ENU-induced mutations reveal quaking functions in embryogenesis and postnatal myelination

Author

Janice K. Noveroske

Date of Award

12-2000

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jeff A. MacCabe

Committee Members

Monica J. Justice, Jim D. Godkin, Ranjan Ganguly

Abstract

Since the discovery of the mouse quaking viable allele (qk^v) in 1964, researchers have attempted to elucidate the function of quaking in postnatal brain myelination. More recently, a critical role for quaking in embryogenesis was revealed, but little has been determined on how the loss of quaking function leads to embryonic death. In the work presented here, we report that the defect in the embryonic lethal qk^k2 allele is a T to A trans version in the KH domain, and that homozygous qk^k2 mutant embryos die from cardiovascular defects. Antibodies against the vascular endothelial marker PECAM-1 reveal that qk^k2 homozygous mutants completely lack the yolk sac vascular network present in wild-type embryos. Hearts of qk^k2/qk^k2 embryos lack the looping morphogenesis of their wild-type littennates, but Nkx2.5, a gene that functions in cardiac looping, is expressed in a wild-type pattern. Cardiac myocytes in mutant embryos also express α-sarcomeric actin protein and whole hearts in organ culture were found to beat at wild-type rates. Real-time quantitative RT-PCR reveals that cardiac developmental markers are expressed at normal levels in qk^k2 mutant hearts but that expression of the vascularization gene, Vegf, is increased two-fold compared to wild-type. In addition, the cell cycle genes Ccnd1, Cdkn1a and Cdkn1c have abnormal expression compared to wild-type embryonic hearts. The neural associated genes Shh, Foxa2, Fgf8, and Pax6 as well as the notochord marker T, are all expressed in wild-type patterns in qk^k2 embryos at the time of death. However, adult carriers of the qk^k2 allele are significantly more susceptible to chemically induced seizures with the convulsant drug pentylenetetrazole than wild-type animals. In an effort to further dissect the function of quaking, we performed a single generation ENU mutagenesis screen to create new alleles. We report here, the generation of the qk^e5 allele which, unlike the previous induced alleles of quaking, is homozygous viable. The qk^e5 phenotype consists of postnatal quaking and seizures, which progresses to severe ataxia and early death. Ultrastructural analysis reveals an almost complete lack of myelin compared to both wild-type and qk^k2/qk^k2 brains. Protein expression analysis in the postnatal brain shows that like qk^k2/qk^k2, qk^e5/qk^e5 mice have low QKI-5 and no QKI-6 and QKI-7 proteins in developing oligodendrocytes. However, oligodendrocytes of both qk^e5 and qk^v homozygous mice express a full range of developmental marker genes indicating that quaking may function relatively late in oligodendrocyte development. We determined by electroencephalogram recordings, that qk^e5/qk^e5 surface cortex activity is abnormal and that seizures result in electrical activity characteristic of a decrimental response. Together these results reveal a previously unsuspected and critical role for quaking in cardiovascular development as well as a more important role in postnatal brain myelination then had been determined from 40 years of studies utilizing the qk^v allele.

Recommended Citation

Noveroske, Janice K., "ENU-induced mutations reveal quaking functions in embryogenesis and postnatal myelination. " PhD diss., University of Tennessee, 2000.
https://trace.tennessee.edu/utk_graddiss/8372