Pathogenesis of bromodeoxyuridine-induced cleft palate in hamster

In the present study, the morphological, histochemical, biochemical, and cellular aspects of the pathogenesis of bromodeoxyuridine (BrdU)-induced cleft palate in hamster fetuses were analyzed. Morphological observations indicated that BrdU interferes with the growth of the vertical shelves and thus induces cleft palate. At an ultrastructural level, BrdU-induced changes were first seen in the mesenchymal cells. Eighteen hours after drug administration, the initial alterations were characterized by swelling of the nuclear membrane and the appearance of lysosomes in the mesenchymal cells of the roof of the oronasal cavity. During the next 6 hr, as the palatal primordia developed, lysosomes were also seen in the overlying epithelial cells. The appearance of lysosomal activity, which was verified by acid phosphatase histochemistry, was temporally abnormal and was interpreted as a sublethal response to BrdU treatment. Later the cellular alterations subsided; 48 hr after BrdU treatment, they were absent in both the epithelial and mesenchymal cells of the vertically developing palatal shelves. Subsequently, unlike controls (in which the palatal shelves undergo reorientation and fusion), the BrdU-treated shelves remained vertical until term. Biochemical determination of DNA synthesis indicated that although there was an inhibition of DNA synthesis at the time of appearance of palatal primordia, a catch-up growth during the ensuing 12 hr may have restored the number of cells available for the formation of a vertical palatal shelf. It was suggested that BrdU affected cytodifferentiation in the palatal tissues during the critical phase of early vertical development to induce a cleft palate.     

D-penicillamine-induced cleft palate in mice

The teratogenic potential of the lathyrogen, D-penicillamine (DP), was assessed in pregnant mice, especially with respect to its ability to produce cleft palate. The dosage and the duration of treatment as they relate to the induction of cleft palate were also studied. Two different doses of DP were administered orally for either 5 or 4 consecutive days during the critical period of palatal closure. D-penicillamine (DP) at a dose level which does not have any apparent maternal toxic effects produced cleft palate in the offspring, and this teratogenic effect depended more upon the duration of treatment than the dosage administered. Inhibitory effects on the formation of bone matrix were observed at the base of the palatal shelf. It is suggested that DP is potentially an osteolathyrogenic agent. The mechanism of induction of cleft palate in DP-treated mice was explored by histological studies using light microscopy. Delayed elevation of the palatal shelves was observed and is considered to be the cause of the induction of cleft palate. No other external malformations could be detected in DP-treated fetuses.     

Corticosteroid-induced cleft palate in short-ear mice

Corticosteroid-induced facial clefting was examined in short-ear mice (inbred strain SEA/GnJ from Jackson Laboratory). They were found to be gentle, prolific breeders (average litter size 7.3 +/- 2.6), with no increased fetal loss due to treatment. Although they have the most "resistant" H-2 haplotype (H-2d), they were found to be highly susceptible to corticosteroid-induced clefting, demonstrating that loci other than H-2 are involved in susceptibility. The short-ear locus itself is a plausible candidate, given that this gene on chromosome 9 leads to defective mesenchymal condensation, which in turn may render these mice extremely sensitive to teratogen exposure. Another gene or genes on chromosome 9 are also possibilities.     

Corticosterone induction of cleft palate in mice dosed with orciprenaline sulfate

Orciprenaline sulfate is a beta-adrenoceptor stimulant chemically described as 1-(3,5-dihydroxyphenyl)-1-hydroxy-2-isopropylaminoethane sulfate (Alupent). The drug has broncho-dilating activity and has been developed in numerous countries since 1961. The purpose of these studies was to investigate the teratogenic potential of orciprenaline and its mode of action in pregnant Jcl:ICR mice, when administered during the period of organogenesis and, more systematically, during the critical period of palate formation. Daily doses of 5, 50, and 500 mg/kg were given orally by gavage to mice on days 6-15, 11-13, or on day 12 of gestation. Additional studies were done to evaluate the maternal cardiotoxic action of orciprenaline and its effects on adrenal cortex and endogenous serum corticosterone. Five mg/kg triamcin-olone acetonide, a glucocorticoid, were given subcutaneously as a positive control causing 100% cleft palate. Myocardial necroses occurred in pregnant mice only after 500 mg/kg orciprenaline had been given, and a significant increase in cleft palate occurred if exposure took place during days 11-13 or day 12 of gestation. This increase in cleft palate can be explained by the teratogenic effect of an elevated maternal serum corticosterone level 1 hr after orciprenaline treatment, about three times the control value.     

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.     

Spontaneous cleft palate in CF#1/Ohu mice]

We found a spontaneous cleft palate in a mouse of CF#1/Ohu (Ohu University, Japan). Further, the frequency of the spontaneous cleft palate in strains of CF#1/Jms (Institute of Medical Science, University of Tokyo, Japan) and CF#1/Jah (National Institute of Animal Health, Japan) were about 3%, respectively. The frequency and the types of spontaneous cleft palate in CF#1 were clearly different from those in A/J and CL/Fr strains which were used as model animals of lip and cleft palate. We think that CF#1 is the new animal models to analyze a cleft palate genetically and biochemically.