Mouse mutagenesis identifies novel roles for left-right patterning genes in pulmonary, craniofacial, ocular, and limb development.

Vertebrate organs show consistent left-right (L-R) asymmetry in placement and patterning. To identify genes involved in this process we performed an ENU-based genetic screen. Of 135 lines analyzed 11 showed clear single gene defects affecting L-R patterning, including 3 new alleles of known L-R genes and mutants in novel L-R loci. We identified six lines (termed "gasping") that, in addition to abnormal L-R patterning and associated cardiovascular defects, had complex phenotypes including pulmonary agenesis, exencephaly, polydactyly, ocular and craniofacial malformations. These complex abnormalities are present in certain human disease syndromes (e.g., HYLS, SRPS, VACTERL). Gasping embryos also show defects in ciliogenesis, suggesting a role for cilia in these human congenital malformation syndromes. Our results indicate that genes controlling ciliogenesis and left-right asymmetry have, in addition to their known roles in cardiac patterning, major and unexpected roles in pulmonary, craniofacial, ocular and limb development with implications for human congenital malformation syndromes.

Defining the cause of skewed X-chromosome inactivation in X-linked mental retardation by use of a mouse model.

Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.

Filamin C interacts with the muscular dystrophy KY protein and is abnormally distributed in mouse KY deficient muscle fibres.

The KY protein has been implicated in a neuromuscular dystrophy in the mouse, but its role in muscle function remains unclear. Here, we show that KY interacts with several sarcomeric cytoskeletal proteins including, amongst others, filamin C and the slow isoform of the myosin-binding protein C. These interactions were confirmed in vitro and because of its central role in skeletal muscle disease, characterized in more detail for filamin C. A role for KY in regulating filamin C function in vivo is supported by the expression analysis of filamin C in the null ky mouse mutant, where distinct irregular subcellular localization of filamin C was found in subsets of muscle fibres, which appears to be a specific outcome of KY deficiency. Furthermore, KY shows protease activity in in vitro assays, and specific degradation of filamin C by KY is shown in transfected cells. Given the enzymatic nature of the KY protein, it is likely that some of the identified partners are catalytic substrates. These results suggest that KY is an intrinsic part of the protein networks underlying the molecular mechanism of several limb-girdle muscular dystrophies, particularly those where interactions between filamin C and disease causing proteins have been shown.

Overlapping and distinct expression domains of Zic2 and Zic3 during mouse gastrulation.

The Zic genes are the vertebrate homologues of the Drosophila Odd-paired gene. Mutations in two of these genes are associated with human congenital genetic disorders. Mutation of human and mouse Zic2 is associated with holoprosencephaly which is caused by a defect of ventral forebrain development and mutation of human and mouse Zic3 is associated with a X-linked heterotaxy syndrome that results from a failure of left-right axis formation. The embryological role of the Zic genes in these disorders is not well understood. Here we show that both of these genes are expressed prior to and throughout gastrulation. The genes show some broad similarities in their expression domains. Both genes however are also uniquely expressed in some tissues and these unique domains correlate with regions that potentially play a role in the aetiology of the respective genetic disorders. During primitive streak stages Zic2 is expressed transiently and uniquely in the node and the head process mesendoderm. The head process is known to be required for the establishment or maintenance of the ventral forebrain, which is the region disrupted in holoprosencephaly. Zic3 is not expressed in the node during primitive streak stages but is expressed in and around the node beginning from the head fold stages of development. This expression of Zic3 correlates well with the first steps in the establishment of the left-right axis. We also examined the expression of the closely related gene, Zic1, and did not detect any transcripts in gastrulation stage embryos.