Expression and requirement of T-box transcription factors Tbx2 and Tbx3 during secondary palate development in the mouse

Developmental Biology 336 (2009) 145–155

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Developmental Biology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / d e v e l o p m e n t a l b i o l o g y

Expression and requirement of T-box transcription factors Tbx2 and Tbx3 during secondary palate development in the mouse Susann Zirzow a, Timo H.-W. Lüdtke a, Janynke F. Brons b, Marianne Petry a, Vincent M. Christoffels b, Andreas Kispert a,⁎ a b

Institut für Molekularbiologie, OE5250, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef 15 L2-108, 1105 AZ Amsterdam, The Netherlands

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Article history: Received for publication 14 May 2009 Revised 27 August 2009 Accepted 15 September 2009 Available online 19 September 2009 Keywords: Secondary palate T-box Mouse Phenotypic analysis Cleft palate Tbx2 Tbx3 Bmp Phenotype Expression CyclinD1

a b s t r a c t Formation of the mammalian secondary palate is a highly regulated and complex process. Impairment of the underlying cellular and molecular programs often results in cleft palate, a common birth defect in mammals. Here we report that Tbx2 and Tbx3, two closely related genes encoding T-box transcription factors, are expressed in the mesenchyme of the mouse palatal structures during development. Mice homozygous mutant for Tbx2 and mice double heterozygous for Tbx2 and Tbx3 exhibit a cleft palate phenotype arguing for an important contribution of Tbx2 and Tbx3 to palatogenesis. In Tbx2-deficient embryos, the bilateral primordial palatal shelves form but are smaller and retarded in the outgrowth process. They do not make contact but retain the potential to fuse. Development of other craniofacial structures appears normal, suggesting that impaired palate formation in Tbx2-mutant mice is caused by a primary defect in the palatal shelf mesenchyme. This is further supported by increased cell proliferation and apoptosis accompanied by increased expression of Bmp4 and CyclinD1 in Tbx2-deficient palatal shelves. Hence, Tbx2 and Tbx3 function overlappingly to control growth of the palatal shelf mesenchyme. © 2009 Elsevier Inc. All rights reserved.

Introduction Cleft palate without or with association of a cleft lip (CP, CL/P) is the most frequent human birth defect. This complex trait is caused by multiple genetic disorders but also by environmental perturbations of the development of the secondary palate (Gritli-Linde, 2007; Murray, 2002). The mammalian secondary palate arises from the medial sides of the maxillary processes that flank the oral cavity. In the mouse, the bilateral palatal shelves (PS) initiate outgrowth at embryonic day (E) 11.5 with a vertical movement down the side of the tongue. With the expansion of the lower jaw, the tongue drops in the oral cavity providing space for an elevation of the PS. With continued growth, the PS oppose in the midline and finally fuse. The multilayered epithelial seam initially present at the midline subsequently degenerates, establishing a continuous mesenchyme across the whole roof of the oral cavity at E15.5 (Ferguson, 1988). PS morphogenesis depends on survival, continued proliferation, migration and differentiation of mesenchymal cells and their inter-

⁎ Corresponding author. Fax: +49 511 5324283. E-mail address: [email protected] (A. Kispert). 0012-1606/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2009.09.020

action with the surrounding epithelial cover. Impairment of any of these processes will delay or inhibit palatal outgrowth resulting in PS non-fusion. The failure to separate the oral from the nasal cavity will lead to early postnatal lethality in mammals (Gritli-Linde, 2007; Rice, 2005). T-box (Tbx) genes encode a conserved family of transcription factors that play important roles in numerous processes during embryonic development including palatogenesis (Naiche et al., 2005). Phenotypic analyses of mice and human patients have identified three family members as critical mediators in this program to date. Tbx1-deficient mice display a wide range of developmental anomalies including CP (Jerome and Papaioannou, 2001; Merscher et al., 2001). The phenotypic spectrum of Tbx1-deficient mice resembles that of heterozygous patients of DiGeorge/velocardiofacial syndrome (DGS/VCFS), a common human disorder, usually associated with deletions of chromosome 22q11 in which TBX1 resides (Shprintzen et al., 2005). Cleft palate in association with ankyloglossia, a rare disorder with X-linked inheritance (CPX) has recently been shown to result from mutations in the T-box gene family member TBX22 (Braybrook et al., 2001). Tbx22-deficient mice have a submucous cleft palate (SMCP) and ankyloglossia, similar to the human phenotype, with a small minority showing overt clefts (Pauws et al.,

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2009). Mice carrying the spontaneous Dancer (Dc) mutation exhibit CL/P in homozygotes and show significantly increased susceptibility to CL/P in heterozygotes. Chromosomal analysis of the Dc locus revealed an insertional mutation that causes ectopic expression of a p23-Tbx10 chimeric transcript indicating that gain of expression of a T-box transcription factor gene can also cause CP pathogenesis (Bush et al., 2004). Tbx2 and Tbx3 encode a closely related pair of T-box proteins whose high sequence conservation, equivalent function as transcriptional repressors on common DNA binding sites and shared expression domains suggest that the two genes arose by a recent gene duplication event during vertebrate evolution (Harrelson et al., 2004; Naiche et al., 2005). Phenotypic analyses of mutant mice have revealed unique functions of Tbx2 in heart and limb formation (Aanhaanen et al., 2009; Harrelson et al., 2004) and of Tbx3 in the development of limbs, yolk sac, mammary gland, heart and liver (Bakker et al., 2008; Davenport et al., 2003; Hoogaars et al., 2007b; Ludtke et al., 2009; Suzuki et al., 2008). Expression analysis and organ culture experiments have implicated Tbx3 in control of cell proliferation in the PS but an in vivo requirement for Tbx3 has not yet been shown (Lee et al., 2007). Unfortunately, analysis of compound mutant embryos to test the functional redundancy at sites of co-expression of Tbx2 and Tbx3 has been hampered by the difficulty of propagating mice double heterozygous for both mutant alleles (Jerome-Majewska et al., 2005). Here, we extend the functional analysis of T-box genes in mouse palatogenesis and show that Tbx2 and Tbx3 are co-expressed during palate formation and are required for the formation of a secondary palate. Mice homozygous mutant for Tbx2 and mice double heterozygous for Tbx2 and Tbx3 exhibit a CP phenotype arguing for an important contribution of Tbx2 and Tbx3 to palatogenesis. We determine the temporal onset of the phenotype and characterize the underlying histochemical and molecular changes. We conclude that formation of the mammalian secondary palate like formation of other organs requires the precise interplay of T-box transcription factor activities and signaling pathways. Materials and methods Mice Mice carrying a null allele of Tbx2 (Tbx2tm1.1(cre)Vmc, synonym: Tbx2cre) (Aanhaanen et al., 2009) or Tbx3 (Tbx3tm1.1(cre)Vmc, synonym: Tbx3cre) (Hoogaars et al., 2007b) were maintained on an NMRI outbred and FvB/N inbred background, respectively. For the generation of Tbx2 or Tbx3 mutant embryos, mice heterozygous for either mutant allele were intercrossed. Mice double heterozygous for Tbx2 and Tbx3 mutant alleles were mated to obtain compound mutant embryos. Embryos for Tbx2 and Tbx3 expression analysis were derived from matings of NMRI wildtype mice. For timed pregnancies, vaginal plugs were checked in the morning after mating, noon was taken as embryonic day (E) 0.5. Embryos were harvested in PBS, fixed in 4% paraformaldehyde overnight and stored in 100% methanol at −20 °C before further use. Wildtype littermates were used as controls. Genomic DNA prepared from yolk sacs or tail biopsies was used for genotyping by PCR (Hoogaars et al., 2007b). Organ culture PS were isolated from E12.5 mouse maxillae and cultured in medium with fetal bovine serum at 37 °C and 5% CO2 for 48 h using a slight modification of the culture method reported by Trowell (Taya et al., 1999). The culture medium (DMEM/F12, Gibco) was supplemented with 20 μg/ml ascorbic acid (Sigma) and 1% penicillin/ streptomycin. Tissues were then fixed and processed for histological analysis.

Histological analysis Heads of the embryos and organ culture tissues were embedded in paraffin wax and sectioned to 5 μm. Sections were stained with hematoxylin and eosin. Cellular assays Cell proliferation in tissues of E12.5 and E14.0 embryos was investigated by the detection of incorporated BrdU on 5-μm sections of paraffin wax-embedded specimens, similar to published protocols (Bussen et al., 2004). Four sections each of anterior and posterior palate were taken from three embryos of each genotype at both stages for quantification. The BrdU-labeling index was defined as the number of BrdU-positive nuclei relative to the total number of nuclei as detected by DAPI counterstain in the mesenchyme of PS tip region. Detection of apoptotic cells in 5-μm paraffin sections of E11.5-14.0 embryos was based on the modification of genomic DNA using terminal deoxynucleotidyl transferase (TUNEL assay) and indirect detection of positive cells by fluorescein-conjugated anti-digoxigenin antibody. The procedure followed exactly the recommendation of the manufacturer (Serologicals Corp.) of the ApopTag kit used. The rate of apoptosis was defined as the number of TUNEL positive nuclei relative to the total number of nuclei as detected by DAPI counterstain in the mesenchyme of PS tip region. Cell densities in the PS were determined by counting DAPI-positive nuclei in a defined square of 60 × 80 μm centered in the PS tip region. PS size was measured using ImageJ software (NIH freeware). Statistical analysis was performed using the two-tailed Student's t-test. Data were expressed as mean +/− standard deviation. Differences were considered significant (⁎) when the P-value was below 0.05 and highly significant (⁎⁎) when the P-value was below 0.005. In situ hybridization analysis Whole mount in situ hybridization analysis was performed with digoxigenin-labeled antisense riboprobes following a standard procedure (Wilkinson, 1992). Stained specimens were transferred into 80% glycerol prior to documentation. In situ hybridization analysis on 10-μm frontal sections of embryo heads followed a previously described protocol (Moorman et al., 2001). Semiquantitative reverse transcription PCR Total RNA was extracted from microdissected E12.5 and E14.0 PS with RNAPure reagent (Peqlab). RNA (100–500 ng) was reverse transcribed with RevertAid M-MuLV Reverse Transcriptase (Fermentas). For semiquantitative PCR, the number of cycles was adjusted to the midlogarithmic phase for each PCR independently. Expression levels were normalised to Hprt expression. Quantification was performed with Quantity One software (Bio-Rad). Assays were performed in duplicates and with two different concentrations of cDNA on three independent pools of four PS each. Statistical analysis was performed using the two-tailed Student's t-test. Data were expressed as mean ± standard deviation. Differences were considered significant (⁎) when the P-value was below 0.05 and highly significant (⁎⁎) when the P-value was below 0.005. Primers and PCR conditions are available on request. Documentation Whole mount specimens were photographed on a Leica M420 microscope with a Fujix digital camera HC-300Z. Sections were photographed using a Leica DM5000 microscope with a Leica DFC300FX digital camera. All images were processed in Adobe Photoshop CS.

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Results

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palate and in the mesenchyme around the midline epithelial seam (Figs. 1U–X).

Tbx2 and Tbx3 are co-expressed in the developing palate Tbx2 and Tbx3 are required for palate development Earlier studies have reported expression of Tbx2 and Tbx3 in facial primordia at E9.5 (Chapman et al., 1996) and of Tbx3 in PS mesenchyme at E13.5 and E14.5 of mouse development (Lee et al., 2007). However, a detailed analysis of expression of the two genes during development of the maxilla and the secondary palate has not yet been documented. We therefore analyzed expression of the two genes during all stages of palate development using in situ hybridization analysis (Fig. 1). Analysis of whole heads revealed high expression of Tbx2 and Tbx3 in all prominences of the developing face, including the fronto-nasal process, the maxillary process and the cranial half of the mandible from E9.5 to E11.5 (Figs. 1A–E, G). At E11.5, PS primordia became first visible as outgrowths of the maxillary processes on the oral side (Figs. 1E–H). Tbx2 and Tbx3 were expressed on all following stages of outgrowth (E11.5–E14.0), elevation (E14.0–E14.5) and fusion (E14.5–E15.5) in the PS (Figs. 1I– X). Expression of Tbx2 and Tbx3 was confined to the mesenchymal core of the PS (Figs. 1F, H, J, L, N, P, R, T) with Tbx3 expression extending slightly more laterally in the posterior PS region than that of Tbx2 (arrows in Figs. 1I, K, M, O, Q, S). After completion of the fusion process at E15.5, expression persisted in the rugae of the

Co-expression of Tbx2 and Tbx3 during palatogenesis argued for a possible functional overlap of the two genes in this process. Previous studies reported embryonic lethality of Tbx2tm1Pa/tm1Pa mice at E14.5 and of Tbx3 tm1Pa/tm1Pa mice between E11.5 and E14.5 due to cardiovascular defects (Davenport et al., 2003; Harrelson et al., 2004), precluding analysis of a requirement of either gene in palatogenesis. We wondered whether embryonic viability could be extended beyond completion of palatogenesis by maintaining null alleles of Tbx2 (Tbx2cre) (Aanhaanen et al., 2009) and Tbx3 (Tbx3cre) (Hoogaars et al., 2007a) on an NMRI outbred rather than on a mixed inbred C57/129/ICR background as used in the previous studies (Davenport et al., 2003; Harrelson et al., 2004). Indeed, Tbx2cre/cre embryos maintained on NMRI background exhibited less severe cardiac defects at E14.5, were viable and represented in the expected Mendelian ratio at E18.5 (Aanhaanen et al., 2009). Tbx2cre/cre mice showed a bilateral hindlimb-specific duplication of digit IV (data not shown), consistent with previous observations in the Tbx2tm1Pa strain (Harrelson et al., 2004). Inspection of whole heads and of frontal sections at E18.5 and E15.5 revealed a complete (isolated) cleft

Fig. 1. Tbx2 and Tbx3 are co-expressed during palate development. RNA in situ hybridization analysis for Tbx2 and Tbx3 expression was carried out on whole heads (A–E, G, I, K, M, O, Q, S, U, W) and on adjacent frontal sections (F, H, J, L, N, P, R, T, V, X). (E, G, I, K, M, O, Q, S, U, W) show ventral views of the upper jaw region, the posterior (p)–anterior (a) axis is indicated. Black bars indicate section planes shown in the adjacent image; (F, H, J, L, N, P, R, T, V, X) sections show higher magnification of the left PS, the dorsal (d)–ventral (v) axis is indicated. Tbx2 and Tbx3 are co-expressed in the mesenchyme of the facial processes and in the PS throughout development. Arrows indicate the spatial extent of Tbx2 (I, M, Q) and Tbx3 (K, O, S) expression in the PS. Ea, ear vesicle; eye, eye vesicle; fnp, frontonasal process; mes, midline epithelial seam; mn, mandible; mx, maxilla; nc, nasal cavity; oc, oral cavity; psp, palatal shelf primordium; ps, palatal shelf; ru, rugue; to, tongue.

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of the secondary palate in 86% of embryos analyzed (18 of 21) (Figs. 2A–D) proving a requirement for Tbx2 in secondary palate development. Mice homozygous for the Tbx3cre null allele maintained on NMRI outbred background, similar to Tbx3tm1Pa/tm1Pa embryos on C57/129/ ICR background, died around E14.5 most likely due to a combination of cardiac, vascular and hepatic defects (Davenport et al., 2003; Ludtke et al., 2009; Suzuki et al., 2008). In the one embryo that survived until E15.5, the PS were fused in the middle region but not anteriorly (Figs. 2E–I) suggesting that PS growth and fusion occur normally but may be delayed due to the general developmental retardation of these embryos. Since a conditional approach is not available to date to stringently test the individual requirement of Tbx3 in palatogenesis, we analyzed embryos compound mutant for Tbx2 and Tbx3 for defects in this process. Mice heterozygous for Tbx2cre or Tbx3cre alleles were phenotypically normal on the NMRI outbred background they were maintained on. In contrast, 38% (5 of 13) of Tbx2cre/+Tbx3cre/+ embryos harvested at E18.5 exhibited a complete CP suggesting a crucial but less important contribution of Tbx3 to palatogenesis than Tbx2 (Figs. 2J–M). The few embryos with three or four mutant alleles of Tbx2 and Tbx3 obtained from intercrosses of Tbx2cre/+Tbx3cre/+ mice died at E10.5 due to hemodynamic failure precluding further analysis of the redundant role of the two genes in secondary palate development. Cardiovascular defects observed in these embryos are in agreement with a redundant function of the two genes in the

formation of the atrioventricular canal and will be presented elsewhere. Mice double heterozygous for null alleles of Tbx2 and Tbx3 that were maintained on an FvB/N genetic background exhibited cleft palate with a penetrance similar to Tbx2 single mutants (85%, 11 of 13) (Figs. 2N–Q) arguing for a strong contribution of the genetic background to CP etiology in Tbx2- and Tbx3-deficient mice. Tbx2-deficient mice exhibit retardation of PS elevation To investigate which step of secondary palate formation is disturbed in Tbx2cre/cre embryos, we carried out a detailed morphological and histological analysis throughout palate development (Fig. 3). At E12.5, no obvious difference in morphology and histology of the PS was observed between wildtype and Tbx2-deficient embryos (Figs. 3A–D). At E13.5, PS of Tbx2-deficient embryos appeared smaller and had moved less down the tongue compared to the wildtype (Figs. 3E–H). PS of wildtype embryos were elevated at E14.25 (Figs. 3I, K) and fused between E14.5 and E15.5 (Figs. 3M, O, Q, S). In contrast, in Tbx2-deficient embryos morphogenesis of the smaller PS was severely retarded and eventually came to a premature hold as evidenced by the appearance of elevated shelves only at E14.5 that failed to undergo the fusion process (Figs. 3J, L, N, P, R, T). The failure of PS fusion may be caused by a direct requirement for Tbx2 in the fusion process or by physical constrain due to the increased distance between the shelves. We tested these possibilities by juxtaposing PS

Fig. 2. Requirement of Tbx2 and Tbx3 in the development of the secondary palate. Analysis of defects in cleft palate formation in embryos by morphological inspection of upper jaw regions at E18.5 (A, B, J, K, N, O) and at E15.5 (E), the posterior (p)–anterior (a) axis is indicated; and by histological staining with hematoxylin and eosin of frontal sections of heads at E18.5 (C, D, L, M, P, Q) and at E15.5 (F–I), the dorsal (d)–ventral (v) axis is shown. Section planes are indicated by black bars in whole upper jaw preparations. Genotypes and genetic backgrounds (NMRI or FvB/N) are as indicated in the figure. 85% of Tbx2cre/cre embryos (B, D) and 38% of Tbx2cre/+Tbx3cre/+ (K, M) embryos on NMRI genetic background, 86% of Tbx2cre/+Tbx3cre/+ on FvB/N genetic background (P, Q) but not Tbx3cre/cre embryos on NMRI background (G, I) show a complete isolated cleft of the secondary palate. ns, nasal septum; oc, oral cavity; p, palate; ps, palatal shelf; to, tongue.

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Fig. 3. Retardation of PS growth in Tbx2-deficient embryos. Morphological (left two columns, the posterior (p)–anterior (a) axis is indicated) and histological (right two columns, the dorsal (d)–ventral (v) axis is shown) analysis of secondary palate formation in wildtype and Tbx2cre/cre mice. Developmental stages and genotypes are as indicated. In Tbx2-deficient embryos, PS are smaller, their outgrowth and elevation is retarded and fusion does not occur. md, mandible; mes, midline epithelial seam; ns, nasal septum; oc, oral cavity; ps, palatal shelf; to, tongue.

isolated from E13.5 Tbx2-deficient embryos in an organ culture system for 48 h. Under these conditions the PS of the Tbx2cre/cre embryos fused as readily as the wildtype control (Fig. 4). Hence, loss of Tbx2 function primarily affects growth but not fusion of the PS. Tbx2 plays a role in survival and proliferation of the PS mesenchyme In agreement with the histological observations (Fig. 3) quantitative analysis of PS size revealed no change at E12.5 but a significant reduction in the anterior region at E14.0 in Tbx2-deficient embryos (Fig. 5A). Cell densities in both anterior and posterior aspects of the PS was not significantly altered at both time points between wildtype and Tbx2cre/cre embryos (Fig. 5B). To investigate the cellular mechanisms that may underlie the decreased size and the retardation in PS morphogenesis in Tbx2-deficient embryos, we analyzed cell prolifer-

ation in the PS mesenchyme using the BrdU incorporation assay (Fig. 5C). We detected a significant increase of the cell proliferation rate from 19.5% to 25.4% in the anterior but not in the posterior PS region of Tbx2-deficient mice at E12.5. At E14.0, cell proliferation was not significantly changed throughout the PS mesenchyme compared to control embryos (Fig. 5C). We further performed TUNEL assays to analyze the role of apoptosis for the defect in shape and morphogenesis of PS in the Tbx2-deficient embryos (Fig. 6). We compared the situation in homozygous Tbx2-mutant embryos with that of wildtype as well as of heterozygous control embryos to exclude a contribution of Cre expression to changes in cell survival (Naiche and Papaioannou, 2007). We detected increased apoptosis throughout the PS mesenchyme at E11.5 and in the anterior PS region at E12.5 of Tbx2cre/cre embryos. At E13.5 and E14.0, apoptosis was unchanged compared to

Fig. 4. Tbx2-deficient PS retain fusion competence. Histological analysis by hematoxylin and eosin stainings of sections of 48 h cultures of juxtaposed PS isolated from E13.5 embryos. Tbx2cre/cre PS undergo fusion as in the control. Arrows indicate position of former midline epithelial seam.

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Fig. 5. Changes of PS size, cell density and proliferation in Tbx2cre/cre embryos. (A) Analysis of PS size, measured on frontal sections of wildtype (black column) and Tbx2deficient embryos (white column) at E12.5 (left panel) and E14.0 (right panel). PS size at E12.5 did not show highly significant differences anteriorly or posteriorly (anterior: wt: 0.0157±0.002 mm2, mut: 0.0187±0.004 mm2, p = 0.03; posterior: wt: 0.0434±0.007 mm2, mut: 0.0397±0.009 mm2, p = 0.19). At E14.0, the PS size was significantly decreased in the anterior and posterior region (anterior: wt: 0.0472±0.0087 mm2, mut: 0.0188±0.0023 mm2, p b 0.0001; posterior: wt: 0.0718±0.0106 mm2, mut: 0.0626± 0.0093 mm2, p b 0.005). (B) Cell densities were not significantly changed at E12.5 (anterior, wt: 13791±1416 cells/mm2, mut: 12416±3024 cells/mm2, p = 0.209; posterior, wt: 13541±1725 cells/mm2, mut: 12041±878 cells/mm2, p = 0.023) and at E14.0 (anterior: wt: 9948±1107 cells/mm2, mut: 10833±1339 cells/mm2, p = 0.294; posterior, wt: 11125±2524 cells/mm2, mut: 11718±1510 cells/mm2, p = 0.547). (C) Analysis of cell proliferation in frontal sections of the palate region of wildtype (wt) and Tbx2deficient (Tbx2cre/cre) embryos at E12.5 (left panel) and E14.0 (right panel) by the BrdU incorporation assay. The PS region used for quantification is shown in the histological sections. Quantification of cell proliferation was performed by the ratio of BrdU-positive cells to total cell number as determined by DAPI counterstain, the BrdU-labeling index, in the analyzed area at an anterior and posterior position in the PS. The BrdU labeling index is significantly increased in the anterior region of the Tbx2-mutant PS mesenchyme at E12.5 (wt: 19.5±0.4, mut: 25.4±0.9, p b 0.0001), while in the posterior region at this stage (wt: 22.5±1.7, mut: 26.0±1.6, p = 0.15), and at E14.0 in the anterior region (wt: 22.2±0.4, mut: 23.0±0.5, p = 0.023) and in the posterior region (wt: 30,3±0.6, mut: 28.0±0.6, p = 0.009) no significant changes were detected.

the controls (data not shown). Thus, changes in cell proliferation and survival precede morphological and histological alteration of PS morphology and growth. Bmp4 and Ccnd1 expression are up-regulated in Tbx2-deficient PS To analyze the molecular changes underlying disturbed PS formation in Tbx2cre/cre embryos, we analyzed expression of a large panel of genes which mutational analyses in the mouse implicated in the palatal growth process (Gritli-Linde, 2007) by in situ hybridization analysis of sections and whole heads between E12.5 and E14.0. Bone morphogenetic protein 2 and 4 (Bmp2 and Bmp4) and their target gene homeobox, msh-like 1 (Msx1) are weakly expressed in the PS mesenchyme where they regulate in an autoregulatory loop proliferation mainly in the anterior region (Zhang et al., 2000, 2002). Notably, Msx1 and Tbx2/Tbx3 are targets genes of Bmp signaling in a variety of tissues including the atrioventricular canal (Yamada et al., 2000) where the encoded proteins form complexes to repress transcription of genes (Boogerd et al., 2008). Expression of Bmp2 and Msx1 as well as distribution of phospho-Smad1,5,8, the intracellular mediator of Bmp signaling, appeared unchanged in the Tbx2-deficient palate (Supplemental Fig. 1). Bmp4 levels appeared slightly elevated in some of our experiments (Fig. 7A). Mesenchymal fibroblast growth factor 10 (Fgf10) signaling regulates proliferation in the PS mesenchyme and epithelium.

Interestingly, Fgf10 activates sonic hedgehog (Shh) expression in the epithelium that in turn signals back into the mesenchyme (Rice et al., 2004). Expression of Shh in the PS epithelium, and of Fgf10, the intracellular Fgf inhibitor gene sprouty homolog 2 (Drosophila) (Spry2), the Fgf target ets variant gene 4 (Etv4 or Pea3) and of the Shh target gene patched homolog 1 (Ptch1) in the PS mesenchyme was unaffected in the mutant arguing against an involvement of Fgf- and Shh-signalling pathways in the etiology of CP in Tbx2-deficient mice (Supplemental Figs. 2 and 3). It was recently shown that loss of Eph receptor B1 (Ephb1) alone or compound loss of Ephb2 and Ephb3 signaling also affects palate growth and elevation (Orioli et al., 1996; Risley et al., 2009). Expression of these genes as well as other members of the families of Eph receptor A and B genes, and the ephrin A and ephrin B ligand genes was unchanged in the mutant PS mesenchyme (Supplemental Figs. 4 and 5). Finally, expression of wingless-related MMTV integration site 5A gene (Wnt5a) that was recently implicated in cellular proliferation and migration in the developing palate (He et al., 2008) was similarly unaffected (Supplemental Fig. 6). PS are regionalized into an anterior and a posterior compartment. Short stature homeobox 2 (Shox2) is expressed in the anterior PS mesenchyme at E12.5 and E13.5 (Hilliard et al., 2005; Yu et al., 2005). In contrast, Tbx22 expression is found in the midposterior mesenchyme at E12.5 and posteriorly at E13.5 (Braybrook et al., 2002; Bush et al., 2002; Herr et al., 2003). Paired box gene 9 (Pax9) is expressed along the anterior-posterior extent of the PS mesenchyme with an

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Fig. 6. Changes of apoptosis in Tbx2cre/cre PS. TUNEL assay on frontal sections through the head detects increased apoptosis in the entire PS mesenchyme of Tbx2-deficient embryos at E 11.5 (A, left panel) (anterior: wt: 0.18±0.18, mut: 3.68±0.79, p = 0.00005; posterior: wt: 1.26±0.38, mut: 1.47±0.51, p = 0.69) and in the anterior part at E12.5 (B, left panel) compared to wildtype and heterozygous controls (wt: 0.21±0.13, mut: 1.55±0.08, p = 0.001). Apoptosis was unchanged in the posterior portion at E12.5 (A, right panel; B, right panel) (wt: 0.17±0.26), mut: 0.53±0.34, p = 0.27) between controls and Tbx2-deficient embryos.

enhanced posterior domain (Peters et al., 1998). Expression of all three markers was unchanged in the mutant arguing that anterior– posterior PS regionalization is not controlled by Tbx2 (Supplemental Fig. 7). Odd-skipped related 1 and 2 (Osr1 and Osr2) encode transcription factors that are expressed and required for proliferation in the PS mesenchyme (Lan et al., 2001, 2004). Both genes showed unaltered expression in the Tbx2-deficient palate at E12.5-E14.0 (Supplemental Fig. 8). We additionally tested expression of genes directly regulating cell cycle progression and proliferation since Tbx2 has been implicated in cell cycle control in other contexts (Bilican and Goding, 2006; JeromeMajewska et al., 2005). Expression of CyclinD2 (CycD2, Ccnd2), CyclinD3 (CycD3, Ccnd3), of cyclin-dependent kinase inhibitor 2A (Cdkn2a or p16Ink), 2C (Cdkn2c or p18), 2D (Cdkn2d or p19Arf) and of cyclindependent kinase inhibitor 1A (Cdkn1a or p21Waf), 1B (Cdkn1b or p27Kip1), 1C (Cdkn1c or p57Kip2) was not detectably altered by loss of Tbx2 in the PS mesenchyme in this assay (Supplemental Fig. 9). Expression of CyclinD1 (CycD1, Ccnd1), however, appeared upregulated (Fig. 7B). Analysis of chondrogenesis by in situ hybridization of the chondrogenic regulator SRY-box containing gene 9 (Sox9) and its target gene collagen type II, alpha 1 (Col2a1) and histochemical analysis of ECM composition by Alcian blue staining revealed no disturbance of chondrogenesis in the Tbx2-deficient PS (Supplemental Figs. 10 and 11). In order to confirm and expand this molecular phenotype analysis, we additionally employed a semiquantitative RT-PCR analysis on mRNA of microdissected whole PS from E12.5 and E14.0 embryos (Figs. 7C, D). In the later case, we used independent pools from anterior and posterior PS regions. We successfully established PCRs for most of the genes that have been implicated or are likely to be involved in growth control of the PS mesenchyme including genes of the Bmp (Bmp2, Bmp4, Msx1), and Fgf pathways (Fgf10, Spry2, Pea3),

Osr genes (Osr1, Osr2), and cell cycle control genes (CycD1, CycD2, CycD3, p16Ink, p19Arf, p21Waf, p27Kip1, p27Kip2). We omitted genes the spatially restricted expression of which cannot be properly reflected in this assay (Pax9, Tbx22, Shox2, Shh, Ptch1). RT-PCR analysis reliably identified significant up-regulation of Bmp4 (1.7 fold) and CyclinD1 (3.2 fold) expression in Tbx2-deficient PS at E12.5 (Fig. 7C) confirming the results of our in situ hybridization assays (Figs. 7A, B). All other genes tested were unchanged at both time points (Figs. 7C, D) again in agreement with our in situ hybridization data (Supplemental Figs. 1, 2, 8, and 9). Thus, our analyses excludes a number of signaling pathways including Fgf, Shh, Wnt and Ephrins as molecular mediators of the defects in secondary palate development in Tbx2-deficient mice. Since CyclinD1 has been characterized as a factor positively influencing cellular proliferation, its elevated expression can explain the observed proliferation increase in the E12.5 anterior PS mesenchyme of Tbx2-deficient embryos. Bmp-signaling affects a large number of cellular processes in diverse embryonic context. Notably, elevated levels of Bmp have been shown to induce apoptosis in a number of tissues but may also affect cellular proliferation (Kiyono and Shibuya, 2006; Koide et al., 2009; Lagna et al., 2006; Mukhopadhyay et al., 2006). Thus, dysregulation of cell proliferation and apoptosis in Tbx2-deficient PS mesenchyme possibly caused by altered levels of CyclinD1 and Bmp4 expression in the early outgrowth phase may translate to altered morphology and morphogenesis of this tissue, finally resulting in a CP phenotype. Discussion CP is the most frequent human birth defect caused by genetic and environmental perturbations of the development of the secondary palate. Recovery of more than 60 gene loci in the mouse the mutation of which causes this phenotype has allowed the assignment of specific molecular and cellular pathways that control palate development

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Fig. 7. Molecular changes in palate development in Tbx2-deficient embryos. (A, B) RNA in situ hybridization analysis for Bmp4 and CyclinD1 expression in PS at E12.5. Images show expression on sections through the anterior, middle and posterior region of PS with dorsal oriented upwards. Bmp4 and CyclinD1 expression appears increased in the anterior region of PS. (C, D) Quantitative RT-PCR analysis detects molecular changes in Tbx2-deficient PS at E12.5 (C) but not at E14.5 (D). QRT-PCR analysis of marker genes was done on mRNA from E12.5 and E14.0 PS as indicated in the figure. Expression levels are relative to the wildtype/heterozygous control pool (100%). Expression of Bmp4 and CyclinD1 is significantly upregulated with a mean ± SD for Bmp4 of 1.798 ± 0.2802, p = 0.0154 and 3.210 ± 0.7005, p = 0.0069 for CyclinD1. All other tested genes remained unchanged in the Tbx2cre/cre embryo.

(reviewed in Gritli-Linde, 2007). Here, we have shown that Tbx2 and Tbx3, two closely related T-box transcription factor genes, are specifically required for PS growth during secondary palate development. Increased apoptosis and altered proliferation may be cellular

mediators of this phenotype, while increased expression of CyclinD1 and Bmp4 may be a molecular cause. Our identification adds to the complexity of the T-box transcription factor network controlling mammalian palate development.

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Genetic control of PS growth Palatogenesis is a multistep process that comprises an initial bilateral expansion of the mandibulary processes followed by a complex growth program of the PS before a fusion process takes place. Developmental analysis of our Tbx2-deficient embryos showed that PS vertical outgrowth and elevation occurred but were severely delayed while the fusion potential of the shelves was unaffected. Disturbed proliferation and apoptosis in the Tbx2deficient palate at E11.5 to 12.5 indicates a requirement for Tbx2 in tissue growth by maintaining the cell number during the initial outgrowth phase. Even slight changes of this cellular parameter are likely to accumulate and translate into a morphogenetic halt as evidenced by the large number of CP phenotypes associated with changes of cellular survival and proliferation (Chou et al., 2004; Gritli-Linde, 2007). Palate development is tightly coupled to the growth and morphogenesis of the whole craniofacial complex and disturbances in the development of the later may perturb PS elevation and result in CP (Chai and Maxson, 2006). Tbx2 and Tbx3 are strongly expressed in all facial primordia, compatible with a wider requirement for these genes in craniofacial development. Yet, our histological analysis has not revealed any gross defects outside the secondary palate pointing to primary role for Tbx2 and Tbx3 in palatogenesis. PS growth and morphogenesis are controlled by a complex interplay of several epithelial and mesenchymal signaling pathways and their downstream transcriptional mediators (Gritli-Linde, 2007). Tbx2 and Tbx3 are specifically expressed in the mesenchyme of the developing secondary palate pointing to regulation by epithelial and/or mesenchymal signals and a function in the mesenchyme itself. Tbx2 and Tbx3 are targets of Bmp-signaling in a number of embryonic contexts including the limb and the atrioventricular canal (Behesti et al., 2006; Ma et al., 2005; Yamada et al., 2000). Notably, Lee et al. (2007) have recently shown that Bmp4 induces Tbx3 expression and that ectopic Tbx3 in turn downregulates Bmp4 in a palate culture system. Our molecular characterization of Tbx2-deficient PS revealed a significant up-regulation of Bmp4 in this tissue supporting the notion that Tbx3 and Tbx2 are involved in a negative feedback loop with Bmp4. Zhang et al. (2002) recently showed that Bmp4 and its target gene Msx1 engage in a positive feedback loop arguing for a complex network to tightly control expression of this potent signaling molecule. Since we did not detect changes of Msx1 expression in our assays, Tbx2/Tbx3 and Msx1 may represent independent and differently sensitive targets of Bmp-signaling in the PS mesenchyme. Unchanged levels of PSmad1,5,8 do not exclude that Bmp-signaling is augmented since alternative pathways (including p38 MAPK) are known to operate downstream of the ligand activated receptors (Xu et al., 2008). Increased proliferation in the anterior PS mesenchyme of E12.5 Tbx2cre/cre embryos may be caused by increased Bmp4 levels since Bmp4 is known to be a powerful mitogen in this tissue (Hilliard et al., 2005; Zhang et al., 2002). However, elevated levels of Bmp4 have also been associated with increased apoptosis, which we detected in early PS outgrowth in Tbx2-deficient embryos (Kiyono and Shibuya, 2006; Koide et al., 2009; Lagna et al., 2006; Mukhopadhyay et al., 2006). A mitogenic effect of Bmp4 may be mediated or further enhanced by increased expression of CyclinD1, a regulatory subunit of CDK4/6 kinases that controls G1-S progression in the cell cycle. Increased expression of CyclinD1 and increased proliferation in Tbx2-deficient PS is unexpected since Tbx2 (and Tbx3) have frequently been associated with the repression of cell cycle inhibitor genes including p19Arf and p21Waf, thus, cell proliferation and senescence bypass (Jacobs et al., 2000; Prince et al., 2004). Lee et al. (2007) have shown that ectopic expression of Tbx3 leads to increased cellular proliferation in cultures of E13.5 and E14.5 PS. The finding that gain of Tbx3 in vitro and loss of Tbx2 in vivo both

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lead to increased proliferation is surprising and cannot be explained at this moment. It remains unclear how exactly altered proliferation and apoptosis in early PS development translates in reduced PS size and retarded outgrowth. At this point, we cannot exclude that the delay in PS elevation in Tbx2-deficient mice may also be caused by changes of ECM composition, cytoskeletal architecture or cell movements. However, histological analysis of the ECM, of Wnt5a expression (He et al., 2008) and Ephrin signaling (Risley et al., 2009) provided no indication of an involvement of these factors and parameters in CP etiology in Tbx2-deficient embryos. It is noteworthy that the phenotypic penetrance of CP in Tbx2/ Tbx3 compound heterozygous mice was strongly dependent on the genetic background, the defect being much more frequent on FvB/N than on NMRI background. We assume that relatively little changes of Tbx2 and Tbx3 activity levels result in changes in cellular parameters. However, since PS growth and morphogenesis is exquisitely sensitive to proliferation changes, even minor changes of cell cycle regulatory molecules may accumulate over time and translate in a delay of outgrowth and eventually cleft palate. T-box genes and palate development T-box genes regulate tissue and organ development in a complex manner that may include hierarchal activities, but also independent, opposing and/or redundant regional functions of individual family members (Hoogaars et al., 2007a; King et al., 2006). Previous studies have identified three T-box genes in human (and mouse) palate development. Tbx1 seems to act early in patterning and outgrowth of the pharyngeal pouches preceding secondary palate development. Tbx22 is expressed in the posterior PS arguing for a role in regionalization or compartmentalization of the palate in anterior and posterior halves (Arnold et al., 2006; Braybrook et al., 2002; Bush et al., 2002; Herr et al., 2003). Expression of Tbx22 depends on the transcription factor Mn1 that is preferentially required for posterior palate growth (Liu et al., 2008). Recent analysis of Tbx22deficient mice revealed submucous cleft palate (SMCP) and ankyloglossia. Only a small minority of the animals showed overt clefts indicating a less important role in palatogenesis in mice compared to humans (Pauws et al., 2009). Tbx2 and Tbx3, as shown in this study, show no regional restricted expression in the palatal mesenchyme but may synergize with Tbx22 in the control of cellular programs important for the elevation process including proliferation and apoptosis. Interestingly, ubiquitous overexpression of Tbx10 in the spontaneous Dancer mutant or in transgenic mice causes CP (Bush et al., 2004) arguing that not only loss but also ectopic expression of T-box genes can result in the same phenotypic consequences. It is unclear whether Tbx10 acts as a transcriptional activator or repressor but it is possible that ectopic Tbx10 competes with endogenous Tbx function in the palate including Tbx1, Tbx2/Tbx3 and Tbx22. A paradigm for this has been established in the heart where the transcriptional repressor Tbx2 acts by competing with the transcriptional activation activity of Tbx5 and Tbx20, explaining the dramatic cardiac arrest phenotype in in vivo misexpression experiments of Tbx2 (Christoffels et al., 2004; Habets et al., 2002). The effect of ectopic Tbx10 in cleft palate induction is strongly dose dependent, being much more frequent in homozygous than in heterozygous conditions (Bush et al., 2004). Our analysis has shown that the function of Tbx2 and Tbx3 is also strongly dose- and background-dependent arguing that even slight interference (e.g., by Tbx10) of endogenous T-box transcription factor function may lead to perturbed palate development. In summary, it seems clear that the precise temporal and spatial control of T-box activity levels is mandatory for normal palatogenesis in mice and man.

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