Pathogenesis of cleft palate in TGF-beta3 knockout mice.

We previously reported that mutation of the transforming growth factor-beta3 (TGF-beta3) gene caused cleft palate in homozygous null (-/-) mice. TGF-beta3 is normally expressed in the medial edge epithelial (MEE) cells of the palatal shelf. In the present study, we investigated the mechanisms by which TGF-beta3 deletions caused cleft palate in 129 x CF-1 mice. For organ culture, palatal shelves were dissected from embryonic day 13.5 (E13.5) mouse embryos. Palatal shelves were placed singly or in pairs on Millipore filters and cultured in DMEM/F12 medium. Shelves were placed in homologous (+/+ vs +/+, -/- vs -/-, +/- vs +/-) or heterologous (+/+ vs -/-, +/- vs -/-, +/+ vs +/-) paired combinations and examined by macroscopy and histology. Pairs of -/- and -/- shelves failed to fuse over 72 hours of culture whereas pairs of +/+ (wild-type) and +/+ or +/- (heterozygote) and +/-, as well as +/+ and -/- shelves, fused within the first 48 hour period. Histological examination of the fused +/+ and +/+ shelves showed complete disappearance of the midline epithelial seam whereas -/- and +/+ shelves still had some seam remnants. In order to investigate the ability of TGF-beta family members to rescue the fusion between -/- and -/- palatal shelves in vitro, either recombinant human (rh) TGF-beta1, porcine (p) TGF-beta2, rh TGF-beta3, rh activin, or p inhibin was added to the medium in different concentrations at specific times and for various periods during the culture. In untreated organ culture -/- palate pairs completely failed to fuse, treatment with TGF-beta3 induced complete palatal fusion, TGF-beta1 or TGF-beta2 near normal fusion, but activin and inhibin had no effect. We investigated ultrastructural features of the surface of the MEE cells using SEM to compare TGF-beta3-null embryos (E 12. 5-E 16.5) with +/+ and +/- embryos in vivo and in vitro. Up to E13.5 and after E15.5, structures resembling short rods were observed in both +/+ and -/- embryos. Just before fusion, at E14.5, a lot of filopodia-like structures appeared on the surface of the MEE cells in +/+ embryos, however, none were observed in -/- embryos, either in vivo or in vitro. With TEM these filopodia are coated with material resembling proteoglycan. Interestingly, addition of TGF-beta3 to the culture medium which caused fusion between the -/- palatal shelves also induced the appearance of these filopodia on their MEE surfaces. TGF-beta1 and TGF-beta2 also induced filopodia on the -/- MEE but to a lesser extent than TGF-beta3 and additionally induced lamellipodia on their cell surfaces. These results suggest that TGF-beta3 may regulate palatal fusion by inducing filopodia on the outer cell membrane of the palatal medial edge epithelia prior to shelf contact. Exogenous recombinant TGF-beta3 can rescue fusion in -/- palatal shelves by inducing such filopodia, illustrating that the effects of TGF-beta3 are transduced by cell surface receptors which raises interesting potential therapeutic strategies to prevent and treat embryonic cleft palate.     

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.     

Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.     

Overexpression of iNOS and down-regulation of BMPs-2, 4 and 7 in retinoic acid induced cleft palate formation

The present work studied the induction of cleft palate formation in embryos developed from pregnant BALB/c mice treated orally with retinoic acid (RA). Previous studies on mature somatic cell types showed that RA exerted inhibitory effects on inducible nitric oxide synthase (iNOS) production. For the first time, our study has shown that RA actually stimulates significant expression of iNOS at specific zones of the affected embryonic palatal tissues at three consecutive stages, from gestation day 13 (GD13) to day 16 (GD16). Enzymatically, iNOS facilitates intracellular nitric oxide (NO) synthesis from L-arginine. When NO reacts with reactive superoxides it may result in irreparable cell injury. NO was also reported to induce apoptosis in some mammalian cell systems. Based on our findings, we propose that such an increase in NO production might be associated with apoptosis in the embryonic palatal tissues in the RA-treated mice. The detrimental effects of NO resulted in a reduction in proliferating palatal cells and therefore disturbed the normal plasticity of the palatal shelves. With iNOS overexpression, our findings also showed that there was significant concomitant down-regulation in the expressions of Bone Morphogenetic Proteins (BMPs) -2, 4, and 7 with regional variations particularly in the palatal mesenchymal cells for those embryos developing cleft palate. Since specific spatial and temporal expressions of BMPs -2, 4, and 7 are critical during normal palatal morphogenesis, any deficiency in the epithelial-mesenchymal interaction may result in retarding growth at the embryonic palatal shelves. Taken together, our study has demonstrated cleft palate formation in the BALB/c embryos involved overexpression of iNOS and down-regulation of BMPs-2, 4 and 7.     

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.     

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.     

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.     

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.     

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.     

Strain differences between C57BL/6 and SWV mice in time of palate closure and induction of palatal slit and cleft palate

Palatal slit, which occurs spontaneously in C57BL/6 (C57BL) mice, is increased in frequency among C57BL fetuses from dams treated with triamcinolone acetonide, but is not induced in SWV fetuses. On the other hand, C57BL is more resistant than SWV to cleft palate induction by triamcinolone. Using these C57BL and SWV mice, the relation of palate stage and chronological age was examined from 1 P.M. on day 14 to 9 A.M. on day 16 in untreated embryos, and the condition of the palate after triamcinolone treatment on day 12 was examined at 9 A.M. on day 16. In untreated embryos, horizontalization and fusion of the palatal shelves occurred earlier in C57BL than in SWV embryos, but fusion of the primary palate with the secondary palate occurred later. After triamcinolone treatment, the development of the palate was delayed in both C57BL and SWV embryos. These results suggest that the times of normal palate closure are related to the differences between C57BL and SWV mice in their susceptibilities to palatal slit and cleft palate induction and that triamcinolone produces palatal slit and cleft palate by delaying palate closure.     

Snail family members and cell survival in physiological and pathological cleft palates

Palate fusion is a complex process that involves the coordination of a series of cellular changes including cell death and epithelial to mesenchymal transition (EMT). Since members of the Snail family of zinc-finger regulators are involved in both triggering of the EMT and cell survival, we decided to study their putative role in palatal fusion. Furthermore, Snail genes are induced by transforming growth factor beta gene (TGF-beta) superfamily members, and TGF-beta(3) null mutant mice (TGF-beta(3)-/-) show a cleft palate phenotype. Here we show that in the wild-type mouse at the time of fusion, Snail is expressed in a few cells of the midline epithelial seam (MES), compatible with a role in triggering of the EMT in a small subpopulation of the MES. We also find an intriguing relationship between the expression of Snail family members and cell survival associated to the cleft palate condition. Indeed, Snail is expressed in the medial edge epithelial (MEE) cells in TGF-beta(3)-/-mouse embryo palates, where it is activated by the aberrant expression of its inducer, TGF-beta(1), in the underlying mesenchyme. In contrast to Snail-deficient wild-type pre-adhesion MEE cells, Snail-expressing TGF-beta(3) mutant MEE cells survive as they do their counterparts in the chick embryo. Interestingly, Slug is the Snail family member expressed in the chick MEE, providing another example of interchange of Snail and Slug expression between avian and mammalian embryos. We propose that in the absence of TGF-beta(3), TGF-beta(1) is upregulated in the mesenchyme, and that in both physiological (avian) and pathological (TGF-beta(3)-/-mammalian) cleft palates, it induces the expression of Snail genes promoting the survival of the MEE cells and permitting their subsequent differentiation into keratinized stratified epithelium.     

Bulging medial edge epithelial cells and palatal fusion

The surface of the medial edge epithelium of embryonic day 12, 13 and 14 mouse palatal shelves was observed utilising Environmental Scanning Electron Microscopy (ESEM). This technique offers the advantage of visualisation of biological samples after short fixation times in their natural hydrated state. Bulging epithelial cells were observed consistently on the medial edge epithelium prior to palatal shelf fusion. Additionally, we have used ESEM to compare the morphology and surface features of palatal shelves from embryonic day 13 to 16 mouse embryos that are homozygous null (TGF-beta3 -/-), heterozygous (TGF-beta3 +/-) or homozygous normal (TGF-beta3 +/+) for transforming growth factor beta-3 (TGF-beta3). At embryonic day 15 and 16 most TGF-beta3 +/- and +/+ embryos showed total palatal fusion, whilst all TGF-beta3 null mutants had cleft palate: the middle third of the palatal shelves had adhered, leaving an anterior and posterior cleft. From embryonic day 14 to 16 abundant cells were observed bulging on the medial edge epithelial surface of palates from the TGF-beta3 +/- and +/+ embryos. However, they were never seen in the TGF-beta3 null embryos, suggesting that these surface bulges might be important in palatal fusion and that their normal differentiation is induced by TGF-beta3. The expression pattern of E-Cadherin, beta-catenin, chondroitin sulphate proteoglycan, beta-Actin and vinculin as assayed by immunocytochemistry in these cells shows specific variations that suggest their importance in palatal shelf adhesion.     

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.     

Processes involved in retinoic acid production of small embryonic palatal shelves and limb defects

All-trans-retinoic acid (RA) is teratogenic to the embryonic mouse, producing malformations in many developing systems, including the limb bud and palate. High incidences of limb defects and cleft palate are induced at doses which are not maternally toxic and do not increase resorptions. Exposure to RA on gestational day (GD) 10 results in small palatal shelves, which fail to make contact on GD 14. The formation of small shelves could be a consequence of increased cell death, reduced proliferation, a combination of these effects, or some other effect such as inhibition of extracellular matrix production. After exposure to 100 mg RA/kg on GD 10, proliferation in mesenchymal cells of the palatal shelves was not reduced from GD 12 to GD 14 and the levels of cell death in control and treated shelves did not differ when observed by light and electron microscopy. The present study examines the effects of RA on cell death and proliferation from GDs 10-12 and compares the effects in palatal shelves and limb buds. Embryonic mice were exposed to RA suspended in corn oil (100 mg/kg on GD 10), a dose that was teratogenic but not maternally toxic or embryolethal. Embryos were collected at 4, 12, 24, 36, or 48 hr postexposure, and tissues which form the palate or limb were dissected from the embryos, stained by a modified Feulgen procedure, and whole mounted on slides. Mitotic index (MI) and percentage dead cells were determined for mesenchymal cells of the first visceral arch, maxillary process, or palatal shelf (depending on stage of development) and forelimb buds. In the palatal tissues from GD 10 to GD 12, RA did not significantly alter MI and percentage dead cells was significantly increased only at 4 hr postexposure. Some whole embryos were prepared for scanning electron microscopy (SEM). At 48 hr (GD 12) a reduction in the size of the shelves was not apparent on SEM. In the limb buds, RA did not increase percentage dead cells, but MI was significantly decreased. A decreasing rate of proliferation was detected in control facial tissues as development progressed, and this agrees with findings in rat and chick. Thus it appears that mesenchymal cell death and reduced proliferation are not responsible for the small palatal shelves seen on GD 14. RA did not increase cell death but inhibited proliferation in the limb bud, and this effect may contribute to the retarded development and malformations occurring in the limb.