MethodsUUO. Smad3-null (Smad3 ex8/ex8 ) mice were generated as described (13) and were used at 6-8 weeks of age and 20-30 g in body weight. In mice sedated by general anes-

Hox genes regulate patterning during limb development. It is believed that they function in the determination of the timing and extent of local growth rates. Here, it is demonstrated that synpolydactyly, an inherited human abnormality of the hands and feet, is caused by expansions of a polyalanine stretch in the amino-terminal region of HOXD13. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. The mutations identify the polyalanine stretch outside of the DNA binding domain of HOXD13 as a region necessary for proper protein function.

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

Synpolydactyly (SPD) is a dominantly inherited congenital limb malformation. Typical cases have 3͞4 finger and 4͞5 toe syndactyly, with a duplicated digit in the syndactylous web, but incomplete penetrance and variable expressivity are common. The condition has recently been shown to be caused by expansions of an imperfect trinucleotide repeat sequence encoding a 15-residue polyalanine tract in HOXD13. We have studied 16 new and 4 previously published SPD families, with between 7 and 14 extra residues in the tract, to analyze the molecular basis for the observed variation in phenotype. Although there is no evidence of change in expansion size within families, even over six generations, there is a highly significant increase in the penetrance and severity of phenotype with increasing expansion size, affecting both hands (P ‫؍‬ 0.012) and feet (P < 0.00005). Affected individuals from a family with a 14-alanine expansion, the largest so far reported, all have a strikingly similar and unusually severe limb phenotype, involving the first digits and distal carpals. Affected males from this family also have hypospadias, not previously described in SPD, but consistent with HOXD13 expression in the developing genital tubercle. The remarkable correlation between phenotype and expansion size suggests that expansion of the tract leads to a specific gain of function in the mutant HOXD13 protein, and has interesting implications for the role of polyalanine tracts in the control of transcription.Dominantly inherited disorders frequently display incomplete penetrance (a normal phenotype in some mutation carriers) and variable expressivity (different degrees of phenotypic severity in affected individuals), the molecular basis for which is generally not understood. One such disorder is the rare dominantly inherited congenital limb malformation, synpolydactyly (SPD; OMIM No. 186000). Mutations in the first exon of HOXD13 have recently been found in three American SPD families (1), expanding a 15-residue polyalanine tract encoded by an imperfect trinucleotide repeat sequence by 7, 8, and 10 additional residues, respectively. Similar 9-residue expansions subsequently have been reported in two Turkish SPD families (2). SPD typically consists of 3͞4 finger and 4͞5 toe syndactyly, with a duplicated digit in the syndactylous web (3). Incomplete penetrance and variable expressivity both between and within affected families are common (4-6). From one to four limbs can be involved, and the severity of involvement ranges from partial skin syndactyly to complete reduplication of a digit, extending as far proximally as the metacarpals͞tarsals. Associated distal limb abnormalities include fifth-finger clinodactyly, camptodactyly, or brachydactyly; variable syndactyly of the second to fifth toes; and middle phalanx hypoplasia͞ aplasia.To investigate the molecular basis for this incomplete penetrance and variable expressivity, we analyzed the genotype and phenotype of 16 new SPD pedigrees, including one with an expansion that almost doub...