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.     

The origin and development of alopecia in mice homozygous for strong's luxoid gene

Hair follicles are initiated in mice homozygous for Strong's luxoid gene at the normal times. The dermis from 16 days of gestation to nine days after birth lags in development. The adipose layer instead of enlarging at the normal time of three days after birth delays until nine days. The growth of the first cycle hairs is inhibited, particularly on dorsal surfaces. Some follicles of all types degenerate. The surviving follicles enter telogen at seven days after birth, after forming only short unpigmented or poorly-pigmented hairs. Many follicles immediately begin a second cycle of growth, in which more normal hairs develop and a substantial adipose layer forms. No alopecia develops on ventral surfaces, but growth of the first cycle ceases and the second cycle commences earlier than normal; the hairs formed are abnormal. Abnormal hair growth in Strong's luxoid homozygotes may be a result of the retarded growth of the dermis or both defects may be secondary to a more fundamental defect.     

Cytokeratin, vimentin and E-cadherin immunodetection in the embryonic palate in two strains of mice with different susceptibility to glucocorticoid-induced clefting

An immunohistochemical study analyzing the pattern of distribution of some intermediate filament proteins, keratin and vimentin and, one adhesion molecule, cadherin in different stages of developing secondary palate in two strains of mice with different H-2 backgrounds was undertaken to investigate differences between a strain that is susceptible to glucocorticoid-induced cleft palate (A/Sn) and one that is resistant to glucocorticoid-induced cleft palate (C57/BL). The heads of embryos were processed by standard immunohistochemistry with antipancytokeratin (KAE1), antikeratins 18 (K18) and 19 (K19), antivimentin, and anti E-cadherin antibodies. Immunostaining with KAE1 antibody showed differences between the strains. The reaction was stronger in the medial edge epithelia of palatal processes in the A/Sn strain at all stages of palatogenesis. The C57/BL strain showed a weak immunostain to KAE1. Antivimentin antibody stained the mesenchymal cells of palatal processes and K18 and K19 showed no reaction in either strain of mice. Anti E-cadherin antibody was detected in the medial palatal epithelium of both strains of mice and in all stages of palate development. No differences were observed in E-cadherin and vimentin immunostain in palatal epithelium between the strains. The different expression of some cytokeratins in the embryonic palatal epithelium suggests that these intermediate filament proteins may be involved in different susceptibility to glucocorticoid-induced cleft palate in the mouse. The decreased immunoreaction of cytokeratins observed in the resistant strain would facilitate the disappearance of this molecule during the transformation from an epithelial to a mesenchymal phenotype that takes place during the development of the palate. These results may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformation in the embryonic palate.     

Physiological loading of the mandibular osteosynthesis: development of an improved mandibular load simulator]

Loading conditions physiologically approximating those acting on the normal masticatory system were incorporated into a new mandibular load simulator. Separate tension wires attached to each ramus of the mandible simulated the resultant force vectors of the masticatory musculature. The muscle insertion points were chosen in accordance with the anatomical situation, and the maximum in vivo forces acting on the joint. In a first application, the stability of a 2.4 mm LC-WDCP was compared with that of a 2.7 mm EDCP in plastic mandible models. It was found that under largely physiological loading, the 2.4 mm LC-EDCP exerted a stabilizing effect similar to that of a 2.7 mm EDCP. Although of smaller dimensions, the 2.4 mm LC-EDCP appears to enable an osteosynthesis of similar stability in the treatment of fractures of the mandibular angle.     

Altered timing of the extracellular-matrix-mediated epithelial-mesenchymal interaction that initiates mandibular skeletogenesis in three inbred strains of mice: development, heterochrony, and evolutionary change in morphology

Subtle changes in embryonic development are a source of significant morphological alterations during evolution. The mammalian mandibular skeleton, which originates from the cranial neural crest, is a complex structure comprising several components that interact late in embryogenesis to produce a single functional unit. It provides a model system in which individual developmental events at the basis of population-level evolutionary change can be investigated experimentally. Inbred mouse strains exhibit obvious morphological differences despite the relatively short time since their divergence from one another. Some of these differences can be traced to small changes in the timing of early developmental events such as the formation of the cellular condensations that initiate skeletogenesis. This paper examines an even earlier event for changes in timing, the epithelial-mesenchymal interaction(s) required to initiate chondrogenesis of Meckel's cartilage and osteogenesis of the dentary bone. Using three inbred strains of mice (CBA, C3H and C57) we found that, within each strain, cartilage and bone are induced at the same time and by the same (mandibular) epithelium, that chondrogenesis and osteogenesis are initiated by a matrix-mediated epithelial-mesenchymal interaction, and that timing of the interactions differs among the three inbred strains. These results are discussed with respect to the possible molecular basis of such temporal shifts in inductive interactions and how such studies can be used to shed light on heterochrony as a mechanism of evolutionary change in morphology.     

Influence of parathyroid hormone and heparin on tooth development in mice homozygous for the microphthalmia mutation]

In mice, homozygous by microphthalmia (mi/mi) gene the disturbance of dental development was due to defects in dental pulp and to the absence of bone resorpion. Administration of heparin or parathyroid hormone stimulated dental eruption in mi/mi mice. Following combined action of heparin and parathyroid hormone the number of erupted teeth was practically the same as after the action of heparin alone. Possibly the level of heparin is insufficient, but secretion of parathyroid hormone is unimpaired in mi/mi mice.     

Metabolism of phenylalanine in mice homozygous for the gene `dilute lethal'

Mice homozygous for d(l) have been suggested as models for phenylketonuria. We found: (1) the concentration of phenylalanine in the blood was normal at all ages examined; (2) phenylalanine hydroxylase activity in the liver in vitro equalled that in unaffected littermates; (3) the apparent K(m) values for phenylalanine and cofactor respectively in d(l)/d(l) mice were the same as in their normal littermates; (4) inhibition of the overall reaction by the particulate fraction, excess of substrate, excess of cofactor or phenylpyruvic acid showed no difference between d(l)/d(l) mice and their unaffected littermates; (5) phenylalanine injected in vivo had equal, small, effects on phenylalanine hydroxylase activity of the liver measured in vitro in the two groups of mice. An explanation of the findings of other workers, based on the natural history of the disease process, is tentatively put forward.     

Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Calsequestrin is a high capacity Ca(2+)-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca(2+)-induced Ca(2+) release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998) J. Clin. Invest. 101, 1385-1393). To investigate whether alterations in intracellular Ca(2+) trigger changes in the beta-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired beta-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total beta-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased beta-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in beta-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.     

Dermal connective tissue development in mice: an essential role for tenascin-X

Deficiency of the extracellular matrix protein tenascin-X (TNX) causes a recessive form of Ehlers‐Danlos syndrome (EDS) characterized by hyperextensible skin and hypermobile joints. It is not known whether the observed alterations of dermal collagen fibrils and elastic fibers in these patients are caused by disturbed assembly and deposition or by altered stability and turnover. We used biophysical measurements and immunofluorescence to study connective tissue properties in TNX knockout and wild-type mice. We found that TNX knockout mice, even at a young age, have greatly disturbed biomechanical properties of the skin. No joint abnormalities were noted at any age. The spatio-temporal expression of TNX during normal mouse skin development, during embryonic days 13–19 (E13–E19), was distinct from tropoelastin and the dermal fibrillar collagens type I, III, and V. Our data show that TNX is not involved in the earliest phase (E10–E14) of the deposition of collagen fibrils and elastic fibers during fetal development. From E15 to E19, TNX starts partially to colocalize with the dermal collagens and elastin, and in adult mice, TNX is present in the entire dermis. In adult TNX knockout mice, we observed an apparent increase of elastin. We conclude that TNX knockout mice only partially recapitulate the phenotype of TNX-deficient EDS patients, and that TNX could potentially be involved in maturation and/or maintenance of the dermal collagen and elastin network.