Quaking is essential for blood vessel development

For nearly 40 years functional studies of the mouse quaking gene (qkI) have focused on its role in the postnatal central nervous system during myelination. However, the homozygous lethality of a number of ENU-induced alleles reveals that quaking has a critical role in embryonic development prior to the start of myelination. In this article, we show that quaking has a previously unsuspected and essential role in blood vessel development. Interestingly, we found that quaking, a nonsecreted protein, is expressed in the yolk sac endoderm, adjacent to the mesodermal site of developing blood islands, where the differentiation of blood and endothelial cells first occurs. Antibodies against PE-CAM-1, TIE-2 and SM-alpha-actin reveal that embryos homozygous for the qk(k2) allele have defective yolk sac vascular remodeling and abnormal vessels in the embryo proper at midgestation, coinciding with the timing of embryonic death. However, these mutants exhibit normal expression of Nkx2.5 and alpha-sarcomeric actin, indicating that cardiac muscle differentiation was normal. Further, they had normal embryonic heart rates in culture, suggesting that cardiac function was not compromised at this stage of embryonic development. Together, these results suggest that quaking plays an essential role in vascular development and that the blood vessel defects are the cause of embryonic death.     

Apoptosis in the chick wing bud and the permanence of FGF-2 rescue

Summary Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The opaque patch (OP) and posterior necrotic zone (PNZ) were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by inhibition of protein synthesis with cycloheximide. Both regions showed rescue with cycloheximide as determined by the chromium release assay and examination of electron micrographs. Also, the permanence of basic fibroblast growth factor (FGF-2) rescue in the OP and PNZ was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. This research shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation and is prevented by inhibition of protein synthesis with cycloheximide, biochemically characterizing this death as apoptosis. It also suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ.     

Contrasting Effects of ENU Induced Embryonic Lethal Mutations of thequakingGene

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal allelesqk^l-1, qk^kt1, qk^k2,andqk^kt3.The mutation inqk^l-1creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains inqk^k2andqk^kt3,respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qk^vdiffer. Compound heterozygousqk^vanimals carryingqk^kt1, qk^k2,andqk^kt3all exhibit a permanent quaking phenotype similar to that ofqk^v/qk^vanimals, whereasqk^v/qk^l-1animals exhibit only a transient quaking phenotype. Theqk^l-1mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed inqk^v/qk^l-1mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.     

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.     

A new ENU-induced allele of mouse quaking causes severe CNS dysmyelination

The mutant allelic series of the mouse quaking gene consists of the spontaneous quaking^viable (qk^v) allele, which is homozygous viable with a dysmyelination phenotype, and four ENU-induced alleles (qk^kt1, qk^k2, qk^kt3/4, and qk^l-1), which are homozygous embryonic lethal. Here we report the isolation of qk^e5, the first ENU-induced viable allele of quaking. Unlike qk^v/qk^v, qk^e5/qk^e5 animals have early-onset seizures, severe ataxia, and a dramatically reduced lifespan. Ultrastructural analysis of qk^e5/qk^e5 brains reveals severe dysmyelination when compared with both wild-type and qk^v/qk^v brains. In addition, Calbindin detection in young adult qk^e5/qk^e5 mice reveals Purkinje cell axonal swellings indicative of neurodegeneration , which is not seen in young adult qk^v/qk^v mice. Although the molecular defect in the qk^e5 allele is not evident by sequencing, protein expression studies show that qk^e5/qk^e5 postnatal oligodendrocytes lack the QKI-6 and QKI-7 isoforms and have reduced QKI-5 levels. The oligodendrocyte developmental markers PDGFαR, NG2, O4, CNP, and MBP are also present in the qk^e5/qk^e5 postnatal brain although CNP and MBP levels are considerably reduced. Because the qk^v allele is a large deletion that affects the expression of three genes, the new neurologic qk^e5 allele is an important addition to this allelic series.