Closure Of Palatal Shelves Research Articles

Tbx22null mice have a submucous cleft palate due to reduced palatal bone formation and also display ankyloglossia and choanal atresia phenotypes

Craniofacial defects involving the lip and/or palate are among the most common human birth defects. X-linked cleft palate and ankyloglossia results from loss-of-function mutations in the gene encoding the T-box transcription factor TBX22. Further studies show that TBX22 mutations are also found in around 5% of non-syndromic cleft palate patients. Although palate defects are obvious at birth, the underlying developmental pathogenesis remains unclear. Here, we report a Tbx22(null) mouse, which has a submucous cleft palate (SMCP) and ankyloglossia, similar to the human phenotype, with a small minority showing overt clefts. We also find persistent oro-nasal membranes or, in some mice a partial rupture, resulting in choanal atresia. Each of these defects can cause severe breathing and/or feeding difficulties in the newborn pups, which results in approximately 50% post-natal lethality. Analysis of the craniofacial skeleton demonstrates a marked reduction in bone formation in the posterior hard palate, resulting in the classic notch associated with SMCP. Our results suggest that Tbx22 plays an important role in the osteogenic patterning of the posterior hard palate. Ossification is severely reduced after condensation of the palatal mesenchyme, resulting from a delay in the maturation of osteoblasts. Rather than having a major role in palatal shelf closure, we show that Tbx22 is an important determinant for intramembranous bone formation in the posterior hard palate, which underpins normal palate development and function. These findings could have important implications for the molecular diagnosis in patients with isolated SMCP and/or unexplained choanal atresia.

Tbx22 null mice have a submucous cleft palate due to reduced palatal bone formation and also display ankyloglossia and choanal atresia phenotypes

Craniofacial defects involving the lip and/or palate are among the most common human birth defects. X-linked cleft palate and ankyloglossia results from loss-of-function mutations in the gene encoding the T-box transcription factor TBX22. Further studies show that TBX22 mutations are also found in around 5% of non-syndromic cleft palate patients. Although palate defects are obvious at birth, the underlying developmental pathogenesis remains unclear. Here, we report a Tbx22null mouse, which has a submucous cleft palate (SMCP) and ankyloglossia, similar to the human phenotype, with a small minority showing overt clefts. We also find persistent oro-nasal membranes or, in some mice a partial rupture, resulting in choanal atresia. Each of these defects can cause severe breathing and/or feeding difficulties in the newborn pups, which results in ∼50% post-natal lethality. Analysis of the craniofacial skeleton demonstrates a marked reduction in bone formation in the posterior hard palate, resulting in the classic notch associated with SMCP. Our results suggest that Tbx22 plays an important role in the osteogenic patterning of the posterior hard palate. Ossification is severely reduced after condensation of the palatal mesenchyme, resulting from a delay in the maturation of osteoblasts. Rather than having a major role in palatal shelf closure, we show that Tbx22 is an important determinant for intramembranous bone formation in the posterior hard palate, which underpins normal palate development and function. These findings could have important implications for the molecular diagnosis in patients with isolated SMCP and/or unexplained choanal atresia.

Timing of Implantation and Closure of the Palatal Shelf in New Zealand White and Japanese White Rabbits

Two specific developmental events, namely implantation and palatal shelf closure, are of specific interest because they define, respectively, the beginning and the end of the treatment period in embryo-fetal developmental toxicity studies for pharmaceutical products. Thus, a detailed evaluation of the timing of implantation and closure of the hard palate is necessary to assure use of the proper exposure window in developmental toxicity studies in rabbits, the nonrodent species most commonly evaluated in regulatory developmental toxicology studies. The purpose of this study was to determine the timeline for implantation and closure of the hard palate in the New Zealand White rabbit, and to determine if this timeline differed in the Japanese White rabbit.To describe the timing of implantation, the uteri from does of the New Zealand White rabbit and the Japanese White rabbit were examined on gestation days (GDs) 5 through 8 for macroscopic evidence of implantation. To assess palatal shelf closure, fetuses were removed on GDs 17, 18, and 19 and fixed in Bouin's solution. The fetuses were then categorized into five stages of palatal shelf closure: open (Stage I); approach of the palatal shelves (Stage II); partial closure of the hard palate (Stage III); full closure of the hard palate (Stage IV); and full closure of the soft palate (Stage V).In both the New Zealand White and Japanese White rabbit strains, implantation was initiated on GD 6.5 and was completed on GD 7. Partial closure of the palate began on GD 17.5, and by GD 19, closure of the hard palate was completed in all fetuses, and closure of the soft palate was completed in 75–96% of the fetuses.The timing of implantation and palatal shelf closure were comparable between the New Zealand White rabbit and the Japanese White rabbit. Therefore, treatment beginning on GD 7 and continuing until GD 19 encompasses the period of major organogenesis and is considered appropriate for use in developmental toxicity studies using either of these two strains of rabbits.

Distinct requirements for Gab1 in Met and EGF receptor signaling in vivo

Gab1 is a multiadaptor protein that has been shown to be required for multiple processes in embryonic development and oncogenic transformation. Gab1 functions by amplifying signal transduction downstream of various receptor tyrosine kinases through recruitment of multiple signaling effectors, including phosphatidylinositol 3-kinase and Shp2. Until now, the functional significance of individual interactions in vivo was not known. Here we have generated knockin mice that carry point mutations in either the P13K or Shp2 binding sites of Gab1. We show that different effector interactions with Gab1 play distinct biological roles downstream of Gab1 during the development of different organs. Recruitment of phosphatidylinositol 3-kinase by Gab1 is essential for EGF receptor-mediated embryonic eyelid closure and keratinocyte migration, and the Gab1-Shp2 interaction is crucial for Met receptor-directed placental development and muscle progenitor cell migration to the limbs. Furthermore, we investigate the dual association of Gab1 with the Met receptor. By analyzing knockin mice with mutations in the Grb2 or Met binding site of Gab1, we show that the requirements for Gab1 recruitment to Met varies in different biological contexts. Either the direct or the indirect interaction of Gab1 with Met is sufficient for Met-dependent muscle precursor cell migration, whereas both modes of interaction are required and neither is sufficient for placenta development, liver growth, and palatal shelf closure. These data demonstrate that Gab1 induces different biological responses through the recruitment of distinct effectors and that different modes of recruitment for Gab1 are required in different organs.

Contractile Properties of Single Permeabilized Muscle Fibers from Congenital Cleft Palates and Normal Palates of Spanish Goats

Analysis of the composition of muscle fibers constituent to a cleft palate could provide significant insight into the cause of velopharyngeal inadequacy. The authors hypothesized that levator veli palatini muscle dysfunction inherent to cleft palates could affect the timing and outcome of cleft palate repair. Single, permeabilized muscle fibers from levator veli palatini muscles of three normal (n = 19 fibers) and three chemically induced congenital cleft palates (n = 21 fibers) of 14-month-old goats were isolated, and contractile properties were evaluated. The maximum isometric force and rate constants of tension redevelopment (ktr) were measured, and the specific force and normalized power were calculated for each fiber. The ktr measures indicate that cleft fibers are predominantly fast-fatigable; normal fibers are slow fatigue-resistant: after a 10-minute isometric contraction, fibers from cleft palates had a loss of force 16 percent greater than that from normal palates (p = 0.0001). The cross-sectional areas of the fibers from cleft palates (2750 +/- 209 microm2) were greater (p = 0.05) than those from normal palates (2226 +/- 143 microm2). Specific forces did not differ between the two groups. Maximum normalized power of fibers from cleft palates (11.05 +/- 1.82 W/l) was greater (p = 0.0001) than fibers from normal palates (1.60 +/- 0.12 W/l). There are clear physiologic differences in single muscle fibers from cleft palates and normal palates: cleft palate fibers are physiologically fast, have greater fatigability, and have greater power production. Detection of functional and/or fiber type differences in muscles of cleft palates may provide preoperative identification of a patient's susceptibility to velopharyngeal inadequacy and permit early surgical intervention to correct this clinical condition.

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.

The fetal cleft palate: II. Scarless healing after in utero repair of a congenital model.

The role of fetal surgery in the treatment of non-life-threatening congenital anomalies remains a source of much debate. Before such undertakings can be justified, models must be established that closely resemble the respective human anomalies, and the feasibility and safety of these in utero procedures must be demonstrated. The authors recently described and characterized a congenital model of cleft palate in the goat. The present work demonstrates the methodology they developed to successfully repair these congenital cleft palates in utero, and it shows palatal healing and development after repair. A surgically created cleft model was developed for comparative purposes. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Six pregnant goats were gavaged twice daily during gestational days 32 to 41 (term, 145 days) with a plant slurry of Nicotiana glauca containing the piperidine alkaloid anabasine; the 12 fetuses had complete congenital clefts of the secondary palate. Repair of the congenital clefts was performed at 85 days of gestation using a modified von Langenbeck technique employing lateral relaxing incisions with elevation and midline approximation of full-thickness, bilateral, mucoperiosteal palatal flaps followed by single-layer closure. Six congenitally clefted fetuses underwent in utero repair, six remained as unrepaired controls. Twelve normal fetuses underwent surgical cleft creation by excision of a 20 x 3 mm full-thickness midline section of the secondary palate extending from the alveolus to the uvula, at 85 days of gestation. Six surgically clefted fetuses underwent concurrent repair of the cleft at that time; six clefted fetuses remained as unrepaired controls. At 2 weeks of age, no congenitally or surgically created clefts repaired in utero demonstrated gross or histologic evidence of scar formation. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. Cross-section through the mid-portion of the repaired congenitally clefted palates demonstrated reconstitution of a bilaminar palate, with distinct oral and nasal mucosal layers, after single-layer repair. In utero cleft palate repair is technically feasible and results in scarless healing of the mucoperiosteum and velum. The present work represents the first in utero repair of a congenital cleft palate model in any species. The use of a congenital cleft palate model that can be consistently reproduced with high predictability and little variation represents the ideal experimental situation. It provides an opportunity to manipulate specific variables, assess the influence of each change on the outcome and, subsequently, extrapolate such findings to the clinical arena with a greater degree of relevance.

The fetal cleft palate: I. Characterization of a congenital model.

Any animal model of a human congenital anomaly established by iatrogenic methods involving intrauterine fetal manipulation has limited clinical applicability. A congenital model that more closely simulates the etiopathogenesis of a human anomaly may provide data that can more readily be extrapolated to that anomaly and, therefore, be used in diagnostic and management strategies. The present work provides a description and characterization of a congenital model of cleft palate in the goat. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Sixteen pregnant goats were gavaged twice daily during gestational days 32 through 41 [term, 145 days] with a plant slurry of Nicotiana glauca containing the piperidine alkaloid teratogen anabasine. Gross analysis and measurement of fetal clefts were performed at 60, 70, and 85 days gestation (four fetuses were studied at each time point). Seventeen clefted kids were sacrificed at specific intervals after birth (2 weeks, and 1, 3, and 6 months); after skull debridement and preparation, they were compared with 12 unclefted control kids. Complete clefting of the secondary palate occurred in 97 percent of the fetuses. In all cases, the cleft extended from the posterior aspect of the alveolar ridge to the uvula; the majority of these clefts were bilateral, with complete detachment of the vomer. Morphologically, these clefts were similar to human clefts. Eighteen percent of clefted newborn kids demonstrated gross maxillary hypoplasia and midfacial retrusion at birth with a relative Class III malocclusion. Direct measurement of the congenital caprine skulls confirmed these findings. The incidence of midfacial growth abnormalities in these clefted animals raises questions regarding the etiopathogenesis of facial dysmorphology that is unrelated to scarring of the maxilla. This congenital cleft palate model is currently being used to explore these questions and others related to craniofacial growth and palatal function after in utero repair.

Chapter 2. Role of Extracellular Matrices in Palatal Shelf Closure

Publisher Summary This chapter focuses on the role of extracellular matrices in palatal shelf closure. The palatal shelves themselves play a direct and active role in their reorientation from a vertical position on either side of the tongue to a horizontal one above it. One of the oldest proposed mechanisms for the motive force underlying palatal shelf movement is a rapid increase in shelf volume due to increased cellular volume in the “connective tissue.” Histochemical and autoradiographic evidence suggested that acid mucopolysaccharides [glycosaminoglycans (GAGS)], principally those that are sulfated, are present in the palatal shelves at the time of shelf reorientation. Biochemical data has confirmed that GAGs are present and synthesized before and during shelf reorientation. Extracellular matrices both within the mesenchymal compartment, probably existing as a GAG–collagen gel–fiber matrix, and at the epithelial–mesenchymal interface, organized as a basal lamina, appear to play vital roles in palatal shelf reorientation. One hypothesis that explains their possible roles is that posterior palatal shelf remodeling results largely from the expansion of hyaluronate-rich gel–fiber network within the mesenchymal compartment.

The role of amniotic fluid on the in-vitro palatal fusion of normal and vitamin A-treated rat foetuses

Excess vitamin A has long been used to produce experimental cleft palate in animals. The mechanism by which it causes cleft palate is still unknown. The present investigation studied the effect of amniotic fluid on the in-vitro closure of palatal shelves of foetuses from normal and vitamin A treated rats. The amniotic fluid was added to the culturing medium after being centrifuged for 20 min at 3500 rpm. The majority of normal explants cultured with normal medium containing amniotic fluid from untreated rats showed mesenchymal fusion. Similarly, more than 50 per cent of the explants from the foetuses of vitamin A treated rats grown with normal amniotic fluid demonstrated palatal closure after an incubation period of three days. Palatal explants from vitamin A treated as well as normal rats did not show mesenchymal fusion when they were cultured in the presence of amniotic fluid obtained from vitamin A treated rats. A fluorometric determination of amniotic fluid from untreated and treated mothers showed no traces of vitamin A. The results demonstrate that amniotic fluid from vitamin A treated rats has the potential to disturb the normal fusion mechanism of the palatal shelves in vitro.

Effect of hydrocortisone on the closure of palatal shelves in vivo and in vitro.

SINCE the first experiments of Fraser and Fainstat1 in 1951, it has become clear that cortisone induces cleft palate in mice when administered before the closure of the palatal shelves. Certain direct and indirect effects on the palatal tissue have been suggested, in cluding the effect of cortisone on the turnover of the amniotic fluid2–4. Harris4 postulated that oligohydramnion, resulting from maternal cortisone treatment, affects the normal development of the palatal region by constricting the embryo. Recently, however, Fraser et al.5 have compared the amount of amniotic fluid in embryos treated with cortisone with and without cleft palate. Ko differences were detected and the results seem to rule out the possibility that oligohydramnion plays a part in the genesis of cleft palate.