The transforming growth factor-beta 3 knock-out mouse: An animal model forcleft palate

The recent report of a transforming growth factor-beta3 (TGF-beta3) knock-o ut mouse in which 100 percent of the homozygous pups have cleft palate rais ed the question as to the potential usefulness of these animals as a model for cleft palate research. The specific aim in this study was to carefully document the anatomy of the cleft palate in the TGF-beta3 knock-out mice as compared with wild type controls. Special attention was paid to the levato r veli palatini muscle, the tensor veli palatini muscle, and their respecti ve innervation. Because the TGF-beta3 knock-out is lethal in the early perinatal period and because the heterozygotes are phenotypically normal, polymerase chain reac tion was required to genotype the animals before mating. Time-mated pregnan cies between proven heterozygotes were then delivered by cesarean section a t gestational day 18.5 to prevent maternal cannibalism of homozygote pups. Ail delivered pups were killed and their tails processed by polymerase chai n reaction to verify genotype. The heads were then fixed and sectioned in a xial, coronal, or sagittal planes. Sections were stained with hematoxylin a nd eosin or processed for immunohistochemistry with nerve specific protein gene product 9.5 and calcitonin gene-related peptide antibodies. Sections w ere analyzed in a serial fashion. Nine wild type control animals were analy zed along with nine TGF-beta3 knock-out homozygotes. Time matings between proven heterozygotes yielded wild type pups, heterozyg ote pups, and homozygote knock-out pups in the expected mendelian ratios (2 8 percent to 46 percent to 26 percent; n = 43). The results demonstrated 10 0 percent clefting in the homozygous TGF-beta3 knock-out pups. Complete cle fting of the secondary palate was seen in four of nine and incomplete cleft ing was seen in five of nine. The levator veli palatini and tensor veli pal atini muscles were demonstrated coursing parallel to the cleft margin in al l cleft mice. The orientation of these muscles differs from the normal tran sverse sling of the levator veli palatini muscle and the normal palatine ap oneurosis of the tensor veli palatini muscle at the soft palate in control animals. Innervation of the levator veli palatini muscle by cranial nerve I X and the tensor veli palatini muscle by cranial nerve V were demonstrated in both cleft and control animals by use of immunohistochemistry with nerve -specific antibodies. Demonstration of a teratogen-free, reproducible animal model of clefting of the palate with a known, single-gene etiology is an important step in the systematic understanding of a congenital defect whose multifactorial etiolo gy has hampered previous research efforts. This study presents a detailed a natomic description of such a model, including a description of the muscula r anatomy and the innervation of the muscles of the palate. Because of earl y perinatal mortality, this model has limited applications for postnatal st udies.