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.     

Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.     

Genetics of cleft lip and cleft palate in China.

During the past 10 years, 60 cases of cleft lip with or without cleft palate [CL(P)] were recorded among 45,072 newborns at Shanghai International Peace Maternity and Infant Hospital, China. The incidence was 1.33 per 1,000 births. The family histories of 163 CL(P) patients were analyzed. The incidences of CL(P) in the first-, second-, and third-degree relatives of CL(P) patients were 11/246 (4.47%), 10/1,032 (0.97%), and 6/1,727 (0.35%), respectively. Of the 163 probands, three had a history of consanguinity of the parents (1.8%), in contrast to 0.77% in the general population. These data are suggestive of multifactorial inheritance. The heritability of CL(P) in our study calculated by Falconer's formula was 77.6%.     

Partial IgA-deficiency with increased Th2-type cytokines in TGF-beta 1 knockout mice.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.     

Development of the lung in mice with bromodeoxyuridine-induced cleft palate

Clinical and laboratory observations show that denial of free communication between the amniotic fluid and lung fluid results in pulmonary hypoplasia. Thus, cleft palate resulting from tongue obstruction to palatal shelf elevation might be associated with disturbed lung development. This association exists in the Pena-Shokeir phenotype. The goal of these experiments was to see what effect bromodeoxyuridine (BUdR)-induced cleft palate had on lung development. LACA mice were injected with 500 mg/kg BUdR on E11 or E11 and E12 of gestation, a treatment known to produce a 25% and 50% incidence of cleft palate, respectively. BUdR had a direct retarding effect on lung growth but, when cleft palate occurred as well, the lungs were more severely affected. Morphometry showed that lungs from fetuses with cleft palate had only one-half the saccular volume of controls or of treated fetuses with normal palates. Although hypoplastic, lungs associated with cleft palate had type I and type II pneumocytes, and the latter were shown by electron microscopy to be capable of producing surfactant. Hence, cellular differentiation had not been affected by the treatment. Fetuses with cleft palate had less amniotic fluid than controls but significantly more than those with normal palates after treatment. Thus, the pattern of abnormalities in this animal model bears some resemblance to that of the human Pena-Shokeir phenotype.