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Plast Reconstr Surg. Author manuscript; available in PMC Mar 1, 2012.
Published in final edited form as:
PMCID: PMC3078688
NIHMSID: NIHMS261203
Role of Indian Hedgehog Signaling in Palatal Osteogenesis
Benjamin Levi, M.D.,1 Aaron W. James, M.D.,1* Emily R. Nelson, B.S.,1* Samantha A. Brugmann, PhD.,1 Michael Sorkin, M.D.,1 Alina Manu,1 and Michael T. Longaker, M.D., M.B.A.1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, California.
2 Corresponding Author
*These authors have contributed equally here within
Introduction
Cleft lip and palate are common birth defects and represent a large biomedical burden. Through the use of animal models, the molecular underpinnings of cleft palate are becoming increasingly clear. Indian Hedgheg (Ihh) has been shown to be associated craniofacial development and active in the palatine bone. We hypothesize that Ihh activity plays a role in osteogenesis within the secondary palate and that defects in this pathway may inhibit the osteogenesis of the secondary palate.
Methods
Palates were isolated from wild type mice during the period of palate development (e9.5-e17.5). qRT-PCR was used for detecting gene expression during osteogenic differentiation and cellular differentiation (Shh, Ihh, Ptc1, Gli1, Gli2, Gli3, Runx2, Alp, Col1a1). Next, palates were analyzed by H&E, Aniline Blue, Pentachrome, and In situ Hybridization to assess osteogenesis of the palatal shelf and expression of hedgehog pathway genes. Finally, the palate of Indian Hedgehog null mice was analyzed to determine the effect of genetic deficiency on palatal development osteogenesis.
Results
Increased Indian Hedgehog and osteogenic signaling coincided with ossification and fusion of the palate in wild-type mice. This included a 5-150 fold peak in expression of Hedgehog elements, including Ihh, at e15.5 as compared to e9.5. Contrarily, loss of Indian Hedgehog by genetic knockout (Ihh −/−) resulted in decreased secondary palate ossification.
Conclusion
Our results suggest a role for Hedgehog signaling during palatal ossification. The hedgehog pathway is activated during palatal fusion and deletion of Indian hedgehog leads to diminished ossification of the secondary hard palate.
Keywords: Palatal Development, Cleft palate, Indian Hedgehog, Osteogenesis
The incidence of cleft lip varies from 1 in 500 to about 1 in 1000 births, while isolated cleft palate has an incidence of approximately 1 in 1000 births.(1) The commonality of clefting creates an enormous biomedical burden. Affected children with cleft lip/palate require multiple operations beginning as early as 3 months of age with the goals of cleft closure and establishing normal facial form, normal midface maxillary position, proper dental occlusion, and preventing hearing loss. The molecular underpinnings of cleft palate are increasingly being understood complicated and clearly multifactorial.
Defects in signaling pathways, such as Wnt and NFAT have been shown to increase the propensity of clefting.(2-4) Another signaling pathway, the Hedgehog pathway is known to play a major role in craniofacial development, however its association with clefting remains unclear.(5) We set out to examine the potential role of Indian Hedgehog (Ihh) signaling pathway in palatal development. We hypothesized that Ihh activity may play a role in osteogenesis within the secondary palate and that defects in this signaling pathway may inhibit the osteogenesis of the secondary palate.
Hedgehog signaling has been shown to modulate mammalian skeletal development. The Hedgehog family of secreted proteins consists of Indian hedgehog (Ihh), Sonic hedgehog (Shh) and Desert hedgehog (Dhh).(6) Hedgehog signaling is initiated by the binding of the Hedgehog ligand to its transmembrane receptor Patched1 (Ptc1), which in turn, relieves suppression of the transmembrane protein Smoothened (Smo). Smo then activates an intracellular cascade that results in activation of Gli transcription factors.(7) Both Ihh and Shh have been shown to play a key role in skeletal development, and mutations in the Hedgehog pathway have been shown to lead to craniofacial malformations such as holoprosencephaly.(8-11)
Ihh has also been shown to play a key role in skeletogenesis, specifically with regards to periosteum induced osteoblast differentiation.(12) Ectopic expression of Ihh has been shown to promote osteoblast differentiation.(13) Most studies on Ihh and skeletal development, however, focus on long bone or endochondral ossification.(13-16) A paucity of research exists with regards to the effect of Ihh on intramembranous ossification, the mechanism active during palatal development. A case report of a child with a gene deletion in the location of the hedgehog gene demonstrated a submucous cleft palate further supporting the idea that the hedgehog pathway may play a crucial role in the skeletal development of the palate rather than in fusion of the mucous portion.(11) Deletion of Smo, has also been shown to result in facial deformities.(17) Further studies of Smo deficient mice demonstrated decreased, proliferation and differentiation of cranial neural crest cells, the progenitors of the palatal bones.(17, 18) Though Smo can be activated by Shh or Ihh, only Ihh was shown in previous studies to be upregulated in the developing palatine bones.(19)
Herein, we analyze the correlation between Ihh and palatal osteogenesis. We quantified Hedgehog signaling expression over palatal development by qRT-PCR. Next, we utilize Ihh−/− transgenic mice to determine the role of Hh signaling in defining the osteogenic capacity of the palatal shelves. The combination of these techniques suggested that Ihh signaling derived from palatal bone may induce palatal ossification.
Animals
Animals were maintained in the Stanford Animal Care Laboratory and received food and water ad libitum. For all experiments, animals were bred over night and the day of vaginal was considered as e0.5 day of gestation. Time-gestations of mice were obtained from Charles River Laboratories (Wilmington, Mass.). Ihh+/− mice were mated to generate Ihh−/− embryos on a C57B6 background. The tails of the embryos were used for genotyping by PCR as previously described (12). To generate Ihh+/−;Ptc-lacZ mice, Ihh+/− mice were crossed with Ptc-lacZ mice that contain an insertion of the lacZ transgene in one patched 1 (Ptc1) locus (20). Ihh+/−;Ptc-lacZ mice were mated with Ihh+/− mice to obtain Ihh−/−; PtclacZ embryos. All procedures were approved by the Stanford University Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals.
Palatal shelf dissection
Wild-type pups were harvested on embryonic (e) days e9.5, e11.5, e13.5, e15.5 and e17.5 of gestation. Embryos were harvested and immediately placed on ice in cold, sterile, phosphate-buffered saline. Microdissection was performed on the individual embryos to isolate and remove the palatal shelves. Once dissected, palatal shelves were snap-frozen in liquid nitrogen for gene expression analysis.
RNA Isolation and Real-Time PCR
Once dissected, wild type palates (e9.5-17.5) were pooled (N=3 distinct pooled samples, consisting of 3-4 microdissected palates per sample), and homogenized by sonification. RNA was isolated per the manufacturer’s instructions (RNeasy Kit, Qiagen Sciences, Maryland), genomic DNA was removed (DNA-free kit, Ambion, Austin, TX), and a total of 1 ug RNA was reverse-transcribed (Taqman Reverse Transcription Reagents, Applied Biosystems, Foster City, CA). Quantitative real-time PCR was carried out using the Applied Biosystems Prism 7900HT Sequence Detection System and Power Sybr Green Mastermix (Applied Biosystems, Foster City, CA). Specific primers were designed based on PrimerBank sequences (http://pga.mgh.harvard.edu/primerbank/). Sequences are shown in Table 1. All reactions were performed in triplicate; relative values were calculated to housekeeping gene (GAPDH) and presented as means and standard deviations.
TABLE 1
TABLE 1
Quantitative PCR Genes and Primer Sequences
Tissue Processing and Histology
For histological analysis, sections were prepared from tissues fixed overnight in 0.4% PFA, decalcified in 19% EDTA (pH 7.4) at 4°C and dehydrated through graded ethanol for paraffin embedding. For histological staining, 5 μm paraffin-embedded tissue sections were stained with Aniline Blue, and Movat’s Pentachrome bone stain. Alkaline phosphatase staining was performed using NTMT ALP buffer (5M NaCl, 1M Tris, 1M MgCl2, Tween20), and nitroblue terazolium (NBT) and 5-bromo-4-chloro-indolyl-phosphatase (BCIP) for color (Roche, Indianapolis, IN). In situ hybridization was performed on select slides for mouse Ptc1, Ihh, Runx-2, Ocn and Bmp-2 as previously described.(21) Non-specific binding was minimized by high stringency hybridization conditions, for all assays sense probes were used side-by-side with minimal background.
Whole mount skull preparation
To evaluate the palate phenotype of Ihh−/− embryos whole mount bone and cartilage staining was performed as previously described.(21) Specimens were fixed in 100% ethanol for 48 h, transferred to acetone for 48 h, stained in 0.15% alcian blue in 20% glacial acetic / 80% ethanol for 24 h, fixed in ethanol for 24 h and cleared in 1% aqueous KOH for 24-31 h prior to staining in 0.005% alizarin red in 1% KOH for 15 h. The tissue was cleared through 20% glycerol 1% KOH for 24-72h, followed by increasing concentrations of glycerol in dH2O up to 70%.
Detection of Ptc-LacZ transgene expression
Expression of the Ptc-lacZ transgene was compared in serial coronal sections between Ihh−/− embryos and Ihh+/+ littermates. Briefly, sections were fixed in 1% formaldehyde, 0.2% glutaraldehyde in PBS for 10 min at 4°C, washed twice in 0.01% sodium deoxycholate, 0.02% Nonidet P-40 in PBS at room temperature, and stained in 1% deoxycholate, 2% Nonidet P-40, 2mM MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mg/mL Xgal in PBS at 37°C.
Statistical analyses
Means and standard deviations were calculated from all numerical data. In graphs, all bars represent means whereas all error bars represent one standard deviation. Statistical analyses were performed using the Welch’s two-tailed t-test when comparing two groups with unequal standard deviations. When comparing multiple groups, a one-way ANOVA test without replication was performed, followed by post-hoc analysis using the Bonferroni correction to adjust for multiple comparisons. *P<0.05 was considered significant, unless otherwise stated.
Osteogenic Gene Expression Increases with Palate Embryonic Development
We first set out to define and quantify osteogenic gene expression during mouse palatal development and ossification. To do this, palatal shelves were harvested at stratified embryonic timepoints (from embryonic age (e)9.5-17.5) by microdissection and qRT-PCR analysis as described in the methods above. Genes associated with early osteogenesis were analyzed, including Runt-related transcription factor-2 (Runx2/Cfba1), Alkaline phosphatase, and Type I Collagen. Overall, a significant increase in gene expression was observed during ossification events, beginning at e13.5 and continuing until e17.5 (Figure 1A,B). Alkaline Phosphatase and Type I Collagen increased between e13.5 and e17.5 (Figure 1A,B). Runx2/Cfba1, the known master transcription factor for osteogenesis, increased overtime starting at e11.5 with a peak in expression at e15.5 (Figure 1B). These gene expression profiles corresponded with secondary palate development, which begins at e11.5, undergoes condensation at e12.5 and ossification at e13.5 and continues through e17.5.
Figure 1
Figure 1
Palatal osteogenic gene expression. (A-B) Quantitative assessment of osteogenic gene expression overtime throughout embryonic palatal development, normalized to Gapdh (including Alkaline Phosphatase, Runx2/Cbfa1, and Type I Collagen, respectively). Asterisks (more ...)
Hedgehog pathway activity colocalizes with Osteogenic Gene Expression during Palatal Development
Next, we analyzed Hedgehog pathway activity during palatal development. First, in situ hybridization for hedgehog responsive element Ptc1 was performed (Figure 2A). Ptc1 expression correlated with newly ossified bone as marked by pentachrome and aniline blue staining in the palatine bones (Figure 2A). To further validate the presence of Hh activity in the developing palatine bones we analyzed Ihh and Shh levels within the palatal mesenchyme via qRT –PCR. Ihh expression was significantly increased between e13.5 and e17.5, with peak expression at e15.5 (Figure 2B), whereas Shh was significantly upregulated at e15.5 and e17.5 (Figure 2C). Upregulation of the Hedgehog ligands was accompanied by increased expression of Hedgehog targets including, Ptc1, Gli1 and Gli2 (Figure 2C). Gli1 and Gli2 demonstrated a significant upregulation between e11.5 and e17.5, with peak expression at e15.5 (Figure 2C), whereas Ptc 1 demonstrated a significant upregulation at e15.5 and e17.5 (Figure 2C). Gli3 did not reach statistical significance (Figure 2C). This is what we expected given that Gli1 and Gli2 are known to be activators and Gli3 is known to be a repressor of Hedgehog pathway activity. In summary, Hedgehog pathway activity was detected in the palatal mesenchyme and developing palatine bones during palatal development and correlated with markers of osteogenic differentiation.
Figure 2
Figure 2
In vivo Hedgehog signaling gene expression. (A) Expression of Ptc1 in palatine bones by in situ. (Left) H&E is provided for orientation with a box signifying the palatine bone (pb) within the palatal shelf. From left to right, H&E, Ptc1 (more ...)
Indian Hedgehog Null Mice have Decreased Osteogenic Gene Expression
Results from our histology, in situ hybridization and RT-PCR as well as previous studies suggest that Ihh might play a role in the developing palatine bones(19). We therefore inquired as to the specific signal of Ihh in secondary palate ossification. To answer this question, we examined the palatal phenotype of the Ihh null mouse (Figure 3).
Figure 3
Figure 3
Histology of Indian Hedgehog −/− phenotype at e16.5. (A) Alkaline phosphatase stain of wild-type (left) and Ihh−/− littermates (right) at e16.5. There is greater alkaline phosphatase activity (more purple staining) in the (more ...)
In order to appreciate differences in osteogenic activity, we assessed alkaline phosphatase activity on histological sections of e16.5 wild-type and Ihh−/− palates (Figure 3A). A notable decrease in alkaline phosphatase activity in the palatal shelves was observed in the Ihh−/− mice when compared to their wild-type littermates.
In addition to the differences we observed in alkaline phosphatase activity, we also found relative differences in level of gene expression of known osteogenic markers at e16.5 by in situ hybridization (Figure 3B-D). Expression of Runx2 (Figure 3B), Osteocalcin (Ocn) (Figure 3C), and Bmp2 (Figure 3D) transcripts within the palatine bone and surrounding areas, were appreciably decreased in Ihh−/− palates when compared to wild-type littermates. In situ hybridization of Ihh was performed, which confirmed that Ihh is expressed in high levels in the palatal shelf and palatine bone in the wild-type mice (Figure 3E, left column). As expected, Ihh was not expressed in the Ihh −/− mice (Figure 3E, right column), further confirming the genotype of the null mutant mice.
Indian Hedgehog Null Mice have Impaired Palatal Osteogenesis
To detect palatal ossification, whole mount bone and cartilage staining was performed (Figure 4A). An appreciable decrease in size of palatine bones was observed in the Ihh−/− mice as compared to Ihh+/+ littermates at e16.5 and e17.0 (Figure 4A). Upon close examination of the e17.0 null mouse, one can see both decreased osteogenesis by alizarin red stain and a cleft in the midline of the palatine bone (second column, Figure 4A).
Figure 4
Figure 4
Ihh−/− palatal phenotype. (A) Bone and cartilage whole mount staining among wild-type (left) and Ihh−/− littermates (right). White dotted lines indicate the location of the palatine bones (pb). Additionally, the basisphenoid (more ...)
To visualize Hh activity in Ihh null mice, we crossed Ihh+/− into the Ptc-LacZ background (20) (Figure 4B). As expected, Xgal staining was reduced in the palate of Ihh−/− Ptc-LacZ mice, most notably at the ossification fronts of the palatine bone. Staining was equivalent between Ihh+/+ and Ihh−/− mice in other areas, such as the oral epithelium (Figure 4B).
In humans, it is estimated that 70% of cases of cleft lip and palate are nonsyndromic with complex inheritance patterns (22). Significant research efforts have been made to identify specific gene loci that are linked to cleft palate formation. Thus far, some progress has been made through the identification of TGF-α, TGF-β3 and MSX1, however, these have not been related to specific genetic variations with identifiable functional consequences(23, 24). MSX1 has led to the most fruitful findings as heterozygous MSX1 mutations have been shown to result in cleft lip and palate as well as tooth agenesis(25). Syndromic causes of cleft palate with osteochondrodysplasias such as Stickler Syndrome have been linked to mutations in COL1A1 and COL1A2(26).
In mice, secondary palatal development begins on e12.5. The maxillary processes give rise to bilateral palatal shelves, which consist of a mesodermal core surrounded by neural crest and subsequently by pharyngeal endoderm and ectoderm. These palatal shelves initially grow vertically downward, flanking the tongue. On e14.5, the initial reflex opening and closing movements of the mouth occur, which subsequently permit descent of the tongue and the horizontal alignment of the palatal shelves. By e15.5, palatal fusion is complete and mesenchymal condensation followed by osteogenic differentiation occurs. Hedgehog ligands such as Ihh have been shown to localize to areas of the palate from e11.5-14.5 (19).
Targets and effectors of the Hedgehog pathway are expressed in the palate epithelium and mesenchyme, and Ihh is specifically expressed in developing palatine bones(19, 27). During skeletogenesis, Indian Hedgehog plays a role in endochondral ossification (6, 28). In addition, Ihh has been implicated in intramembranous ossification, although disagreement exists on their precise roles (29, 30). Specifically, Ihh−/− mice have been shown to demonstrate cranial base synchondrosis and brachycephaly but there was no comment on abnormal mandibular development or tongue descent that might prevent palatal fusion (28).
Though previous studies have shown that Shh inhibition causes a cleft palate and that Ihh is localized in the palatine bone (19), previous studies have not investigated the effect of a genetic knockout of Ihh on palatal clefting(27). We hope this current study sheds some light on the involvement of the hedgehog pathway, and specifically Ihh in the secondary hard palate.
Hedgehog signaling exerts pleiotropic effects through regulation of the cell cycle,(31) cell differentiation,(32) and alteration of cell survival(33) which have been shown to play a key role in limb development (13-16). Increased Hedgehog signaling promotes osteogenesis in various bone-forming cells in vitro(34-37). We present an analysis of the effects of Hedgehog signaling in intramembranous ossification as seen in palatal development. First, we demonstrated osteogenic gene expression during normal palatal development. Next we showed an increase in Hedgehog activity during palatal development in vivo. Finally, we demonstrate mice lacking Ihh have deficiencies in osteogenic gene expression and osteogenesis within the secondary palate. Taken together, these data provide evidence that Ihh signaling favors the differentiation of palatal tissue to an osteogenic lineage and suggest a mechanism whereby cell fate can be directed to further study palatal development. Submucous cleft palate, a subset of a cleft palate where there is fusion of the soft palate but incomplete fusion of the hard palate that may represent a similar phenotype to the one seen in our Ihh null mice.
Conclusion
Osteogenic genes are upregulated during palatal development and this coincides with an upregulation of Ihh. Ihh may play a role in the osteogenic differentiation of the secondary palate and the Ihh null mouse represents a model of incomplete hard palate ossification.
Acknowledgments
Sources of Support: This study was supported by National Institutes of Health, National Institute of Dental and Craniofacial Research grant 1R21DE018727-01, the Oak Foundation and Hagey Laboratory for Pediatric Regenerative Medicine and the National Endowment for Plastic Surgery to M.T.L. B.L was supported by the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases grant 1F32AR057302-02.
Footnotes
Financial Disclosure: None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.
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