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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Dev Dyn. Author manuscript; available in PMC 2010 December 28.
Published in final edited form as:
PMCID: PMC3010755
NIHMSID: NIHMS146670

Mice With an Anterior Cleft of the Palate Survive Neonatal Lethality

Abstract

Many genes are known to function in a region-specific manner in the developing secondary palate. We have previously shown that Shox2-deficient embryos die at mid-gestation stage and develop an anterior clefting phenotype. Here, we show that mice carrying a conditional inactivation of Shox2 in the palatal mesenchyme survive the embryonic and neonatal lethality, but develop a wasting syndrome. Phenotypic analyses indicate a delayed closure of the secondary palate at the anterior end, leading to a failed fusion of the primary and secondary palates. Consistent with a role proposed for Shox2 in skeletogenesis, Shox2 inactivation causes a significantly reduced bone formation in the hard palate, probably due to a down-regulation of Runx2 and Osterix. We conclude that the secondary palatal shelves are capable of fusion with each other, but fail to fuse with the primary palate in a developmentally delayed manner. Mice carrying an anterior cleft can survive neonatal lethality.

Keywords: palatogenesis, cleft palate, Shox2, mice

INTRODUCTION

The cleft lip and/or palate (CLP) represents one of the major groups of congenital birth defects in humans. It exemplifies the complex birth defects with contributions from multiple genetic and environmental factors (Murray, 2002). The mammalian definite palate, which separates the oropharynx with its various functions from the nasopharynx, is formed by the fusion of the primary palate of the frontonasal process and two bilateral maxillary palatal shelves that will form the secondary palate. Clinically, human patients with nonsyndromic CLP are viable but require surgical repair. However, a complete cleft palate phenotype is inevitably lethal to newborn rodents.

The development of the secondary palate, which is composed of the cranial neural crest derived mesenchymal cells and the pharyngeal epithelium, is a complex and multiple-step process, including palatal shelf growth, elevation, fusion between paired shelves, and disappearance of the midline epithelial seam (Ferguson, 1988; Ito et al., 2003). In the developing mouse embryo, the proliferation of the cranial neural crest derived mesenchymal cells within the maxillary processes results in the outgrowth of the palatal shelf primordia at embryonic day (E) 11.5. Subsequently, the palatal shelf primordia grow vertically downward beside the tongue (around E12.5–E13.5), followed by a rapid elevation/reorientation of the palatal shelves, which brings the two processes into horizontal apposition above the tongue around E14.0. The initial contact between the bilateral shelves is believed to take place around the third rugae within the anterior third of the palate, and the fusion extends bidirectionally, anteriorly and posteriorly (Schupbach, 1983; Sakamoto et al., 1989; Kaufman and Bard, 1999; Sperber, 2001).

Recent studies using molecular genetics approaches have implicated many genes during palatogenesis (Gritli-Linde, 2007). Of interest, many of these genes are found to be expressed in a regionally specific manner in the developing palate along the anterior–posterior (A-P) axis (Hilliard et al., 2005; Okano et al., 2006, Gritli-Linde, 2007). It has been demonstrated that several key molecules, including Msx1, Shh, Bmp2, Bmp4, Fgf10, and Shox2, are expressed specifically in the anterior palate and form a regulatory genetic network that controls palatal growth (Zhang et al., 2002; Alappat et al., 2005; Hilliard et al., 2005; Yu et al., 2005). In contrast, several other genes, such as Tbx22 and Meox-2, exhibit a restricted expression pattern in the posterior palate (Herr et al., 2003; Jin and Ding, 2006; Li and Ding, 2007). The anterior and posterior palatal tissues not only exhibit differential gene expression patterns, but also show distinct molecular and cellular responses to exogenous growth factors, indicating the heterogeneous property of the palatal tissue (Zhang et al., 2002; Hilliard et al., 2005). This heterogeneity appears to be established through the epithelial–mesenchymal interactions (Hilliard et al., 2005; Yu et al., 2005).

Shox2 belongs to the short stature homeobox (SHOX) gene family. Humans have both the SHOX and SHOX2 genes, but rodents do not have a SHOX orthologue (Rovescalli et al., 1996; Clement-Jones et al., 2000). A zooblot analysis of SHOX and SHOX2 reveals that both genes are present only in the vertebrates and not in the invertebrates that have been studied, suggesting their central role in the development of the internal skeleton and its related structures (Clement-Jones et al., 2000). Indeed, in humans, mutations in SHOX are associated with the idiopathic growth retardation, Turner syndrome, Leri-Weill dyschondrosterosis, and the Langer mesomelic dyspalsia (Ellison et al., 1997; Rao et al., 1997; Belin et al., 1998; Shears et al., 1998; Zinn et al., 2002). Although SHOX2 has not been linked to any known syndromes so far, studies using mouse gene-targeting models have demonstrated an essential role for Shox2 in the limb skeletogenesis, palatogenesis, and cardiovascular development (Yu et al., 2005, 2007; Cobb et al., 2006; Blaschke et al., 2007).

We have previously reported that Shox2-deficiency causes the formation of anterior clefting in mice (Yu et al., 2005), mimicking a very rare type of incomplete cleft palate phenotype in humans (Fara, 1971; Mitts et al., 1981). Under these circumstances, the hard palate is cleft with soft palate integrity, suggesting that the palate closure in both the anterior and posterior domains is not dependent on one another. However, because Shox2-deficient mice die during mid-gestation due to severe cardiovascular defects (Blaschke et al., 2007), it remains to be determined whether the anterior clefting phenotype observed in Shox2 mutants represents a delayed closure of the anterior palate. It is also of great interest to know if mice with an anterior clefting can survive neonatal lethality. To address these issues, we used mice carrying floxed Shox2 allele and the Wnt1-Cre transgenic mice to generate Shox2 conditional inactivation mice. Mice bearing a specific deletion of Shox2 in the palatal mesenchyme exhibit indeed an anterior clefting defect but survive the embryonic and neonatal lethality. A failure of the fusion between the primary and secondary palates results in the cleft palate formation. Although the anterior palate does not initially fuse due to its small size, it can do so later when the anterior palatal shelves approximate to each other. They demonstrate that the epithelial seam is still competent to fuse. However, the anterior palate is unable to fuse with the primary palate at these later stages.

RESULTS AND DISCUSSION

Shox2 Conditional Inactivation Mice (Wnt1-Cre; Shox2F/−) Survive Neonatal Lethality

Mice deficient in Shox2 die around E12 due to severe cardiovascular defects (Cobb et al., 2006; Blaschke et al., 2007). However, a small portion of the mutant mice could survive up to E17, exhibiting an anterior clefting of the palate (Yu et al., 2005). To circumvent this early embryonic lethality and further study the role of Shox2 in palate development, we conditionally inactivated Shox2 in the palatal mesenchyme by using the floxed Shox2 allele and the Wnt1-Cre transgenic mouse line. Mice carrying Wnt1-Cre and Shox2F/− should have inactivated Shox2 in the cranial neural crest derived palatal mesenchyme. Indeed, in situ hybridization assays showed a complete absence of Shox2 expression in the developing palate of Wnt1-Cre; Shox2F/− mice (Fig. 1A,B). Similar to Shox2 conventional inactivation mice (Yu et al., 2005), ectopic Fgf10 expression was detected in the anterior palatal mesenchyme of Wnt1-Cre;Shox2F/− mice (Fig. 1C,D), thus confirming the identical molecular outcomes in the both Shox2-decifient models. In addition, we also found that Sox9, a HMG box gene whose mutation also leads to a cleft palate phenotype in mice (Bi et al., 1999; Mori-Akiyama et al., 2003), is down-regulated in the mutant palatal mesenchyme (Fig. 1E,F). These results demonstrate an efficient ablation of Shox2 in the cranial neural crest derived palatal mesenchyme of Wnt1-Cre;Shox2F/− mice. Our results also place Shox2 upstream of Sox9 during palate development, which differs from the observation in the developing limbs where Sox9 expression is unaltered in the absence of Shox2 (Yu et al., 2007).

Fig. 1
Efficient ablation of Shox2 in the palatal mesenchyme of Wnt1-Cre;Shox2F/− embryos. A: Shox2 expression is detected in the mesenchyme of the anterior palate of an embryonic day (E) 13.5 wild-type embryo. B: No detectable Shox2 expression is seen ...

The genotyping analysis of the newborn offspring from the crosses of Wnt1-Cre;Shox2+/− and Shox2F/F mice identified surviving Wnt1-Cre;Shox2F/− mice, indicating that the conditional inactivation of Shox2 indeed circumvents embryonic lethality. These mutant mice appeared indistinguishable from their control littermates before postnatal day (P) 3. However, from P4 on, the mutants began to develop a wasting syndrome, appearing smaller in body size as compared with their wild-type and heterozygous littermates (Fig. 2). The wasting syndrome became more severe as the mice grew and, eventually, led to death of the mice. Of 15 mutant mice that were closely monitored, 13 died before or at P15, and the other 2 were killed at P15 due to their sick appearance.

Fig. 2
Wnt1-Cre;Shox2F/− mice develop a wasting syndrome. A,B: Comparison of wild-type mice (Wt) and the mutants (Mt) at postnatal day 4 (A) and postnatal day 15 (B) reveals a significantly reduced body size of the mutants.

Wnt1-Cre;Shox2F/1 Mice Exhibit Anterior Clefting

We have previously reported that Shox2-deficient embryos develop an anterior clefting of the secondary palate (Yu et al., 2005). To our surprise, a gross examination of Wnt1-Cre;Shox2F/− mice (n = 6) at P15 under the dissecting microscope revealed no recognizable defects in the palate (Fig. 3G,H). To determine whether a cleft palate defect never develops in Wnt1-Cre;Shox2F/− mice or if a cleft palate defect appears at the early embryonic stage but is recovered during late embryonic development and postnatal growth, we examined the palate of Wnt1-Cre; Shox2F/− mice at different developmental stages by scanning electron microscopy. At E14.5, when the paired secondary palatal shelves have closed and fused at the midline in the wild-type control (Fig. 3A), a large gap between the paired secondary palatal shelves was present in the anterior region (Fig. 3B), which is identical to the phenotype that has been reported previously in Shox2-deficient embryo at the same stage (Yu et al., 2005). At E17.5, similarly to the results reported previously, the cleft, although smaller in appearance, remained in the anterior region of the mutant palate (Fig. 3D). While in the wild-type control at the same stage, the primary and secondary palates have almost fused, leaving two small holes known as orifices of incisive canal (Fig. 3C). Interestingly, at P0, the cleft appeared extremely small in the mutant, being only visible under the higher magnification (Fig. 3E,F). Histological examination confirmed a failure of the fusion between the primary and secondary palates in Wnt1-Cre;Shox2F/− embryos and newborn mice, which results in a cleft palate defect in the anterior extreme of the palate (data not shown). The clefting phenotype was found in all postnatal samples examined (n = 15), including six P15 mice (Fig. 3I,J).

Fig. 3
Wnt1-Cre;Shox2F/− mice exhibit a delayed closure of the secondary palate but a failed fusion between the primary and the secondary palate. A,C,E: Scanning electron microscopy (SEM) images show ventral view of the palate from wild-type mice at ...

Unlike human patients with a nonsyndromic cleft palate defect who are viable, rodents with a cleft palate defect die inevitably at birth. Our results show that mice bearing an anterior clefting defect, although relatively mild, can survive neonatal lethality. These mice were indeed able to suck milk, as evidenced by the presence of milk in the stomach (data not shown). However, all mutant mice developed a wasting syndrome after P3 and eventually died. The underlying mechanisms for the development of the wasting syndrome are not clear, but an inefficient sucking action due to the presence of the clefting could be a cause. Furthermore, a developmental defect found in the temporomandibular joint of the mutants could also account for an inefficient food-taking (Gu et al., manuscript submitted for publication).

Our observations also revealed a delayed closure and fusion of the secondary palatal shelves in Wnt1-Cre; Shox2F/− mice. The anterior portion of the mutant secondary palatal shelves was able to grow continuously and fuse eventually, although it was delayed in development. In the wild-type controls, the developing palatal shelves maintain a relatively high level of cell proliferation during the growth stage (Fig. 4A). After palate closure, the palatal mesenchymal cells begin to differentiate, and cell proliferation rate drops accordingly (Fig. 4C). Interestingly, although the cell proliferation rate was significantly reduced in the mutant palatal mesenchyme as compared with that in the wild-type controls at E13.5 (Fig. 4B), the proliferation rate was increased in the anterior portion of the palatal shelves that had not yet made contact at E15.5 (Fig. 4D,E), reaching a level comparable to that found in the E13.5 wild-type palatal shelves (Fig. 4E). This elevated level of cell proliferation in the mutant palatal mesenchyme could account for, at least partially, the late catch-up of the delayed palate closure. In contrast to the ability of a delayed fusion between the secondary palatal shelves, the secondary palate was unable to fuse with the primary palate in a developmentally delayed manner, resulting in the formation of an anterior cleft. Thus, the contact between the primary and secondary palates at the appropriate developmental timing appears to be critical for the palatal fusion.

Fig. 4
Bromodeoxyuridine (BrdU) labeling of cell proliferation in the developing palate. A,B: A section through the anterior palate shelf of embryonic day (E) 13.5 mutant embryo shows a lower level of cell proliferation (B), as compared with a wild-type control ...

Reduced Osteogenesis in the Hard Palate of Wnt1-Cre;Shox2F/− Mice

After the closure of the palate, the anterior two-thirds of the palate undergo intramembranous ossification and form the hard palate, while the posterior third represents the bone-free region of the soft palate (Greene and Pratt, 1976; Ferguson, 1988). Because the SHOX genes have been implicated in skeletogenesis in humans and rodents (Clement-Jones et al., 2000; Zinn et al., 2002; Cobb et al., 2006; Yu et al., 2007), we set out to examine potential osteogenic defects in the hard palate of Wnt1-Cre;Shox2F/− mice at the age of P15 by using the Azon red/Anilin blue staining method, which stains bone matrix in red, and in situ hybridization assays for the expression of osteocalcin, an osteoblast-specific gene. In the wild-type controls, ossificated bone matrix and osteocalcin expression could be readily detected (Fig. 5A,C). In contrast, a very small amount of bone was found in the mutant (Fig. 5B). Accordingly, Osteocalcin expression was also barely detected (Fig. 5D). These results demonstrate a dramatically reduced level of osteogenesis in the hard palate of Wnt1-Cre;Shox2F/− mice, and support a role for Shox2 in skeletogenesis. In addition, the mutant hard palate appeared much thinner as compared with the wild-type controls (Fig. 5). In addition, no increased muscle mass was found in the mutant palate (data not shown).

Fig. 5
The hard palate of Wnt1-Cre;Shox2F/− mice exhibit significantly reduced bone formation. A: Large amount of bone (arrows) is seen in a section through the hard palate of a postnatal day (P) 15 wild-type mouse. B: A much smaller amount of bone is ...

Runx2, a Runt-domain transcription factor, is essential for the osteoblast differentiation and bone formation (Ducy et al., 1997; Komori et al., 1997; Otto et al., 1997). Runx2 expression was noticed in the dorsal-nasal aspect of the developing palate at E13.5 (Yamashiro et al., 2002), and its inactivation led to cleft palate formation in the majority of Runx2 mutants (Aberg et al., 2004), implicating Runx2 in palatogenesis. Furthermore, Shox2 has been shown to regulate Runx2 expression in the developing limbs (Cobb et al., 2006; Yu et al., 2007). We therefore asked if Shox2 regulates Runx2 expression in the developing palate. In the wild-type controls at E12.5 and E13.5, Runx2 expression is seen in the palatal mesenchyme in the dorsal-nasal aspect of the palatal shelves (Fig. 6A, data not shown). However, its expression domain is restricted in the posterior region of the developing palate, with its anterior boundary falling into the posterior third of the first molar. The Runx2 expression domain is thus complementary to that of Shox2 along the A-P axis (Yu et al., 2005). The expression of Osterix, a known Runx2 downstream gene that is also essential for the osteoblast differentiation and bone formation (Nakashima et al., 2002), was detected in an overlapping domain with that of Runx2 in the palatal shelves (Fig. 6C). Despite that the expression of Runx2 and Osterix does not overlap with that of Shox2, a dramatically down-regulated expression was observed for both Runx2 and Osterix in the palate Wnt1-Cre;Shox2F/− embryo (Fig. 6B,D), which could account for the significantly reduced osteogenesis.

Fig. 6
Altered expression of osteogenic genes in the palatal shelves of Wnt1-Cre;Shox2F/− embryo. A,C: In the wild-type controls, the expression of Runx2 (A) and Osterix (C) is seen in the dorsal-nasal aspect in the posterior palate of embryonic day ...

Because Shox2 expression does not overlap with that of Runx2 and Osterix in the developing palate, Shox2 apparently does not directly regulate Runx2 and Osterix. A diffusible factor(s) regulated by Shox2 may be responsible for this regulation. This holds true for the developing limbs where Shox2 regulates Runx2 expression by controlling Bmp4 expression (Yu et al., 2007). In the absence of Shox2, the elevated and ectopic Bmp4 expression was thought to be responsible for the repression of Runx2 in the adjacent tissue of the developing limbs. Both Bmp2 and Bmp4 are expressed in the palatal mesenchyme, in a pattern overlapping with Shox2 (Zhang et al., 2002, reviewed in Gritli-Linde, 2007). We subsequently surveyed the expression of Bmp2 and Bmp4 in the developing palatal shelves and confirmed an unaltered Bmp4 expression in the mutants (Yu et al., 2005, data not shown). However, we indeed observed a changed expression pattern for Bmp2. In the anterior palate of the mutant, Bmp2 expression was highly elevated in the palatal mesenchyme as compared to the wild-type control (Fig. 6E,F). In the middle region, at a section plane through the first molar, Bmp2 was ectopically activated in the palatal mesenchyme in the mutant, as compared to the wild-type control where Bmp2 expression was barely detectable or was just above the background level (Fig. 6G,H). Given the fact that excess bone morphogenetic protein-4 (BMP4) inhibits Runx2 expression in the developing limbs (Yu et al., 2007), it is conceivable that a similar mechanism is used in the developing palate. To test this hypothesis, we applied exogenous BMP2 to the wild-type palatal shelves in organ culture, and examined the expression of Runx2 and its downstream gene Osterix. Our results demonstrate that BMP2-soaked (100 ng/μl) beads were indeed able to inhibit the expression of both Runx2 (four of five) and Osterix (four of four) in the palate (Fig. 7A,C). As controls, bovine serum albumin beads (100 ng/μl) failed to repress the expression of these two genes (eight of eight, Fig. 7B,D).

Fig. 7
Repression of Runx2 and Osterix by exogenous bone morphogenetic protein-2 (BMP2) in the palatal shelves. A–D: Reduced expression of Runx2 (A) and Osterix (C) is seen in the palatal shelves implanted with BMP2 beads, as compared with their controls ...

As it was shown in previous studies, many transcription factors and growth factors have been implicated in the normal palatogenesis (Gritli-Linde, 2007). In the developing palate, which relies heavily on the epithelial–mesenchymal interactions, transcription factors have been proposed to control the interactions by regulating the expression of growth factors. This is exemplified by the regulation of Fgf10 and Bmp2 expression by Shox2. In addition, Shox2 expression in the palate requires BMP activities (Yu et al., 2005). Considering the fact that Msx1, Bmp4, Bmp2, Shh, and Fgf10 form a complex genetic network to regulate palate development, Shox2 appears to be a critical component in this network (reviewed in Hilliard et al., 2005; Gritli-Linde, 2007).

The observations that the secondary palate is able to recover from a delayed anterior palatal closure, despite that fact that it fails to fuse with the primary palate, indicate that Shox2 is not absolutely required for the closure and fusion of the secondary palatal shelves. Although the mutant palatal shelves failed to make contact in the anterior domain on time due to their reduced size, they were able to grow continuously, and eventually to make contact and to fuse in a posterior-to-anterior sequence as the embryo develops. The increased cell proliferation rate in the uncontacted portion of the palatal shelves may contribute, at least partially, to the eventual closure. The results indicate that the fusion of the secondary palatal shelves can occur in a developmentally delayed manner. However, this is not true for the fusion between the primary and secondary palates. The failed fusion leads to the formation of an anterior cleft, which apparently does not cause neonatal lethality. However, this anterior cleft may attribute, at least partially, to the development of a wasting syndrome, which eventually leads to the death of the mutant mice.

The existence of a genetic heterogeneity along the A-P axis of the developing palate may provide differential regulatory mechanisms for the growth, patterning, and differentiation of the anterior vs. posterior region of the palate. However, it is not clear if cross-talk between the anterior and posterior palate exists. The fact that the expression of Runx2 and Osterix is altered in the palate of Shox2 mutants provides evidence to support the hypothesis that genetic communications occur between the anterior and posterior palate. These communications are likely mediated by signaling molecules. Shox2 thus appears to control, in the anterior palate, the expression of diffusible factors that can travel and exert their effects long distant. However, we can not rule out the possibility that the posterior palatal cells are derived from the Shox2-expressing cells in the anterior domain. Although these posterior cells do not express Shox2, they may have already been imprinted with Shox2 activity. This hypothesis appears to be at odds with a previously proposed model, in which the formation of the anterior secondary palate was thought to involve a recruitment of cells from the posterior region (Li and Ding, 2007). Nevertheless, it warrants future research on whether or not and how the cross-talk occurs between the anterior and posterior palate during palatogenesis.

EXPERIMENTAL PROCEDURES

Animals

Mice carrying a conventionally inactivated Shox2 allele or a floxed Shox2 allele have been described previously (Yu et al., 2005; Cobb et al., 2006). The Wnt1-Cre transgenic mice (Danielian et al., 1998) were obtained from the Jackson Laboratories. To obtain Wnt1-Cre; Shox2F/− mice, the mice carrying both Wnt1-Cre and Shox2+/− alleles are crossed with Shox2F/F mice. The embryonic age was defined as the E0.5 in the morning of the day when the vaginal plug was detected. The genotypes of these mice were determined by polymerase chain reaction–based methods, as described previously (Chai et al., 2000; Yu et al., 2005; Cobb et al., 2006), using genomic DNAs isolated from the tails.

Histology and Scanning Electron Microscopy

For a histological analysis, mouse heads were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4°C overnight. Heads older than E17.5 were decalcified in 10% ethylenediaminetetraacetic acid in fixative for various days (from 4 to 30 days, depending on the age of the mouse) before further processing. Paraffin-embedded samples were sectioned at 6 μm and subjected to hematoxylin/eosin staining or Azon red/Anilin blue staining, as described (Presnell and Schreibman, 1997). For scanning electron microscopy, samples were fixed in 2.5% glutaradehydrate/0.1 M sodium cacodylate at 4°C for 12 hr, followed by post-fixation in 1% osmium tetroxide/0.1 M sodium cacodylate. Samples were further dehydrated, trimmed, dried, coated with gold, and examined under a scanning electron microscope, as previously described (Zhang et al., 2002).

In Situ Hybridization and Detection of Cell Proliferation

The samples used for in situ hybridization were fixed in 4% PFA/PBS. For section in situ hybridization, samples were ethanol-dehydrated, paraffin embedded, and sectioned at 10 μm. For whole-mount in situ hybridization, samples were bleached in 6% H2O2 after fixation, dehydrated through a graded methanol series. Nonradioactive RNA probes were generated by in vitro transcription labeling with digoxigenin-UTP following the manufacturer’s instruction. In situ hybridization was performed as described previously (Zhang et al., 1999). For the detection of cell proliferation, the samples were labeled with bromodeoxyuridine (BrdU) for 2 hr by means of intraperitoneal injection of BrdU labeling reagent into timed pregnant mice at a dose of 1.5 ml of labeling reagent/100 g body weight using BrdU labeling and Detection Kit II from Roche. The samples were fixed, dehydrated, sectioned at 5 μm, and subjected to immunodetection as described previously (Zhang et al., 2002).

In Vitro Organ Culture and Bead Implantation

The secondary palatal shelves from E12.5 wild-type embryos were micro-dissected in cold PBS, and placed on filter in the Trowell type organ culture. Affi-Gel blue agarose beads (from Bio-Rad, Hercules, CA) were soaked with BMP2 protein (from R&D Systems, Minneapolis, MN) at the concentration of 100 ng/μl, and were then implanted into the palatal tissues on the filter, as previously described (Chen et al., 1996). Samples were cultured in DMEM supplemented with 10% fetal bovine serum for 24 hr before being harvested for whole-mount in situ hybridization analysis.

Acknowledgments

We thank Drs. Dennis Duboule and John Cobb for providing Shox2 floxed mice, and Dr. Yang Chai for mouse cDNA probes. We also thank Mr. Catalin Anghelina of the Chen laboratory for his excellent editing work on the manuscript. Y.P.C. was funded by the NIH, and S.G. was funded by the National Natural Science Foundation of China.

Grant sponsor: NIH; Grant number: R01DE12329; Grant number: R01DE14044; Grant sponsor: National Natural Science Foundation of China; Grant number: 30671022.

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