Decrease in excitatory neurons, astrocytes and proliferating progenitors in the cerebral cortex of mice lacking exon 3 from the Fgf2 gene.

BACKGROUND: The Fgf2 gene is expressed in the brain neuroepithelium during embryonic development and in astroglial cells throughout life. Previous knockout studies suggested that FGF2 plays a role in the proliferation of neural progenitors in the embryonic cerebral cortex. These studies exclusively used knockout alleles lacking the Fgf2 exon 1. However, the description of putative alternative exons located downstream from the canonical exon 1 raised the possibility that alternatively spliced transcripts may compensate for the lack of the canonical exon 1 in the Fgf2 -/- mice. RESULTS: We generated and characterized a new line of Fgf2 knockout mice lacking the expression of exon 3, which is conserved in all Fgf2 transcripts and contains essential heparin and receptor binding interfaces. The expression of Fgf2 exon 3 was prevented by inserting a transcriptional STOP cassette in the Fgf2 genomic locus. These mice demonstrate a phenotype in the adult neocortex characterized by decreased density and number of cortical excitatory neurons and astrocytes, which is virtually identical to that of the Fgf2 -/- mice lacking exon 1. In addition, we also show that the Fgf2 exon 3 knockout mice have decreased proliferation of precursors in the adult cerebral cortex, which had not been previously investigated in the other mutant lines. CONCLUSION: The results demonstrate that the phenotype of two completely different Fgf2 KO mouse lines, lacking exon 1 or exon 3, is remarkably similar. The combined results from these KO models clearly indicate that FGF2 plays a role in cortical cell genesis during embryonic development as well as in adulthood. Thus, FGF2 may be required for the maintenance of the pool of adult cortical progenitor cells.

Mutations in apoptosis-related gene, PDCD10, cause cerebral cavernous malformation 3.

OBJECTIVE: To identify the CCM3 gene in a population of 61 families with a positive family history of cerebral cavernous malformations (CCM), 8 of which had suggestive linkage to the CCM3 locus. METHODS: We searched for mutations within the CCM3 interval using a high-throughput screening technique, temperature-gradient capillary electrophoresis. Mutations detected by this device were subsequently sequenced, and the results were analyzed. RESULTS: A recent study by Bergametti et al. established Programmed Cell Death 10 (PDCD10) as the gene responsible for CCM3. We hereby confirm PDCD10 as the CCM3 gene by reporting four novel mutations in 61 CCM families. Two of these mutations were identical and produced a stop codon in exon 7. Another two resulted in frameshift mutations in exon 6, although the mutations occurred at different points along the exon. The last mutation caused a frameshift in exon 9. Of note, mutations in these families completely cosegregated with the trait. Three of the five families had prior linkage data suggestive of the CCM3 locus, whereas the remaining two were identified in index patients with a positive family history but no linkage data. CONCLUSION: Our data establish PDCD10 as the gene responsible for CCM in families linking to the CCM3 locus. The discovery of the third gene involved in inherited forms of CCM, after KRIT1 and Malcavernin, is an important step toward dissecting the molecular pathophysiology of this disease.

Fibroblast growth factor receptor 1 is required for the proliferation of hippocampal progenitor cells and for hippocampal growth in mouse.

Fibroblast growth factor receptor 1 (Fgfr1) is expressed at high levels by progenitor cells of the ventricular zone (VZ) within the hippocampal primordium. To investigate the role of Fgfr1 in these cells, in vivo Cre recombination of "floxed" Fgfr1 alleles was directed to cells of the radial glial lineage by using the human glial fibrillary acidic protein promoter. Radial glial-like cells of the hippocampal VZ are the progenitors of pyramidal neurons and granule cells of hippocampal dentate gyrus (DG). Mice carrying null Fgfr1 alleles (Fgfr1(Deltaflox)) in cells of this lineage showed a dramatic loss of Fgfr1 gene expression throughout the embryonic dorsal telencephalon. These Fgfr1(Deltaflox) mice exhibited a approximately 30% decrease in dividing radial glial progenitor cells in the hippocampal VZ and DG in the late embryonic period, progressing to a approximately 50-60% loss at birth, without any changes in cell survival. In addition, no FGF2-sensitive neural stem cells could be isolated from the Fgfr1(Deltaflox) hippocampal neuroepithelium, whereas epidermal growth factor-sensitive neural stem cells were not affected. The number of hippocampal pyramidal neurons and DG granule cells was approximately 30-50% decreased from the perinatal period through adulthood, and the number of parvalbumin-containing interneurons was similarly decreased in both the DG and pyramidal cell fields. We conclude that Fgfr1 is necessary for hippocampal growth, because it promotes the proliferation of hippocampal progenitors and stem cells during development.

Loss of glutamatergic pyramidal neurons in frontal and temporal cortex resulting from attenuation of FGFR1 signaling is associated with spontaneous hyperactivity in mice.

Fibroblast growth factor receptor (FGFR) gene products (Fgfr1, Fgfr2, Fgfr3) are widely expressed by embryonic neural progenitor cells throughout the CNS, yet their functional role in cerebral cortical development is still unclear. To understand whether the FGF pathways play a role in cortical development, we attenuated FGFR signaling by expressing a tyrosine kinase domain-deficient Fgfr1 (tFgfr1) gene construct during embryonic brain development. Mice carrying the tFgfr1 transgene under the control of the Otx1 gene promoter have decreased thickness of the cerebral cortex in frontal and temporal areas because of decreased number of pyramidal neurons and disorganization of pyramidal cell dendritic architecture. These alterations may be, in part, attributable to decreased genesis of T-Brain-1-positive early glutamatergic neurons and, in part, to a failure to maintain radial glia fibers in medial prefrontal and temporal areas of the cortical plate. No changes were detected in cortical GABAergic interneurons, including Cajal-Retzius cells or in the basal ganglia. Behaviorally, tFgfr1 transgenic mice displayed spontaneous and persistent locomotor hyperactivity that apparently was not attributable to alterations in subcortical monoaminergic systems, because transgenic animals responded to both amphetamine and guanfacine, an alpha2A adrenergic receptor agonist. We conclude that FGF tyrosine kinase signaling may be required for the genesis and growth of pyramidal neurons in frontal and temporal cortical areas, and that alterations in cortical development attributable to disrupted FGF signaling are critical for the inhibitory regulation of motor behavior.

Mutational analysis of 206 families with cavernous malformations.

OBJECT: A gene contributing to the autosomal-dominant cerebral cavernous malformation (CCM) phenotype, KRIT1 (an acronym for Krev Interaction Trapped 1), has been identified through linkage analysis and mutation screening. The authors collected blood samples from 68 patients with familial CCM and 138 patients with apparently sporadic CCM as well as from their families, in an effort to characterize the prevalence and spectrum of disease-causing sequence variants in the KRIT1 gene. METHODS: The authors used single-strand conformational polymorphism analysis to identify genomic variants in KRIT1, which were sequenced to determine the specific mutation. Among 43 Hispanic-American kindreds who immigrated to the southwestern US from northern Mexico, 31 share an identical founder mutation. This Q455X mutation is found in 18 (86%) of 21 persons with a positive family history and in 13 (59%) of 22 persons with apparently sporadic CCM. This mutation was not found among 13 persons with CCM who were recruited from Mexico. These findings establish the key role of a recent founder mutation in Hispanic persons with CCM who live in the US. Although nearly all Hispanic families in the US in which there are multiple CCM cases linked to the CCM1 locus, only 13 of 25 non-Hispanic CCM-carrying families have displayed evidence of linkage to the CCM1 locus. Among these 13 families, the authors identified eight independent mutations in nine kindreds. They identified four additional mutations among 22 familial CCM kindreds with no linkage information, bringing the total number of independent mutations to 12. Inherited KRIT1 mutations were not detected among 103 non-Hispanic persons in whom a family history of CCM was rigorously excluded. CONCLUSIONS: All mutations were nonsense mutations, frame-shift mutations predicting premature termination, or splice-site mutations located throughout the KRIT1 gene, suggesting that these are genetic loss-of-function mutations. These genetic findings, in conjunction with the clinical phenotype, are consistent with a two-hit model for the occurrence of CCM.

KRIT1, a gene mutated in cerebral cavernous malformation, encodes a microtubule-associated protein.

Mutations in Krev1 interaction trapped gene 1 (KRIT1) cause cerebral cavernous malformation, an autosomal dominant disease featuring malformation of cerebral capillaries resulting in cerebral hemorrhage, strokes, and seizures. The biological functions of KRIT1 are unknown. We have investigated KRIT1 expression in endothelial cells by using specific anti-KRIT1 antibodies. By both microscopy and coimmunoprecipitation, we show that KRIT1 colocalizes with microtubules. In interphase cells, KRIT1 is found along the length of microtubules. During metaphase, KRIT1 is located on spindle pole bodies and the mitotic spindle. During late phases of mitosis, KRIT1 localizes in a pattern indicative of association with microtubule plus ends. In anaphase, the plus ends of the interpolar microtubules show strong KRIT1 staining and, in late telophase, KRIT1 stains the midbody remnant most strongly; this is the site of cytokinesis where plus ends of microtubules from dividing cells overlap. These results establish that KRIT1 is a microtubule-associated protein; its location at plus ends in mitosis suggests a possible role in microtubule targeting. These findings, coupled with evidence of interaction of KRIT1 with Krev1 and integrin cytoplasmic domain-associated protein-1 alpha (ICAP1 alpha), suggest that KRIT1 may help determine endothelial cell shape and function in response to cell-cell and cell-matrix interactions by guiding cytoskeletal structure. We propose that the loss of this targeting function leads to abnormal endothelial tube formation, thereby explaining the mechanism of formation of cerebral cavernous malformation (CCM) lesions.