1602932	uc	nr
Snai2	Snail family zinc finger 2	[140]	1602150	Piebaldism	nr
				Waardenburg syndrome, type 2D
Sox5	SRY (sex determining region Y)-box 5	[141]	1604975	nr	nr
Sox9 (heterozygous)	SRY (sex determining region Y)-box 9	[142,143]	1608160	Acampomelic campomelic dysplasia	r
(Wnt1-Cre-mediated ablation)
Sox11	SRY (sex determining region Y)-box 11	[144]	1600898	Mental retardation, autosomal dominant, 27	nr
Sp8	Sp8 transcription factor	[145]	1608306	nr	nr
Tshz1	Teashirt zinc finger family member 1	[146]	1614427	Aural atresia, congenital	nr
Tbx1	T-box 1	[4,147]	1602054	DiGeorge syndrome	r
(knockout) (KRT14-Cre-mediated ablation)				Velocardiofacial syndrome
				Conotruncal anomaly face syndrome
				Tetralogy of Fallot
Tbx2	T-box 2	[148]	1600747	nr	nr
Tbx22	T-box 22	[149]	1300307	Cleft palate with ankyloglossia	r
				submucous cleft palate (SMCP)
Tcof1	Treacher Collins-Franceschetti syndrome 1	[150]	1606847	Treacher-Collins syndrome	r
(heterozygous)
Tfap2A	Transcription factor AP-2 alpha	[151]	1107580	Branchio-oculo-facial syndrome	r
(Wnt1-Cre-mediated ablation)
Trp63/Tp63	Transformation related protein p63	[152]	1603273	Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3	r
				Orofacial cleft 8
				Hay-Wells syndrome
				Limb-mammary syndrome
Vax1	Ventral anterior homeobox 1	[153]	1604294	Microphthalmia, syndromic 11	r
Whsc1	Wolf-Hirschhorn syndrome candidate 1	[154]	1602952	nr	nr
Zeb1	Zinc finger E-box binding homeobox 1	[155]	1189909	Corneal dystrophy	nr
Zfp640/Mzf6d	Zinc finger protein 640	[87]	nr	nr	nr
Zic3	Zinc finger protein of the cerebellum 3	[156]	1300265	Congenital heart defects, nonsyndromic	r
				Heterotaxy, visceral, 1
				VACTERL association
Cytoplasmic proteins
Akap8/Akap95	A kinase (PRKA) anchor protein 8	[157]	1604692	nr	nr
Apaf1	Apoptotic peptidase activating factor 1	[158]	1602233	nr	nr
B9d1	B9 protein domain 1	[159]	1614144	Meckel syndrome 9	nr
Cask	Calcium/calmodulin-dependent serine protein kinase	[160]	1300172	FG syndrome 4	r
				Mental retardation and microcephaly with pontine and cerebellar hypoplasia
Cdkn1c/p57kip2	Cyclin-dependent kinase inhibitor 1C	[161,162]	1600856	Beckwith-Wiedemann syndrome	r
				IMAGe syndrome
Chuk/Ikk1/Tcf16	Conserved helix-loop-helix ubiquitous kinase	[163]	1600664	Cocoon syndrome	nr
Crk	v-crk sarcoma virus CT10 oncogene homolog	[164]	1164762	nr	nr
Ctnnb1	Catenin (cadherin-associated protein), beta 1,	[165,166]	1116806	Mental retardation, autosomal dominant 19	nr
(KRT14-Cre-mediated ablation)
Cyp26B1	Cytochrome P450, family 26, subfamily b, polypeptide 1	[167]	1605207	Craniosynostosis with radiohumeral fusions and other skeletal and craniofacial anomalies	nr
Cyp51	Cytochrome P450, family 51	[168]	1601637	nr	nr
Dhcr7	7-dehydrocholesterol reductase	[169,170]	1602858	Smith-Lemli-Opitz syndrome	r
Dhrs3	Dehydrogenase/reductase (SDR family) member 3	[171,172]	1612830	nr	nr
Dicer1	Dicer 1, ribonuclease type III	[29]	1606241	Rhabdomyosarcoma, embryonal, 2	nr
(Pax2-Cre-mediated ablation)				Goiter, multinodular 1
				Pleuropulmonary blastoma
Dlg1/Dlgh/Sap97	Discs large 1	[173]	1601014	nr	nr
Fuz	Fuzzy planar cell polarity protein	[174]	1610622	Neural tube defects	nr
Gab1	Growth factor receptor bound protein 2-associated protein 1	[175]	1604439	nr	nr
Gad1/Gad67	Glutamate decarboxylase 1	[176,177]	1605363	Cerebral palsy, spastic quadriplegic, 1	r
Glce	Glucuronyl C5-epimerase	[178]	1612134	nr	nr
Glg1	Golgi apparatus protein 1	[179]	1600753	nr	nr
Grb2	Growth factor receptor bound protein 2	[180]	1108355	nr	nr
Gsk3b	Glycogen synthase kinase 3 beta	[181]	1605004	nr	nr
Hs2st1	Heparan sulfate 2-O-sulfotransferase 1	[182]	1604844	nr	nr
Hspb11/Ift25	Heat shock protein family B (small), member 11	[183]	1604844	nr	nr
Ilk	Integrin linked kinase	[184]	1602366	nr	nr
(Col2a1-Cre-mediated ablation)
Impad1/Jaws	Inositol monophosphatase domain containing 1	[185]	1614010	Chondrodysplasia with joint dislocations, GRAPP type	r
Inpp5e	Inositol polyphosphate-5-phosphatase E	[186]	1613037	Joubert syndrome 1	nr
				Mental retardation, truncal obesity, retinal dystrophy, and micropenis
Kif3a	Kinesin family member 3A	[187]	1604683	nr	nr
(Wnt1-Cre-mediated ablation)
Map3k7/Tak1	Mitogen-activated protein kinase kinase kinase 7	[188,189]	1602614	nr	nr
(Wnt1-Cre-mediated ablation)
Nprl3	Nitrogen permease regulator-like 3	[190]	1600928	nr	nr
Ofd1	Oral-facial-digital syndrome 1 gene homolog (human)	[191]	1300170	Joubert syndrome 10	r
(CAG-Cre-mediated ablation)				Orofaciodigital syndrome I
				Simpson-Golabi-Behmel syndrome, type 2
Pdss2	Prenyl (solanesyl) diphosphate synthase, subunit 2	[192]	1610564	Coenzyme Q10 deficiency, primary, 3	nr
(Pax2-Cre-mediated ablation)
Piga	Phosphatidylinositol glycan anchor biosynthesis, class A	[193]	1311770	Multiple congenital anomalies-hypotonia-seizures syndrome 2 Paroxysmal nocturnal hemoglobinuria, somatic	nr
(EIIa-Cre-mediated ablation)
Pkdcc/Vlk	Protein kinase domain containing, cytoplasmic	[194,195]	1614150	nr	nr
(Sox2-Cre-mediated ablation)
Prickle1	Prickle homolog 1	[196]	1608500	Epilepsy, progressive myoclonic 1B	nr
Rad23b	RAD23b homolog (S. cerevisiae)	[197]	1600062	nr	nr
Rspo2	R-spondin 2 homolog (Xenopus laevis)	[198,199]	1610575	nr	nr
Schip1	Schwannomin interacting protein 1	[87]	nr	nr	nr
Sdccag8	Serologically defined colon cancer antigen 8	[200]	1613524	Bardet-Biedl syndrome 16	nr
				Senior-Loken syndrome 7
Slc32a1/Viaat	Solute carrier family 32, member 1	[201,202]	nr	nr	nr
Spry1	Sprouty homolog 1	[203]	1602465	nr	nr
(Wnt1-Cre-mediated ablation)
Spry2	Sprouty homolog 2	[204]	1602466	nr	nr
Sumo1	SMT3 suppressor of mif two 3 homolog 1 (yeast)	[205]	1601912	Orofacial cleft 10	r
(heterozygous)
Ugdh	UDP-glucose dehydrogenase	[206]	1603370	nr	nr
(Wnt1-Cre-mediated ablation)
Wdpcp	WD repeat containing planar cell polarity effector	[207]	1613580	uc	nr
Extracellular proteins
Col2a1	Collagen, type II, alpha 1	[208]	2120140	Achondrogenesis, type II	r
				Stickler syndrome, type I
				Kniest dysplasia
Hspg2	Heparan sulfate proteoglycan 2, perlecan	[209,210]	1142461	Dyssegmental dysplasia	nr
				Schwartz-Jampel syndrome, type 1
Serpinh1/Hsp47	Serpine peptidase inhibitor, clade H, member 1	[211]	1600943	Osteogenesis imperfecta, type X	nr
(Col2a1-Cre-mediated ablation)
Smoc1	SPARC related modular calcium binding 1	[212]	1608488	Microphthalmia with limb anomalies	r

¹Before an OMIM entry number indicates a gene;

²Before an OMIM entry number indicates that the entry includes a description of a gene of known sequence and a phenotype. OMIM: Online Mendelian Inheritance in Man (http://omim.org); CL/P: Cleft lip and/or palate; r: Reported; nr: Not reported; uc: Unclarified.

Table 2.

Classification of genes associated with cleft palate in mice

	Genes
Signaling pathway
TGF-beta signaling pathway	Acvr1/Alk2, Acvr2a, Bmp41, Bmp7, Bmpr1a/Alk3, Cdc42, Chrd, Crebbp/Cbp, Cited2, Foxc2/Mfh11, Foxd3, Foxe1/Titf2/Fkhl151, Foxf2, Fst, Inhba, Gdf11/Bmp11, Map3k7/Tak1, Pitx2, Smad4, Smad7, Tgfb21, Tgfb31, Tgfbr1/Alk51, Tgfbr21
Hedgehog signaling pathway	Bmp41, Bmp7, Crebbp/Cbp, Gli21, Gli31, Gsk3b, Ptch1/Ptc11, Shh1, Smo/Smoh, Wnt5a1, Wnt9b
Wnt signaling pathway	Acvr1/Alk2, Ctnnb1, Crebbp/Cbp, Edn11, Fzd2, Gsk3b, Lrp6, Map3k7/Tak1, Prickle1, Smad4, Smo/Smoh, Wnt5a1, Wnt9b
FGF signaling pathway	Fgf10, Fgf18, Fgf9, Fgfr11, Fgfr21, Grb2, Spry1, Spry2
MAPK signaling pathway	Cdc42, Chuk/Ikk1/Tcf16, Egfr, Fgf10, Fgf18, Fgf9, Fgfr11, Fgfr21, Grb2, Map3k7/Tak1, Pdgfra1, Tgfb21, Tgfb31, Tgfbr1/Alk51, Tgfbr21, Crk, Itgb1
Cytokine-cytokine receptor interaction	Acvr1/Alk2, Acvr2a, Bmp7, Bmpr1a/Alk3, Egfr, Inhba, Pdgfra1, Pdgfc1, Tgfb21, Tgfb31, Tgfbr1/Alk51, Tgfbr21, Vegfa
CBL mediated ligand- induced downregulation of EGF receptors	Egfr, Grb2, Pdgfra1
Sprouty regulation of tyrosine kinase signals	Egfr, Grb2, Spry2, Spry1
NFkB activation	Crebbp/Cbp, Chuk/Ikk1/Tcf16, Map3k7/Tak1, Smad4, Tgfbr1/Alk51, Tgfbr21
Adherens junction	Crebbp/Cbp, Ctnnb1, Cdc42, Egfr, Fgfr11, Map3k7/ Tak1, Smad4, Snai2, Tgfbr1/Alk51, Tgfbr21
Focal adhesion	Ctnnb1, Cdc42, Col2a11, Crk, Egfr, Gsk3b, Grb2, Itga5, Itgb1, Itgb8, Ilk, Pdgfra1, Pdgfc1, Vegfa
Steroid biosynthesis	Cyp51, Dhcr71, Sc5d/Sc5dl
Cell cycle	Crebbp/Cbp, Cdkn1c/p57kip21, Gsk3b, Smad4, Tgfb21, Tgfb31
Regulation of actin cytoskeleton	Cdc42, Crk, Egfr, Fgf9, Fgf10, Fgf18, Fgfr11, Fgfr21, Itga5, Itgb1, Itgb8, Pdgfra1, Pdgfc1
Axon guidance	Cdc42, Efna5, Efnb11, Efnb2, Gsk3b, Itgb1
Endocytosis	Cdc42, Egfr, Fgfr21, Pdgfra1, Tgfbr1/Alk51, Tgfbr21
Angiogenesis	Ctnnb1, Crk, Efnb11, Efnb2, Fgfr11, Fgfr21, Fzd2, Gsk3b, Grb2, Jag1, Jag2, Pdgfra1, Pdgfc1, Vegfa, Wnt5a1
Family
Homeobox protein	Alx11, Barx1, Alx31, Alx41, Dlx1, Dlx2, Dlx51, Gbx2, Gsc, Hoxa21, Msx11, Msx21, Pax9, Prrx11, Pitx11, Pitx2, Rax, Shox2, Vax11
Tgf-beta receptor type I and II	Acvr1/Alk2, Acvr2a, Bmpr1a, Tgfbr1/Alk51, Tgfbr21
Tgf-beta family	Bmp41, Bmp7, Gdf11, Inhba, Tgfb21, Tgfb31
Tyrosine protein kinase	Egfr, Fgfr11, Fgfr21, Pdgfra1, Ror21, Ryk
Ephrin	Efna5, Efnb11, Efnb2
Zinc finger protein	Gli21, Gli31, Zic31, Hic1, Snai2
Forkhead protein	Foxc21, Foxd3, Foxe11, Foxf2
T-box protein	Tbx11, Tbx2, Tbx221
Sox transcription factor	Sox5, Sox91, Sox11
Heparin-binding growth factor family member/FGF	Fgf9, Fgf10, Fgf18
Sprouty	Spry1, Spry2
Smad	Smad4, Smad7
Integrin beta subunit	Itgb1, Itgb8
Frizzled	Fzd2, Smo
Wnt related	Wnt5a1, Wnt9b
Serine-threonine protein kinase	Ilk, Mpa3k7/Tak1
LIM domain containing protein	Lhx81, Lhx7, Prickle1
EGF-like domain protein	Jag1, Jag2

¹Genes involved in human cleft lip and/or palate. TGF: Transforming growth factor; FGF: Fibroblast growth factor; MAPK: Mitogen-activated protein kinase; CBL: Casitas B-lineage lymphoma; EGF: Epidermal growth factor.

Figure 3.

Gene ontology analysis of genes associated with cleft palate in mice. Gene ontology analysis of genes associated with cleft palate in mice was performed using the PANTHER classification system (http://pantherdb.org). The most significantly enriched molecular function was the “transcription factor” (P = 1.2 × 10^-12). A P value less than 0.05 was considered statistically significant.

To analyze mutant mice with cleft palates, the defects in palatal shelf development were divided into the following six categories (Table 3), which were modified from a previously published classification[3]. The first category is the failure of the palatal shelf formation. The gene mutations affect the initial development of the palatal shelf. The second one is the abnormal fusion of the palatal shelves and the mandible or tongue. Oral fusions between the palatal shelves and the tongue or mandible are rare. In Tbx1 (T-box 1) knockout mice, the posterior part of the palatal shelves fuse to the mandible, inhibiting the elevation of the palatal shelves[4]. The third category is the failed or delayed palatal shelf elevation. Ablation of Pax9 (paired box gene 9), Pitx1 (paired-like homeodomain 1), Gli2 (GLI family zinc finger 2), or Osr2 (Odd-skipped related transcription factor 2) in the palatal mesenchyme results in the failed palatal shelf elevation[6-9], suggesting crucial roles for these transcription factors in controlling the mesenchymal cells during palatal shelf elevation. The fourth one is the failure of the palatal shelf development after elevation. The loss of Msx1 (msh homeobox 1) and Lhx8 (LIM homeobox gene 8) and the conditional ablation of Tgfbr2 (transforming growth factor, beta receptor II) in the neural crest or Shh (sonic hedgehog) in the epithelium result in failure of the palatal shelf development[10-13]. The fifth category is the persistence of medial edge epithelial (MEE) cells. The palatal epithelia are regionally divided into three parts: oral, nasal, and MEE. The MEE cells are removed from the fusion line by epithelial cell migration, apoptosis, and epithelial-mesenchymal transdifferentiation[14]. Tgfb3 (transforming growth factor, beta 3) or Egfr (epidermal growth factor receptor) knockout mice lack the adhesive interactions between the palatal shelves because the fate of MEE cells is altered[15-18]. In the last category, the cleft palate arises as a secondary defect, due to tongue or bone anomalies during development. For example, Hoxa2 (homeobox A2) knockout mice exhibit CP, because depression of the tongue is inhibited by the abnormal attachment of the hyoglossus muscle to the greater horn of the hyoid[19,20].

Table 3.

Six categories of defects that result in cleft palate in mutant mice

	Defects	Knockout mice
(1)	Failure of the palatal shelf formation (small palatal shelves)	Acvr2a[34,50], 1Fgfr2[13], 1Lhx8[11], Pitx2[126], Itga5[65], Fst[46]
(2)	Abnormal fusion of palatal shelves and tongue or the mandible	Jag2[70], 1Irf6[109,110], 1Tbx1[4], Fgf10[41]
(3)	Failure or delayed palatal shelf elevation	Pax9[6], 1Pitx1[7], 1Osr2[9], 1Gli2[8], 1Tgfb2[55], 1Pdgfc[51], Dhrs3[172]
(4)	Failure of the palatal shelf development after the elevation	1Msx1[10], 1Lhx8[11], 1Tgfbr2 (Wnt1-Cre-mediated ablation)[12]
(5)	Persistence of medial edge epithelial cells	Apaf1[158], 1Tgfb3[18], Egfr[17], Ctnnb1 (K14-Cre-mediated ablation)[166]
(6)	Secondary defect	1Hoxa2[19,20], 1Satb2[135], Acvr1/Alk2 (Wnt1-Cre-mediated ablation)[33]

¹Genes involved in human cleft lip and/or palate.

There is molecular heterogeneity along the medial-lateral and anterior-posterior axes of palatal shelves. Regionally restricted expression of molecules provides distinct regulatory mechanisms for the development of palatal shelves. For instance, Msx1, Shox2 (short stature homeobox 2), Fgf10 (fibroblast growth factor 10), Bmp2 (bone morphogenetic protein 2), and Bmp4 (bone morphogenetic protein 4) are exclusively expressed in the anterior region of the palatal shelves[4,13,21,22]. The ablation of Msx1 in mice results in cell proliferation alterations in the anterior palatal mesenchyme and cleft palate[23]. Shox2 shows restricted expression patterns in the anterior palatal mesenchyme and the ablation of Shox2 in mice results in anterior cleft palates[22]. Fgf10 is also expressed in the anterior palatal mesenchymal cells and induces Shh expression through its receptor Fgfr2 (fibroblast growth factor receptor 2) in the palatal epithelium[13]. On the other hand, Pax9 is expressed in the posterior palatal shelves. Ablation of Pax9 results in cleft palates because of a palatal shelf development defect[6,21]. Even though it is known that Tbx1 induces the expression of Pax9 in the posterior part of palatal shelves[4], the mechanism of Tbx1-induced Pax9 expression during palatogenesis remains unknown. There is also molecular heterogeneity along the medial-lateral axis of the palatal shelf. For instance, Osr2 expression in the palatal shelf is characterized by a medial-lateral gradient. Loss of Osr2 results in the failure of palatal shelf elevation because of the delayed development of the medial part of palatal shelf[9].

MOLECULAR PATHOGENESIS OF CLEFT PALATES

Since most of the studies in mice focus on complete CP, the pathogenesis of other CP phenotypes is not well understood. Tbx1 is expressed in the developing palatal shelves in mice[4], highlighting the crucial function of Tbx1 in regulating palatal development. Loss of Tbx1 results in the abnormal fusion of the oral epithelia, which induces CP by preventing the elevation of palatal shelves[4]. The phenotypic variation in the Tbx1^-/- palates strongly suggests that Tbx1 is involved in modifier genes and/or stochastic factors. Tgfb3^-/- mice also exhibit either incomplete or complete CP[15,16]. Ablation of Shox2 results in anterior cleft palates[22]. Knockout mice of Sall3 (spalt-like transcription factor 3), which is expressed in the palatal mesenchyme, show hypoplasia of the soft palate and epiglottis[24]. These mice are unique models for studying the etiopathogenesis underlying the variety of CP phenotypes in humans.

A comprehensive list of molecules associated with CL/P in mice and their classification should provide insights into the genetic etiology of CL/P; however, the phenotype of knockout mice does not always recapitulate the phenotype in humans (Table 1). Since Table 1 includes the genes associated with tissue-specific conditional knockout mice, mutations of these genes may induce the phenotype of embryonic lethality in humans. Haploinsufficiency mutations of the TBX1 mutation are associated with CP[25]; however, heterozygous mice with Tbx1 are phenotypically normal, and Tbx1^-/- mice have CP phenotypes[4], thereby suggesting a species-specific requirement for Tbx1 dosage. Mutations of the PVRL1 (poliovirus receptor-related 1 or Nectin 1) cause CL/P-ectodermal dysplasia syndrome and nonsyndromic CL/P (OMIM #225060), whereas Pvrl1^-/- mice do not develop CP[26]. Lack of palatal phenotypes in mice may be a consequence of functional redundancy of Pvrl genes. Interestingly, Smad4, Smad7, Fgf9, Fgf10, and Fgf18 are involved in CP in mice (Table 1), whereas SMAD3 (OMIM *613795), FGF8 (OMIM *600483), and FGF17 (OMIM *603725) are involved in CP in humans.

Candidate genes for nonsyndromic CP in human must show a relevant spatio-temporal gene expression pattern in the developing palatal shelves, and induce a specific cleft palate phenotype when deleted[1]. Disease genes responsible for Mendelian forms of syndromic CP are also important in the etiology of nonsyndromic CP[27]. TBX1 mutations have been found in patients with incomplete CP without clinical diagnosis of del22q11.2 syndrome[25]. TBX1 is also one of the disease genes of conotruncal anomaly face syndrome (OMIM #217095), which is often associated with cleft palates, particularly submucosal CP, and bifid uvula. Tbx1^-/- palatal phenotype in mice makes Tbx1 a potential candidate gene for nonsyndromic CP, especially submucosal CP and incomplete CP in humans.

RECENT ADVANCES IN PALATOGENESIS

Even though many genes associated with CP have been identified, little is known about how the environment influences gene expression in palatogenesis, and palatal phenotype. Epigenetics, such as DNA methylation and chromatin remodeling, and the microRNA (miRNA) regulation could change gene expression profiles and phenotypes. Hundreds of miRNAs, small non-coding RNAs that modulate gene expression at the post-transcriptional level are expressed in murine embryonic craniofacial tissue[28]. Conditional knockout mice of Dicer1 (dicer 1, ribonuclease type III), which regulates the generation of miRNA, resulted in disrupted palatogenesis[29], suggesting that the miRNA function may be important in mammalian palatogenesis. miR-140, which modulates BMP signaling, regulates palatogenesis in mice[30] and miR-17-92 modulates Tbx1 and Tbx3 (T-box 3) activity, resulting in orofacial clefting[31]. Interestingly, transcription of Dicer1 is regulated by TP63 (transformation related protein p63)[32], whose mutations are associated with cleft palate phenotypes (Table 1). Since genes involved in miRNA generation and individual CP genes can both be modulated by several miRNAs, it is conceivable that complex gene-miRNA interactions exist during palatogenesis. Genetically engineered mice with miRNAs, which modulate CP genes, may provide new information on the gene interactions underlying the palatogenesis. Further studies on miRNA and methylated genes involved in palatogenesis are necessary to understand the environmental factors contributing to CP.

CONCLUSION

Studies with genetically engineered mice with CP reveal the importance of regulated molecular functions in palatogenesis and provide the opportunity to discover new genes implicated in palatogenesis. However, there is still much to learn about transcriptional regulation and molecular networks in palatogenesis. The interactions between environmental/stochastic factors and genes in the etiopathogenesis of CL/P require further studies. Teratogenic effects of dioxins and retinoic acid have been reported in mice[1]. Mutant mice with CP can also be used as models to assess environmental effects or gene-environment interactions. Epithelial abnormal fusion could be one of the stochastic causes that induce a variety of CP phenotypes in mice. Understanding the palatal epithelial functions during palatogenesis may also lead to the discovery of novel therapeutic methods for CL/P.