Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.

Chromatin structure and function are for a large part determined by the six members of the structural maintenance of chromosomes (SMC) protein family, which form three heterodimeric complexes: Smc1/3 (cohesin), Smc2/4 (condensin) and Smc5/6. Each complex has distinct and important roles in chromatin dynamics, gene expression and differentiation. In yeast and Drosophila, Smc6 is involved in recombinational repair, restarting collapsed replication forks and prevention of recombination in repetitive sequences such as rDNA and pericentromeric heterochromatin. Although such DNA damage control mechanisms, as well as highly dynamic changes in chromatin composition and function, are essential for gametogenesis, knowledge on Smc6 function in mammalian systems is limited. We therefore have investigated the role of Smc6 during mammalian spermatogonial differentiation, meiosis and subsequent spermiogenesis. We found that, during mouse spermatogenesis, Smc6 functions as part of meiotic pericentromeric heterochromatin domains that are initiated when differentiating spermatogonia become irreversibly committed toward meiosis. To our knowledge, we are the first to provide insight into how commitment toward meiosis alters chromatin structure and dynamics, thereby setting apart differentiating spermatogonia from the undifferentiated spermatogonia, including the spermatogonial stem cells. Interestingly, Smc6 is not essential for spermatogonial mitosis, whereas Smc6-negative meiotic cells appear unable to finish their first meiotic division. Importantly, during meiosis, we find that DNA repair or recombination sites, marked by γH2AX or Rad51 respectively, do not co-localize with the pericentromeric heterochromatin domains where Smc6 is located. Considering the repetitive nature of these domains and that Smc6 has been previously shown to prevent recombination in repetitive sequences, we hypothesize that Smc6 has a role in the prevention of aberrant recombination events between pericentromeric regions during the first meiotic prophase that would otherwise cause chromosomal aberrations leading to apoptosis, meiotic arrest or aneuploidies.