A Comprehensive Study of Soft Palate Development in Mice

Cleft palate is one of the most common congenital birth defects. Tremendous efforts have been made over the last decades towards understanding hard palate development. However, little is known about soft palate morphogenesis and myogenesis. Finding an appropriate surgical repair to restore physiological functions of the soft palate in patients with cleft palate is a major challenge for surgeons, and complete restoration is not always achievable. Here, we first analyzed the morphology, orientation and attachments of the four muscles of the murine soft palate and found that they are very similar to their counterparts in humans, validating the use of mus musculus as a model for future studies. Our data suggests that muscle differentiation extends from the lateral region to the midline following palatal fusion. We also detected an epithelial seam in the fusing soft palatal shelves, consistent with the process of fusion of the posterior palatal shelves, followed by degradation of the epithelial remnants. Innervation and vascularization are present mainly in the oral side of the soft palate, complementing the differentiated muscles. Cell lineage tracing using Wnt1-Cre;Zsgreen ^fl/fl mice indicated that all the tendons and mesenchyme embedding the soft palate muscles are neural crest-derived. We propose that the posterior attachment of the soft palate to the pharyngeal wall is an interface between the neural crest- and mesoderm-derived mesenchyme in the craniofacial region, and thus can serve as a potential model for the study of boundaries during development. Taken together, our study provides a comprehensive view of the development and morphology of the murine soft palate and serves as a reference for further molecular analyses.     

Experimental induction of palate shelf elevation in glutamate decarboxylase 67-deficient mice with cleft palate due to vertically oriented palatal shelf

BACKGROUND: Gamma-aminobutyric acid is an inhibitory neurotransmitter, synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65 and -67. Unexpectedly, inactivation of GAD67 induces cleft palate in mice. Reduction of spontaneous tongue movement resulting from decreased motor nerve activity has been related to the development of cleft palate in GAD67(-/-) fetuses. In the present study, development of cleft palate was examined histologically and manipulated with culture of the maxilla and partial resection of fetal tongue. METHODS: GAD67(-/-) mice and their littermates were used. Histological examination and immunohistochemistry were performed conventionally. Organ culture of the maxilla was carried out as reported previously. Fetuses were maintained alive under anesthesia and tips of their tongues were resected. RESULTS: Elevation of palatal shelves, the second step of palate formation, was not observed in GAD67(-/-) mice. In wild-type mice, GAD67 and gamma-aminobutyric acid were not expressed in the palatal shelves, except in the medial edge epithelium. During 2 days of culture of maxillae dissected from E13.5-E14.0 GAD67(-/-) fetuses, elevation and fusion of the palatal shelves were induced. When E13.5-15.5 mutant fetuses underwent partial tongue resection, the palatal shelves became elevated within 30 min. CONCLUSIONS: These results suggest that the potential for palate formation is maintained in the palatal shelves of GAD67(-/-) fetuses, but it is obstructed by other, probably neural, factors, resulting in cleft palate.     

A unique mouse strain expressing Cre recombinase for tissue-specific analysis of gene function in palate and kidney development

Mammalian palate development is a multistep process, involving initial bilateral downward outgrowth of the palatal shelves from the oral side of the maxillary processes, followed by stage-specific palatal shelf elevation to the horizontal position above the developing tongue and subsequent fusion of the bilateral palatal shelves at the midline to form the intact roof of the oral cavity. While mutations in many genes have been associated with cleft palate pathogenesis, the molecular mechanisms regulating palatal shelf growth, patterning, and elevation are not well understood. Genetic studies of the molecular mechanisms controlling palate development in mutant mouse models are often complicated by early embryonic lethality or gross craniofacial malformation. We report here the development of a mouse strain for tissue-specific analysis of gene function in palate development. We inserted an IresCre bicistronic expression cassette into the 3' untranslated region of the mouse Osr2 gene through gene targeting. We show, upon crossing to the R26R reporter mice, that Cre expression from the Osr2(IresCre) knockin allele activated beta-galactosidase expression specifically throughout the developing palatal mesenchyme from the onset of palatal shelf outgrowth. In addition, the Osr2(IresCre) mice display exclusive Cre-mediated recombination in the glomeruli tissues derived from the metanephric mesenchyme and complete absence of Cre activity in other epithelial and mesenchymal tissues in the developing metanephric kidney. These data indicate that the Osr2(IresCre) knockin mice provide a unique tool for tissue-specific studies of the molecular mechanisms regulating palate and kidney development.     

An in vitro mouse model of cleft palate: defining a critical intershelf distance necessary for palatal clefting

It is unclear whether cleft palate formation is attributable to intrinsic biomolecular defects in the embryonic elevating palatal shelves or to an inability of the shelves to overcome a mechanical obstruction (such as the tongue in Pierre Robin sequence) to normal fusion. Regardless of the specific mechanism, presumably embryonic palatal shelves are ultimately unable to bridge a critical distance and remain unapproximated, resulting in a clefting defect at birth. We propose to use a palate organ culture system to determine the critical distance beyond which embryonic palatal shelves fail to fuse (i.e., the minimal critical intershelf distance). In doing so, we hope to establish an in vitro cleft palate model that could then be used to investigate the contributions of various signaling pathways to cleft formation and to study novel in utero treatment strategies.Palatal shelves from CD-1 mouse embryos were microdissected on day 13.5 of gestation (E13.5; term = 19.5 days), before fusion. Using a standardized microscope ocular grid, paired palatal shelves were placed on a filter insert at precisely graded distances ranging from 0 (in contact) to 1.9 mm (0, 0.095, 0.19, 0.26, 0.38, 0.48, 0.57, 0.76, 0.95, and 1.9 mm). A total of 68 paired palatal shelves were placed in serum-free organ culture for 96 hours (n = 68). Sample sizes of 10 were used for each intershelf distance up to and including 0.48 mm (n = 60). For intershelf distances of 0.57 mm and greater, two-paired palatal shelves were cultured (n = 8). All specimens were assessed grossly and histologically for palatal fusion.Palatal fusion occurred in our model only when intershelf distances were 0.38 mm or less. At 0.38 mm, eight of 10 palates appeared grossly adherent, whereas six of 10 demonstrated clear fusion histologically with resolution of the medial epithelial seam and continuity of the palatal mesenchyme. None of the 18 palates fused when placed at intershelf distances of 0.48 mm or greater.Using our selected intershelf distances as a guideline, we have established an approximate minimal critical intershelf distance (0.48 mm) at which we can reliably expect no palatal fusion. Culturing palatal shelves at intershelf distances of 0.48 mm or greater results in nonfusion or clefting in vitro. This model will allow us to study biomolecular characteristics of unfused or cleft palatal shelves in comparison with fused shelves. Furthermore, we plan to study the efficacy of grafting with exogenous embryonic mesenchyme or candidate factors to overcome clefting in vitro as a first step toward future in utero treatment strategies.     

Retinoic acid alters epithelial differentiation during palatogenesis.

Retinoids are teratogenic in humans and animals, producing a syndrome of craniofacial malformations that includes cleft palate. This study investigates the mechanism through which retinoic acid induces cleft palate. Murine palatogenesis after exposure to retinoic acid in utero is compared to normal development and to alterations observed after exposure in organ culture to retinoic acid or epidermal growth factor (EGF). Human embryonic palatal shelves were placed in the organ culture system and the responses to retinoic acid and EGF were compared to those of the murine palatal shelves. Growth factors play a role in normal development and are found in the embryonic palate. In other cell culture systems, retinoids alter the expression of EGF receptors. Our results suggest that in the medial epithelial cells of the palate, retinoic acid sustains the expression of the EGF receptor and the binding of EGF at a time when the expression in control medial cells has declined, and these control cells subsequently undergo programmed cell death. The continued DNA synthesis, proliferation, survival, and shift in phenotype of the medial cells is believed to interfere with the adhesion and fusion of opposing palatal shelves, resulting in cleft palate.     

The inductive role of Wnt-β-Catenin signaling in the formation of oral apparatus

Proper patterning and growth of oral structures including teeth, tongue, and palate rely on epithelial–mesenchymal interactions involving coordinated regulation of signal transduction. Understanding molecular mechanisms underpinning oral–facial development will provide novel insights into the etiology of common congenital defects such as cleft palate. In this study, we report that ablating Wnt signaling in the oral epithelium blocks the formation of palatal rugae, which are a set of specialized ectodermal appendages serving as Shh signaling centers during development and niches for sensory cells and possibly neural crest related stem cells in adults. Lack of rugae is also associated with retarded anteroposterior extension of the hard palate and precocious mid-line fusion. These data implicate an obligatory role for canonical Wnt signaling in rugae development. Based on this complex phenotype, we propose that the sequential addition of rugae and its morphogen Shh, is intrinsically coupled to the elongation of the hard palate, and is critical for modulating the growth orientation of palatal shelves. In addition, we observe a unique cleft palate phenotype at the anterior end of the secondary palate, which is likely caused by the severely underdeveloped primary palate in these mutants. Last but not least, we also discover that both Wnt and Shh signalings are essential for tongue development. We provide genetic evidence that disruption of either signaling pathway results in severe microglossia. Altogether, we demonstrate a dynamic role for Wnt-β-Catenin signaling in the development of the oral apparatus.     

Temporal and Spatial Expression of Hoxa-2 During Murine Palatogenesis

1. Mice homozygous for a targeted mutation of the Hoxa-2 gene are born with a bilateral cleft of the secondary palate associated with multiple head and cranial anomalies and these animals die within 24 hr of birth (Gendron-Maguire et al., 1993; Rijli et al., 1993; Mallo and Gridley, 1996). We have determined the spatial and temporal expression of the Hoxa-2 homeobox protein in the developing mouse palate at embryonic stages E12, E13, E13.5, E14, E14.5, and E15.2. Hoxa-2 is expressed in the mesenchyme and epithelial cells of the palate at E12, but is progressively restricted to the tips of the growing palatal shelves at E13.3. By the E13.5 stage of development, Hoxa-2 protein was found to be expressed throughout the palatal shelf. These observations correlate with palatal shelf orientation and Hoxa-2 protein may play a direct or indirect role in guiding the palatal shelves vertically along side the tongue, starting with the tips of the palatal shelves at E13, followed by the entire palatal shelf at E13.5.4. As development progresses to E14, the stage at which shelf elevation occurs, Hoxa-2 protein is downregulated in the palatal mesenchyme but remains in the medial edge epithelium. Expression of Hoxa-2 continues in the medial edge epithelium until the fusion of opposing palatal shelves.5. By the E15 stage of development, Hoxa-2 is downregulated in the palate and expression is localized in the nasal and oral epithelia.6. In an animal model of phenytoin-induced cleft palate, we report that Hoxa-2 mRNA and protein expression were significantly decreased, implicating a possible functional role of the Hoxa-2 gene in the development of phenytoin-induced cleft palate.7. A recent report by Barrow and Capecchi (1999), has illustrated the importance of tongue posture during palatal shelf closure in Hoxa-2 mutant mice. This along with our new findings of the expression of the Hoxa-2 protein during palatogenesis has shed some light on the putative role of this gene in palate development.     

Palate development after fetal tongue removal in cortisone-treated mice

Morphological studies of cortisone-induced cleft palate have shown retardation in the rotation of palatine shelves from a sagittal to a transverse plane. Cortisone also reduces fetal muscular movements, which may explain why displacement of the tongue from between the palatine shelves is delayed. Previous work with extrauterine development of control fetuses demonstrated that fetal membranes and tongue were major obstacles to shelf rotation. Thus, removal of these obstacles might permit rotation and fusion of palatine shelves in cortisone-treated fetuses. In the present experiment, fetuses from cortisone-treated strain CD-1 mice were released from uterus and membranes and allowed to develop for eight hours in a fluid medium with the umbilical cord left intact. Compared to 4% fusion in utero, there was palatal fusion in 20% of fetuses released from membranes. When the fetal tongue was removed during extrauterine development, the frequency of fusions increased to 61%. Fusion appeared normal by the criteria applicable through light microscopy. Thus, cortisone induces cleft palate primarily through interference with shelf rotation. The palatine shelves of treated fetuses retain their ability to fuse when they can come in contact during the normal time for palate closure.     

Nonneuronal expression of the GABA(A) beta3 subunit gene is required for normal palate development in mice

Cleft palate is one of the most common birth defects in humans, in which both genetic and environmental factors are involved. In mice, loss of the GABA(A) receptor beta3 subunit gene (Gabrb3) or the targeted mutagenesis of the GABA synthetic enzyme (Gad1) leads to cleft palate. These observations indicate that a GABAergic system is important in normal palate development. To determine what cell types, neuronal or nonneuronal, are critical for GABA signaling in palate development, we used the neuron-specific enolase promoter to express the beta3 subunit in Gabrb3 mutant mice. Expression of this construct was able to rescue the neurological phenotype, but not the cleft palate phenotype. Combined with the previous observation demonstrating that ubiquitous expression of the beta3 subunit rescued the cleft palate phenotype, a nonneuronal GABAergic system is implicated in palate development. Using immunohistochemistry, we detected GABA in the developing palate, initially in the nasal aspect of palatal epithelium of the vertical shelves; later in the medial edge epithelium of the horizontally oriented palatal shelves and in the epithelial seam during fusion. Based on these observations, we propose that GABA, synthesized by the palatal epithelium, acts as a signaling molecule during orientation and fusion of the palate shelves.     

Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1(-/-) palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.     

Bmpr1a signaling plays critical roles in palatal shelf growth and palatal bone formation

Cleft palate, including submucous cleft palate, is among the most common birth defects in humans. While overt cleft palate results from defects in growth or fusion of the developing palatal shelves, submucous cleft palate is characterized by defects in palatal bones. In this report, we show that the Bmpr1a gene, encoding a type I receptor for bone morphogenetic proteins (Bmp), is preferentially expressed in the primary palate and anterior secondary palate during palatal outgrowth. Following palatal fusion, Bmpr1a mRNA expression was upregulated in the condensed mesenchyme progenitors of palatal bone. Tissue-specific inactivation of Bmpr1a in the developing palatal mesenchyme in mice caused reduced cell proliferation in the primary and anterior secondary palate, resulting in partial cleft of the anterior palate at birth. Expression of Msx1 and Fgf10 was downregulated in the anterior palate mesenchyme and expression of Shh was downregulated in the anterior palatal epithelium in the Bmpr1a conditional mutant embryos, indicating that Bmp signaling regulates mesenchymal-epithelial interactions during palatal outgrowth. In addition, formation of the palatal processes of the maxilla was blocked while formation of the palatal processes of the palatine was significantly delayed, resulting in submucous cleft of the hard palate in the mutant mice. Our data indicate that Bmp signaling plays critical roles in the regulation of palatal mesenchyme condensation and osteoblast differentiation during palatal bone formation.     

Temporal requirement of Hoxa2 in cranial neural crest skeletal morphogenesis

Little is known about the spatiotemporal requirement of Hox gene patterning activity in vertebrates. In Hoxa2 mouse mutants, the hyoid skeleton is replaced by a duplicated set of mandibular and middle ear structures. Here, we show that Hoxa2 is selectively required in cranial neural crest cells (NCCs). Moreover, we used a Cre-ERT2 recombinase system to induce a temporally controlled Hoxa2 deletion in the mouse. Hoxa2 inactivation after cranial NCC migration into branchial arches resulted in homeotic transformation of hyoid into mandibular arch skeletal derivatives, reproducing the conventional Hoxa2 knockout phenotype, and induced rapid changes in Alx4, Bapx1, Six2 and Msx1 expression patterns. Thus, hyoid NCCs retain a remarkable degree of plasticity even after their migration in the arch, and require Hoxa2 as an integral component of their morphogenetic program. Moreover, subpopulations of postmigratory NCCs required Hoxa2 at discrete time points to pattern distinct derivatives. This study provides the first temporal inactivation of a vertebrate Hox gene and illustrates Hox requirement during late morphogenetic processes.     

The Etiology of Cleft Palate Formation in BMP7-Deficient Mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.     

Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus

Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. Here, we use an inducible system to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. We uncover stage-specific effects of Hoxa2 gain-of-function suggesting a multistep patterning process for hindbrain NC. Most interestingly, we show that Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, normally devoid of Hox expression, towards hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws.