No abstract

Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea-induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat-containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells Reprints and permissions information is available online at

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency. The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects, and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5' exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of mouse models for MTHFR deficiency.

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog ( Hh ) signaling pathway are among the genes identified in the screen, including Gli2 , which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo , a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk–condyle separation and provide a molecular framework for future studies of the TMJ.