This study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the experiments involving animals described in this study were approved by the Animal Care and Use Committee of the University of Michigan-Ann Arbor (protocol number: PRO00004320).

The 2xcHS4-hsp68-lacZ-PA-2xcHS4 vector was generated by replacing a SacII-SacI fragment from the pUbC-SH-Gm-4xcHS plasmid (kindly provided by R. Behringer) with the hsp68-lacZ-PA cassette. A unique NotI site just upstream of the hsp68 minimal promoter was generated by using the Quikchange-II site-directed mutagenesis kit (Agilent). Regions of interest were PCR-amplified using SuperMix High Fidelity polymerase (Invitrogen) (primer sequences shown in Table 1), and the generated fragments inserted into the NotI site using the In-Fusion HD cloning kit (Clontech). Plasmid DNAs were purified using endonuclease-free Maxi-Prep columns (Qiagen) and the purified DNAs were linearized by SalI for microinjection.

Multi-species sequence comparisons around the Tgfb3 gene were performed using the UCSC genome browser (http://genome.ucsc.edu) and VISTA tools for Comparative Genomics (http://genome.lbl.gov/vista) using the global pair-wise and multiple alignment (LAGAN) program. The threshold used for evolutionary conservation was 70% sequence similarity within 100 bp region of DNA sequence. Predicted transcription factor binding sites were identified by using RankVISTA and TRANSFAC matrices.

The transgenic mouse lines and transient transgenics were generated in the Transgenic Animal Model Core facility at the University of Michigan—Ann Arbor.

We generated epithelium-specific Tgfb3 mutants by crossing mice heterozygous for the floxed Tgfb3 allele (Tgfb3^FXWT) (Doetschman et al., 2012) and carrying the epithelial K14-Cre driver (Andl et al., 2004) with homozygous floxed Tgfb3 (Tgfb3^FXFX) mice. R26R-YFP reporter mice were obtained from the Jackson Laboratories, and generation of Tgfb3-Cre mice has been previously described (Yang et al., 2008).

To detect expression of β-galactosidase encoded by the lacZ reporter gene, embryos were collected, washed and fixed in freshly prepared 4% para-formaldehyde-0.5% glutaraldehyde for 20 min, washed 3 × 20 min in the detergent wash solution and stained from 4 h to overnight in X-Gal staining solution as described (Behringer et al., 2003). The stained samples were examined using a Leica MZ95 dissecting microscope and photographed using an Olympus DP71 camera and DP controller and manager software. Selected samples were processed for paraffin embedding using Histoclear, sectioned, rehydrated and mounted in Immumount (Fisher) or couterstained with eosin or Nuclear Fast Red and mounted in DPX.

For paraffin embedding, embryos were harvested and fixed in 4% para-formaldehyde for 24 h at +4°C, washed, dehydrated and embedded in Leica Histowax. Sections (7 μm) were stained with hematoxylin and eosin using standard protocols. For immunohistochemistry, the paraformaldehyde fixed samples were allowed to sink in sterile 10% sucrose in PBS, then in 7% gelatin/15% sucrose in PBS, oriented and embedded in fresh 7% gelatin/15% sucrose in PBS on ice, then dry ice, and stored at −80°C. Cryosections (10 μm) were cut and stored at −80°C. The sections were stained with αSSEA-1 (MC-480 from DSHB) and αGFP (A11122 from Life Technology) antibodies, which were detected by Alexafluor-594 and Alexafluor-488 secondary antibodies (Invitrogen) respectively. The stained sections were mounted with Vectashield mounting medium containing DAPI (Vector Labs Inc). Sections were viewed using an Olympus BX51 microscope and documented using an Olympus DP71 digital camera as described above.

Comparison of the palatal phenotypes of global Tgfb3 knockout mice (Tgfb3^−/−) and epithelium-specific Tgfb3 (Tgfb3:K14-Cre) mice revealed that, despite the efficient recombination in the MEE, the germline mutants consistently displayed a more severe phenotype than the tissue-specific mutants (Figures 1A–I): Tgfb3^−/− mice had a complete cleft of the secondary palate (Kaartinen et al., 1995; Proetzel et al., 1995), while Tgfb3:K14-Cre mice had a cleft anteriorly, but superficial or complete fusion in the mid-palate, and an aberrant posterior epithelial bridge. Since the Tgfb3:K14-Cre palatal phenotype was practically identical to that observed in the epithelium-specific TGF-β receptor mutants (both Tgfbr1:K14-Cre and Tgfbr2:K14-Cre) (Dudas et al., 2006; Xu et al., 2006), we wondered whether this milder palatal phenotype was caused by an inability of the K14-Cre driver line (Andl et al., 2004) to induce recombination in peridermal cells. To address this question we harvested tissues from K14-Cre, R26R-YFP reporter embryos at E13.5, and assessed the Cre-induced recombination in MEE and peridermal cells (Figures 1J,K). Our results showed that while the MEE was efficiently recombined, we could not detect reporter expression in the adjacent periderm. In contrast, Tgfb3 was strongly and specifically expressed both in the periderm and underlying MEE as demonstrated by both in situ hybridization, and R26R-lacZ reporter expression in the Tgfb3-Cre^KI mouse line (Yang et al., 2008) (Figures 1L,M).

Since Tgfb3 is strongly and specifically expressed in peridermal and MEE cells, we reasoned that identification of cis-regulatory elements controlling palate-specific Tgfb3 expression would be invaluable for development of new improved genetic tools to examine palatal epithelial fusion in vivo. The Tgfb3 gene, composed of 7 evolutionarily conserved exons, is located on mouse chromosome 12 between the Ift43 (intraflagellar transporter 43) and Ttl5 (tubulin tyrosine ligase-like 5) genes, which both lie in the opposite orientation to the Tgfb3 gene (Figure 2A). The intergenic flanking sequences are remarkably short (3–3.5 kb) but the neighboring genes are not expressed in pre-fusion palatal shelves (Figures 4A,B).

To assess large regions upstream and downstream of the Tgfb3 gene for regulatory elements, we obtained two overlapping BACs. The 5′ BAC (RP23-76M13) contained a 289-kb region from −263 to +26 kb [defining Tgfb3 transcriptional start site (TSS) as 0] which included the Tgfb3, Ift43, and Gpatch2l genes (Figure 2A). The 3′ BAC (RP24-299H18) contained the 189-kb region from −35 to +154 kb which included the Tgfb3 gene and the Ttll5 (variant 4) gene (Figure 2A). The sequences of the two BACs overlapped by 61 kb, which includes all the Tgfb3 exons and some of those of the neighboring genes.

To prepare reporter constructs, we inserted an SA-lacZ-pA cassette into Tgfb3 exon 1 of each BAC using standard recombineering techniques (Warming et al., 2005). These recombinant lacZ reporter BACs were used to generate transgenic mice and β-galactosidase activity assessed at E14.0 using X-Gal staining as Tgfb3 is usually strongly expressed in palatal shelf MEE and nasal septal epithelium around this stage. Both 5′ and 3′ lacZ-BACs were able to target reporter activity correctly to the palatal midline and nasal septal tissues (Figures 2B,F). The 5′ lacZ-BAC transgenic embryos were also stained in mammary placodes, whisker follicles, nostrils, and vasculature (Figures 2C–E), while the 3′lacZ-BAC embryos showed additional staining principally in vasculature (Figures 2G–I, 3, which illustrates the positions of all DNA fragments tested for enhancer activity in this study and summarizes expression data). Stable transgenic mouse lines carrying 3′ lacZ-BAC did not show detectable reporter activity until E12.0–E12.5 (Figure 2J), when staining was seen first in blood vessels and soon afterwards in the tips of the posterior palatal shelves (Figures 2K,L). At E14.5 strong staining occurred along the entire A-P axis of the palatal shelf tips, and continued during and after palatal epithelial fusion when it could still be detected in the degrading midline seam and in vasculature at E16.0 (Figures 2M,N).

As the 5′ and 3′ BAC sequences overlapped, and both the 5′ and 3′ lacZ-BAC reporters drove expression in the palatal shelf tips, we hypothesized that sequences in the region common to each BAC may be responsible. We tested this by making a reporter lacZ-BAC containing only this sequence, from −35 to +26 kb (Figures 3A, 4Aa). This 61-kb fragment of mouse genomic DNA consistently drove highly specific reporter expression in the MEE and the adjacent periderm, and in peridermal cells covering the nasal septum where anterior secondary palatal fusion occurs (Figures 4B,F–H). The only other tissue showing detectable though weak X-Gal staining was the lens (Figures 4D,E).

We analyzed the 61-kb overlapping region using the UCSC Genome Browser (http://genome.ucsc.edu) (Figures 4Ac–e) to identify non-coding evolutionarily conserved regions (ECRs), which are likely to include tissue-specific enhancers and found four: ECR-I (2 kb upstream of TSS), ECR-II and ECR-III (in Tgfb3 intron 1) and ECR-IV (in Tgfb3 intron 3) (Figure 5). As these are all highly conserved in placental mammals, which develop a complete secondary palate, but not in avians (or fish), which do not express Tgfb3 in tips of palatal shelves and do not develop the fused secondary palate (Sun et al., 1998), they were good candidates to regulate palate-specific expression. To test this, the ECRs (I-IV) were PCR-amplified and subcloned upstream of the minimal hsp68 promoter and lacZ-PA reporter, and the resulting ECR-(I-IV)–hsp68-lacZ-PA cassette cloned between concatamerized pairs of genomic insulators (cHS4), which have been shown to reduce positional effects of transgenes and alleviate promoter interference (Yahata et al., 2007; Griswold et al., 2011) (Figure 5). Transient transgenic embryos were generated and analyzed for reporter activity at E14.0. While the ECRs were consistently able to target the reporter activity to several tissues (teeth, whisker follicles, nostrils and forebrain), no staining was seen in the MEE (n = 15) (Figure 5). We also modified the 61-kb lacZ-BAC by deleting sequences from −35 to −3 kb. This 28-kb BAC was also unable to direct lacZ reporter expression to the MEE (Figure 3). These data suggest that the sequences from −3.0 to +26 kb, including the entire Tgfb3 gene and ECRs I-IV, are not responsible for the MEE/ periderm-specific gene expression in mouse embryos during palatogenesis.

In addition to the noncoding ECRs I-IV, the 61-kb region from −35 to +26 kb contains three additional highly conserved regions in intron 2 of the Ift43 gene: ECR-V at position −(7.9 to 7.6)kb, ECR-VI at −(13.0 to 12.5)kb and ECR-VII at −(26.9 to 24.0)kb. To assess these regions for the possible presence of MEE/periderm-specific cis-regulatory elements, we first subcloned the ECRs -V and -VI into the 2xcHS4-hsp68-lacZ-2xcHS4 vector as a single 7.6-kb fragment (Figure 5A) and ECR-VII as a 2.9-kb fragment (Figure 5A). Analysis of X-Gal-stained transient transgenic embryos at E14 revealed that the region surrounding ECR-VII targeted the reporter activity to vascular and skeletal structures, but did not direct reporter activity in the MEE (Figure 3, and data not shown). In contrast, the 7.6-kb region containing both ECRs -V and -VI was able to drive expression not only in the MEE/periderm with high efficiency (7/14) (Figures 3B, 6B,C,V,W), but also in the vasculature (including palatal vessels), nostrils and whisker follicles (Figures 6C–F). Within this region, ECR-V is conserved only in mammals but ECR-VI is conserved in both mammals and avians suggesting that ECR-V would be more likely to contain palate-specific control elements. Indeed, this was the case, since the sequences between −9.6 and −6.1 kb including ECR-V targeted reporter activity to the MEE/periderm (Figure 3 and Figures 6G–K,X,Y), while ECR-VI and surrounding sequences (from −13.7 to −9.7 kb) did not (Figure 3 and data not shown). To narrow down the regions within −9.7 and −6.1 kb that contained putative cis-regulatory modules, we next examined a 0.3-kb fragment that encompassed the highly conserved ECR-V (from −7.9 to −7.6 kb), and an adjacent conserved 0.8-kb region from −7.4 to −6.6 kb (Figure 6A). Each region drove reporter expression in the tips of palatal shelves but relatively weakly (Figures 3, 6L–U) and with far less specifically than the larger (−13.7 to −6.1 kb) fragment. These results suggest that the 3.5-kb region in Ift43 intron 2 contains two or more cis-regulatory elements independently able to direct the reporter activity in the MEE and adjacent periderm, but they are needed in combination to drive expression efficiently.

Since the overall conservation of the intergenic region between the Ift43 and Tgfb3 genes is relatively high, we examined whether this region could also contribute to MEE/periderm-specific expression (Figure 7A). First we cloned the 9.8-kb region from −6.1 to +3.7 kb (i.e., Ift43 intron 1, Ift43 exon 1, intervening sequences between the Ift43 and Tgfb3 genes and Tgfb3 exon 1) between concatamerized pairs of cHS4 insulators, and inserted a lacZ-PA cassette in frame into the Tgfb3 exon 1 (Figure 7B). This construct, driven by the endogenous Tgfb3 promoter, directed reporter activity specifically in the palatal midline region in transient transgenic embryos, although with a relatively low frequency (2/5) (Figures 3B, 7B–F). To further define the important region within this 9.8 kb fragment, we subcloned the 5.3-kb region from −6.1 to −0.8 kb into the 2xcHS4-hsp68-lacZ-2xcHS4 vector, as it lacks the endogenous Tgfb3 promoter (Figure 7G). Two of the three resulting transgenic embryos showed reporter activity in the palatal midline (Figure 7H) though also in several other tissues (Figures 3, 7I–N), suggesting that the shorter sequence lacked elements necessary for highly regionally specific regulation. Intron 1 of Ift43 (from −6.1 to −3.7 kb) alone, or the intervening sequence between Ift43 and Tgfb3 (from −3.7 to −0.8 kb), did not direct β–galactosidase activity in the tips of palatal shelves (n = 5 in each case) (Figure 3B and data not shown). These data imply that a fragment from Ift43 intron 1 to Tgfb3 exon 1 contains a putative proximal MEE/periderm enhancer, which is dependent on DNA sequences separately located in smaller fragments.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.