Transgenic complementation of MARCKS deficiency with a nonmyristoylatable, pseudo-phosphorylated form of MARCKS: evidence for simultaneous positive and dominant-negative effects on central nervous system development.

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.

Widespread neuronal ectopia associated with secondary defects in cerebrocortical chondroitin sulfate proteoglycans and basal lamina in MARCKS-deficient mice.

Mice deficient in MARCKS, a prominent neural substrate for protein kinase C (PKC), die before or shortly after birth. They exhibit high frequencies of exencephaly, universal agenesis of forebrain commissures, and abnormalities of cerebral cortical and retinal lamination. We show here that these mice have wide-spread and severe neuronal ectopia in the outer layers of the developing forebrain, manifested by the migration of clusters of developing neuroblasts through the basal lamina and often through the pial membrane and into the subarachnoid space. This abnormality became apparent by Embryonic Day (E) 13 or 14, shortly after the formation of the early marginal zone. MARCKS deficiency was associated with decreased staining for marginal zone chondroitin sulfate proteoglycans; this decrease was detectable earlier in development than the neuronal ectopia. Later in development, there was also marked disruption of the basal lamina at the pial-glial interface, as evidenced by gross abnormalities in laminin and reticulin staining; however, the basal lamina appeared normal at E9.5. These data indicate that MARCKS is required for the prevention of neuronal ectopia during development. Potential mechanisms responsible for the neuronal ectopia in the MARCKS-deficient mice include decreased expression or increased proteolytic destruction of basal lamina proteins and marginal zone chondroitin sulfate proteoglycans in the developing brain.

Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency.

The roles of protein kinase C and its substrates in development are poorly understood. Recently, we disrupted the mouse gene for a major cellular substrate for protein kinase C, the MARCKS protein (Proc. Natl. Acad. Sci. USA, 92, 944-948, 1995). The resulting phenotype consisted of universal perinatal lethality, agenesis of the corpus callosum and other forebrain commissures, and neuronal ectopia and other cortical and retinal lamination disturbances. These mice also had high frequencies of exencephaly (25% overall, 35% in females). In the present study, we have examined the normal expression of MARCKS and the various isozymes of protein kinase C at the time of cranial neural tube closure, in an attempt to correlate MARCKS expression in time and anatomical location with the exencephaly characteristic of MARCKS deficiency. Failure of neural tube closure occurred at various sites in the cranial neural tube, suggesting a cellular functional defect that was not limited to a specific location. Non-exencephalic MARCKS-deficient embryos appeared to be anatomically normal on embryonic day (E) 8.5-9.5. MARCKS and PKC alpha were expressed at the plasma membrane of the neuroepithelial cells comprising the future neural tube, as well as in the surface ectoderm and underlying mesenchyme. Endogenous protein kinase C species, comprising either or both alpha and delta, were capable of phosphorylating MARCKS in intact E8.5 embryos. Thus, MARCKS is expressed at the plasma membranes of the specific cell types involved in cranial neurulation; its deficiency presumably results in a still-to-be-elucidated functional defect in these cells that leads to exencephaly in a high proportion of cases.

Nonmyristoylated MARCKS complements some but not all of the developmental defects associated with MARCKS deficiency in mice.

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, is a widely expressed, prominent substrate for protein kinase C. Although the exact function of MARCKS has not been elucidated, targeted disruption of the MARCKS gene (Macs) in mice has shown that MARCKS plays a crucial role in the development of the central nervous system. Mice deficient in MARCKS exhibited universal perinatal death with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (Stumpo et al., Proc. Natl. Acad. Sci. USA 92, 944-948, 1995). In the present studies, a transgene consisting of approximately 3.4 kb of promoter from the human MARCKS gene (MACS), with an epitope tag sequence inserted at the carboxyl terminus of the MARCKS coding region, was able to complement completely MARCKS deficiency in mice. Thus, the human transgene contained all of the elements necessary for normal developmental expression of MARCKS. To test the importance of MARCKS myristoylation to its developmental role, an otherwise identical transgene was constructed in which the glycine at the amino terminus of MARCKS was mutated to an alanine. This mutation, which resulted in the expression of nonmyristoylated MARCKS, was successful in partially rescuing the Macs null phenotype. Specifically, about 25% of these mice survived the perinatal period; these survivors appeared to develop normally except for slightly decreased body size. In both the survivors and the nonsurvivors, all of the known anatomical defects associated with MARCKS deficiency were corrected by expression of the nonmyristoylated human protein. These results indicate that myristoylation of MARCKS is not required for the protein to correct many of the developmental abnormalities characteristic of its deficiency.

MARCKS deficiency in mice leads to abnormal brain development and perinatal death.

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development. To evaluate a possible developmental role for MARCKS, we disrupted its gene in mice by using the techniques of homologous recombination. Pups homozygous for the disrupted allele lacked detectable MARCKS mRNA and protein. All MARCKS-deficient pups died before or within a few hours of birth. Twenty-five percent had exencephaly and 19% had omphalocele (normal frequencies, < 1%), indicating high frequencies of midline defects, particularly in cranial neurulation. Nonexencephalic MARCKS-deficient pups had agenesis of the corpus callosum and other forebrain commissures, as well as failure of fusion of the cerebral hemispheres. All MARCKS-deficient pups also displayed characteristic lamination abnormalities of the cortex and retina. These studies suggest that MARCKS plays a vital role in the normal developmental processes of neurulation, hemisphere fusion, forebrain commissure formation, and formation of cortical and retinal laminations. We conclude that MARCKS is necessary for normal mouse brain development and postnatal survival.

Chromosomal mapping of the human (MACS) and mouse (Macs) genes encoding the MARCKS protein.

The myristoylated, alanine-rich C-kinase substrate, or MARCKS protein, is a major cellular substrate for protein kinase C that is also a high-affinity calmodulin-binding protein. In addition, it is the prototype of a small family of myristoylated, calmodulin-binding protein kinase C substrate proteins. We isolated a phage clone from a mouse genomic library that spanned the entire coding sequence of the mouse MARCKS protein. The first 612 bp of the putative promoter was 89% identical to a corresponding region of the human promoter, and contained at least 59 potential transcription factor binding sites in analogous locations; both human and mouse promoters lacked TATA boxes. The mouse genomic probe was used to localize the mouse gene to chromosome 10, in the middle of a linkage group that corresponds to a region on human chromosome 6q. These data strongly suggested that the human gene would localize to 6q21. This was confirmed by studies of DNA from a patient with del(6)(q21), in which expression of the human gene encoding MARCKS, MACS, was only about 50% of normal; MARCKS mRNA expression in lymphoblast RNA from this patient was only 22% of normal. These studies confirm that the mouse and human MARCKS proteins are products of the same genes in their respective species; differences in their primary sequence can therefore be attributed to species variation rather than to the existence of related genes.

Decreased levels of hepatic epidermal growth factor receptors in obese hyperglycemic rodents.

Stimulation of epidermal growth factor (EGF) receptor autophosphorylation by EGF and phosphorylation of a Mr 52,000 protein endogenous to the membrane extracts were decreased 6-12-fold in liver membrane extracts from mice homozygous for either the ob/ob or db/db mutation when compared to controls. Liver membranes from the mutant mice bound 4-5-fold less 125I-EGF/unit of protein than did their normal littermates, but exhibited normal EGF binding affinity. Similar decreases in EGF binding were noted in liver membranes from homozygous fa/fa Zucker rats, another obese, hyperinsulinemic animal model, when compared to values from control animals. We also immunoprecipitated hepatic EGF receptors from mice injected with [35S]methionine, and found that livers from db/db mice contained approximately 35% of the labeled EGF receptors found in control animals. Both ob/ob and db/db mice had serum immunoreactive EGF levels similar to or lower than those found in unaffected littermates, suggesting that ligand-mediated down-regulation of receptors was not the cause of the decreased EGF binding. In one mutant, db/db, the decreased binding was associated with a 6-fold decrease in the levels of liver EGF receptor mRNA transcripts; in the ob/ob mice, at most a 2-fold decrease in the level of liver EGF receptor transcripts was observed. EGF binding to cultured peritoneal fibroblasts derived from db/db mice was normal, suggesting that the abnormality in the mutant mice might result from altered environmental or tissue-specific factors rather than an abnormal receptor gene. This was supported by Southern blot analysis of DNA from these animals, which showed identical restriction fragment patterns for the EGF receptor gene in both control and mutant animals. These data indicate that three distinct strains of obese hyperglycemic rodents have decreased levels of hepatic EGF receptors, and suggest that this decrease may result from altered environmental stimuli or tissue-specific factors rather than a primary defect in the EGF receptor gene.