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Transgenic complementation of MARCKS deficiency with a nonmyristoylatable, pseudo-phosphorylated form of MARCKS:
H S Kim1, S L Swierczynski, J S Tuttle
1Office of Clinical Research and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709, USA.
Abstract:
MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure-function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, "pseudo-phosphorylated" form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.
Insights
Modified MARCKS protein partially corrected brain defects in knockout mice but worsened retinal abnormalities, suggesting a dominant-negative effect on related proteins.
Area of Science:
- Neuroscience
- Developmental Biology
- Molecular Biology
Background:
- MARCKS protein is crucial for prenatal central nervous system development in mice.
- MARCKS deficiency leads to perinatal mortality and severe brain/retina abnormalities.
Purpose of the Study:
- To investigate the role of specific MARCKS protein domains in correcting neurodevelopmental defects.
- To assess the impact of a nonmyristoylatable, pseudo-phosphorylated MARCKS form on these anomalies.
Main Methods:
- Interbreeding MARCKS knockout mice with transgenic mice expressing a modified human MARCKS transgene.
- Evaluating cerebral and retinal anatomical abnormalities in the resulting offspring.
Main Results:
- The modified MARCKS transgene largely corrected cerebral anatomical abnormalities.
- A novel, severe retinal ectopia phenotype was observed, suggesting a worsened condition.
- The retinal anomaly was present in a subset of original knockout mice and detectable early in development.
Conclusions:
- A nonmyristoylatable, pseudo-phosphorylated MARCKS form can rescue most cerebral defects.
- This modified form may exacerbate retinal phenotypes, potentially via a dominant-negative effect on MARCKS homologues.