Transforming growth factor-beta 3 is required for secondary palate fusion

Abstract

Mice lacking TGF-beta 3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-beta 3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.

Fig. 1

Generation of TGF-β3-deficient mice, a, Gene targeting strategy. TGF-β3 exons are indicated by filled boxes, the neomycin gene by a shaded box. Restriction sites are EcoRI (E), HincII (H), KpnI (K), NsiI (N) and SacI (S). Probe A (open box) is a PCR-derived fragment containing exon 4, and lies outside the targeting vector. Probe B is a 1.1-kb BstXI-EcoRI fragment containing exon 7 sequences, which are included in the targeting vector. Both probes were used to test for targeted ES cells and to genotype mice, b, Southern blot analysis of genomic DNA from ES cells. Lanes 1, 3, 5, 7, 9 contain DNA from untransfected E14-1 ES cells, lanes 2, 4,6, 8,10 contain DNA prepared from ES ceil clone I98 showing homologous recombination. Enzymes used: EcoRI in lanes 1,2; NsiI in lanes 3, 4,5,6; HincII in lanes 7, 8 and SacI in lanes 9 and 10. Probe A: EcoRI, 10.5-kb and 8.6-kb band with the wild-type and mutated allele, respectively. Similarly, NsiI, a 14-kb and a 21-kb band, respectively. Probe B: NsiI gave a 7-kb and a 21-kb band with wild-type and null allele DNA, respectively; HincII, a 9-kb and a 10-kb band, respectively; SacI, 8.8-kb and 9.8-kb band, respectively, c, RT-PCR analysis of whole day 11.5 embryo RNA derived from wild-type (+/+), heterozygous (+/−) and homozygous (−/−) litter mate embryos.

Fig. 2

Cross-sections of heads showing secondary palate, a–c, 129/Ola × CF-1 background; embryonic d 18.5. a, Wild-type (+/+) embryonic head with fused palate, b, TGF-β3-null (−/−) embryo with a cleft in the posterior palate, c, TGF-β3-null (−/−) embryo with a cleft in the anterior palate region, d, Day 1 −/− neonate with complete cleft palate; 129/Ola × C57BL/6 background. Heads were Bouin´s-fixed.

Fig. 3

Developmental study of palate formation in TGF-β3-null mice (−/−) compared with wild-type (+/+) litter mates, a–o, Mid-palate region, a, b, +/+, d 12.5. c, d, −/−, d 13.5. e, f, +/+, d 14.5. g, h, −/−, d 14.5, two different embryos. i, k, +/+, d 15.5. l–o, −/−, d 15.5, two different embryos, shown to indicate variable expressivity of phenotype. p–s, Posterior palate region, p, q, +/+, d 15.5. r, s, −/−, d 15.5. The embryos were fixed in 4% paraformaldehyde, sectioned and stained with hematoxylineosin-alcian blue. Bar = 0.5 mm for a, c, e, i, l, n, p, r. Bar = 0.1 mm for b, d, f–h, k, m, o, q, s.