Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

Estimation of the incidence of a rare genetic disease through a two-tier mutation survey.

Recent attempts to detect mutations involving single base changes or small deletions that are specific to genetic diseases provide an opportunity to develop a two-tier mutation-screening program through which incidence of rare genetic disorders and gene carriers may be precisely estimated. A two-tier survey consists of mutation screening in a sample of patients with specific genetic disorders and in a second sample of newborns from the same population in which mutation frequency is evaluated. We provide the statistical basis for evaluating the incidence of affected and gene carriers in such two-tier mutation-screening surveys, from which the precision of the estimates is derived. Sample-size requirements of such two-tier mutation-screening surveys are evaluated. Considering examples of cystic fibrosis (CF) and medium-chain acyl-CoA dehydrogenase deficiency (MCAD), the two most frequent autosomal recessive disease in Caucasian populations and the two most frequent mutations (delta F508 and G985) that occur on these disease allele-bearing chromosomes, we show that, with 50-100 patients and a 20-fold larger sample of newborns screened for these mutations, the incidence of such diseases and their gene carriers in a population may be quite reliably estimated. The theory developed here is also applicable to rare autosomal dominant diseases for which disease-specific mutations are found.

Mutation and association analysis of the PVR and PVRL2 genes in patients with non-syndromic cleft lip and palate.

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.

Mutation analysis of the PVRL1 gene in caucasians with nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, MIM 119530) is perhaps the most common major birth defect. Homozygous PVRL1 loss-of-function mutations result in an autosomal recessive CL/P syndrome, CLPED1, and a PVRL1 nonsense mutation is associated with sporadic nsCL/P in Northern Venezuela. To address the more general role of PVRL1 variation in risk of nsCL/P, we carried out mutation analysis of PVRL1 in North American and Australian nsCL/P cases and population-matched controls. We identified a total of 15 variants, 5 of which were seen in both populations and 1 of which, an in-frame insertion at Glu442, was more frequent in patients than in controls in both populations, though the difference was not statistically significant. Another variant, which is specific to the PVRL1 beta (HIgR) isoform, S447L, was marginally associated with nsCL/P in North American Caucasian patients, but not in Australian patients, and overall variants that affect the beta-isoform were significantly more frequent among North American patients. One Australian patient had a splice junction mutation of PVRL1. Our results suggest that PVRL1 may play a minor role in susceptibility to the occurrence of nsCL/P in some Caucasian populations, and that variation involving the beta (HIgR) isoform might have particular importance for risk of orofacial clefts. Nevertheless, these results underscore the need for studies that involve very large numbers when assessing the possible role of rare variants in risk of complex traits such as nsCL/P.

Genetic and hormonal factors modulate spreading depression and transient hemiparesis in mouse models of familial hemiplegic migraine type 1.

Familial hemiplegic migraine type 1 (FHM1) is an autosomal dominant subtype of migraine with aura that is associated with hemiparesis. As with other types of migraine, it affects women more frequently than men. FHM1 is caused by mutations in the CACNA1A gene, which encodes the alpha1A subunit of Cav2.1 channels; the R192Q mutation in CACNA1A causes a mild form of FHM1, whereas the S218L mutation causes a severe, often lethal phenotype. Spreading depression (SD), a slowly propagating neuronal and glial cell depolarization that leads to depression of neuronal activity, is the most likely cause of migraine aura. Here, we have shown that transgenic mice expressing R192Q or S218L FHM1 mutations have increased SD frequency and propagation speed; enhanced corticostriatal propagation; and, similar to the human FHM1 phenotype, more severe and prolonged post-SD neurological deficits. The susceptibility to SD and neurological deficits is affected by allele dosage and is higher in S218L than R192Q mutants. Further, female S218L and R192Q mutant mice were more susceptible to SD and neurological deficits than males. This sex difference was abrogated by ovariectomy and senescence and was partially restored by estrogen replacement, implicating ovarian hormones in the observed sex differences in humans with FHM1. These findings demonstrate that genetic and hormonal factors modulate susceptibility to SD and neurological deficits in FHM1 mutant mice, providing a potential mechanism for the phenotypic diversity of human migraine and aura.

Severe aortic and arterial aneurysms associated with a TGFBR2 mutation.

BACKGROUND: A 24-year-old man presented with previously diagnosed Marfan's syndrome. Since the age of 9 years, he had undergone eight cardiovascular procedures to treat rapidly progressive aneurysms, dissection and tortuous vascular disease involving the aortic root and arch, the thoracoabdominal aorta, and brachiocephalic, vertebral, internal thoracic and superior mesenteric arteries. Throughout this extensive series of cardiovascular surgical repairs, he recovered without stroke, paraplegia or renal impairment. INVESTIGATIONS: CT scans, arteriogram, genetic mutation screening of transforming growth factor beta receptors 1 and 2. DIAGNOSIS: Diffuse and rapidly progressing vascular disease in a patient who met the diagnostic criteria for Marfan's syndrome, but was later rediagnosed with Loeys-Dietz syndrome. Genetic testing also revealed a de novo mutation in transforming growth factor beta receptor 2. MANAGEMENT: Regular cardiovascular surveillance for aneurysms and dissections, and aggressive surgical treatment of vascular disease.

THE MECHANISM OF TUMORIGENESIS IN THE IMMORTALIZED HUMAN PANCREATIC CELL LINES: CELL CULTURE MODELS OF HUMAN PANCREATIC CANCER

The mechanism of tumorigenesis in the immortalized human pancreatic cell lines: cell culture models of human pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in the world. The most common genetic lesions identified in PDAC include activation of K-ras (90%) and Her2 (70%), loss of p16 (95%) and p14 (40%), inactivation p53 (50-75%) and Smad4 (55%). However, the role of these signature gene alterations in PDAC is still not well understood, especially, how these genetic lesions individually or in combination contribute mechanistically to human pancreatic oncogenesis is still elusive. Moreover, a cell culture transformation model with sequential accumulation of signature genetic alterations in human pancreatic ductal cells that resembles the multiple-step human pancreatic carcinogenesis is still not established. In the present study, through the stepwise introduction of the signature genetic alterations in PDAC into the HPV16-E6E7 immortalized human pancreatic duct epithelial (HPDE) cell line and the hTERT immortalized human pancreatic ductal HPNE cell line, we developed the novel experimental cell culture transformation models with the most frequent gene alterations in PDAC and further dissected the molecular mechanism of transformation. We demonstrated that the combination of activation of K-ras and Her2, inactivation of p16/p14 and Smad4, or K-ras mutation plus p16 inactivation, was sufficient for the tumorigenic transformation of HPDE or HPNE cells respectively. We found that these transformed cells exhibited enhanced cell proliferation, anchorage-independent growth in soft agar, and grew tumors with PDAC histopathological features in orthotopic mouse model. Molecular analysis showed that the activation of K-ras and Her2 downstream effector pathways –MAPK, RalA, FAK, together with upregulation of cyclins and c-myc were involved in the malignant transformation. We discovered that MDM2, BMP7 and Bmi-1 were overexpressed in the tumorigenic HPDE cells, and that Smad4 played important roles in regulation of BMP7 and Bmi-1 gene expression and the tumorigenic transformation of HPDE cells. IPA signaling pathway analysis of microarray data revealed that abnormal signaling pathways are involved in transformation. This study is the first complete transformation model of human pancreatic ductal cells with the most common gene alterations in PDAC. Altogether, these novel transformation models more closely recapitulate the human pancreatic carcinogenesis from the cell origin, gene lesion, and activation of specific signaling pathway and histopathological features.

DYSREGULATION OF MEOX2 FOLLOWING WT1 MUTATION IN KIDNEY DEVELOPMENT AND WILMS TUMORIGENESIS

Wilms tumor (WT) is a childhood tumor of the kidney and a productive model for understanding the role of genetic alteration and interactions in tumorigenesis. The Wilms tumor gene 1 (WT1) is a transcriptional factor and one of the few genes known to have genetic alterations in WT and has been shown be inactivated in 20% of WTs. However, the mechanisms of how WT1 mutations lead to Wilms tumorigenesis and its influence on downstream genes are unknown. Since it has been established that WT1 is a transcriptional regulator, it has been hypothesized that the loss of WT1 leads to the dysregulation of downstream genes, in turn result in the formation of WTs. To identify the dysregulated downstream genes following WT1 mutations, an Affymetrix GeneChip Human Genome Array was previously conducted to assess the differentially expressed genes in the WT1-wildtype human and WT1-mutant human WTs. Approximately 700 genes were identified as being significantly dysregulated. These genes were further prioritized based on their statistical significance, fold change, chromosomal region, spatial pattern of gene expression and known or putative cellular functions. Mesenchyme homeobox 2 (MEOX2) was one of the most significantly upregulated genes in WT1-mutant WT. MEOX2 is known to play a role in cell proliferation, apoptosis, and differentiation. In addition to its biological roles, it is expressed during early kidney development in the condensed mesenchyme similar to WT1. Furthermore, the use of the Match® web-based tool from the BIOBASE Biological Data base identified a significant predicted WT1 binding site within the first intron of MEOX2. The similarity in spatial gene expression in the developing kidney and the significant predicted WT1 binding site found in the first intron of MEOX2 lead to the development of my hypothesis that MEOX2 is upregulated via a WT1-dependent manner. Here as a part of my master’s work, I have validated the Affymetrix GeneChip Human Genome Array data using an independent set of Wilms tumors. MEOX2 remained upregulated in the mutant WT1 Wilms tumor by 41-fold. Wt1 and Meox2 gene expression were assessed in murine newborn kidney; both Wt1 and Meox2 were expressed in the condensed, undifferentiated metanephric mesenchyme. I have shown that the in vivo ablation of Wt1 during embryonic development at embryonic day (E) 13.5 resulted in the slight increase of Meox2 gene expression by two fold. In order to functionally demonstrate the effect of the loss of Wt1 on Meox2 gene expression in undifferentiated metanephric mesenchyme, I have generated a kidney mesenchymal cell line to genetically ablate Wt1 in vitro by adenoviral infection. The ablation of Wt1 in the kidney mesenchymal cell line resulted in the upregulation of Meox2 by 61-fold. Moreover, the upregulation of Meox2 resulted in the significant induction of p21 and Itgb5. In addition to the dysregulation of these genes the ablation of Wt1 in the kidney mesenchymal cells resulted in decrease in cell growth and loss of cellular adherence. However, it is uncertain whether the upregulation of Meox2 caused this particular cellular phenotype. Overall, I have demonstrated that the upregulation of Meox2 is Wt1-dependent during early kidney development.

Detection and sequence analysis of a nickel-induced mutation in a retroviral model system

We have developed a novel way to assess the mutagenicity of environmentally important metal carcinogens, such as nickel, by creating a positive selection system based upon the conditional expression of a retroviral transforming gene. The target gene is the v-mos gene in MuSVts110, a murine retrovirus possessing a growth temperature dependent defect in expression of the transforming gene due to viral RNA splicing. In normal rat kidney cells infected with MuSVts110 (6m2 cells), splicing of the MuSVts110 RNA to form the mRNA from which the transforming protein, p85$\sp{\rm gag-mos}$, is translated is growth-temperature dependent, occurring at 33 C and below but not at 39 C and above. This splicing "defect" is mediated by cis-acting viral sequences. Nickel chloride treatment of 6m2 cells followed by growth at 39 C, allowed the selection of "revertant" cells which constitutively express p85$\sp{\rm gag-mos}$ due to stable changes in the viral RNA splicing phenotype, suggesting that nickel, a carcinogen whose mutagenicity has not been well established, could induce mutations in mammalian genes. We also show by direct sequencing of PCR-amplified integrated MuSVts110 DNA from a 6m2 nickel-revertant cell line that the nickel-induced mutation affecting the splicing phenotype is a cis-acting 70-base duplication of a region of the viral DNA surrounding the 3$\sp\prime$ splice site. These findings provide the first example of the molecular basis for a nickel-induced DNA lesion and establish the mutagenicity of this potent carcinogen. ^

Estimation of male-to-female ratios of mutation rate in primates and rodents

Haldane (1935) developed a method for estimating the male-to-female ratio of mutation rate ($\alpha$) by using sex-linked recessive genetic disease, but in six different studies using hemophilia A data the estimates of $\alpha$ varied from 1.2 to 29.3. Direct genomic sequencing is a better approach, but it is laborious and not readily applicable to non-human organisms. To study the sex ratios of mutation rate in various mammals, I used an indirect method proposed by Miyata et al. (1987). This method takes advantage of the fact that different chromosomes segregate differently between males and females, and uses the ratios of mutation rate in sequences on different chromosomes to estimate the male-to-female ratio of mutation rate. I sequenced the last intron of ZFX and ZFY genes in 6 species of primates and 2 species of rodents; I also sequenced the partial genomic sequence of the Ube1x and Ube1y genes of mice and rats. The purposes of my study in addition to estimation of $\alpha$'s in different mammalian species, are to test the hypothesis that most mutations are replication dependent and to examine the generation-time effect on $\alpha$. The $\alpha$ value estimated from the ZFX and ZFY introns of the six primate specise is ${\sim}$6. This estimate is the same as an earlier estimate using only 4 species of primates, but the 95% confidence interval has been reduced from (2, 84) to (2, 33). The estimate of $\alpha$ in the rodents obtained from Zfx and Zfy introns is ${\sim}$1.9, and that deriving from Ube1x and Ube1y introns is ${\sim}$2. Both estimates have a 95% confidence interval from 1 to 3. These two estimates are very close to each other, but are only one-third of that of the primates, suggesting a generation-time effect on $\alpha$. An $\alpha$ of 6 in primates and 2 in rodents are close to the estimates of the male-to-female ratio of the number of germ-cell divisions per generation in humans and mice, which are 6 and 2, respectively, assuming the generation time in humans is 20 years and that in mice is 5 months. These findings suggest that errors during germ-cell DNA replication are the primary source of mutation and that $\alpha$ decreases with decreasing length of generation time. ^

Functional analysis of MRF4 during mouse embryogenesis

MRF4 is one of four skeletal muscle specific regulatory genes, (the other three genes being MyoD, myf5, and myogenin), each of which has the unique ability to orchestrate an entire program of muscle-specific transcription when introduced into diverse cell types. These findings have led to the notion that these factors function as master regulators of muscle cell fate. Analysis of mice lacking MyoD, myf5, and myogenin have further defined their roles in the commitment and differentiation of myotomal progenitor cells. Current data strongly supports the model that MyoD and myf5 share functional redundancy in determining the muscle cell lineage, while myogenin acts downstream of MyoD and myf5, to initiate myoblast differentiation. Unlike other myogenic bHLH genes, MRF4 is expressed predominantly in the adult, suggesting that it may function to regulate adult muscle maturation and maintenance. To test this hypothesis and to eventually incorporate MRF4 into a general model for muscle specification, differentiation, maturation and maintenance, I deleted the MRF4 gene. MRF4-null mice are viable and fertile, however, they show mild rib anomalies. In addition, the expression of myogenin is dramatically upregulated only in the adult, suggesting that myogenin may compensate for the loss of MRF4 in the adult, and MRF4 may normally suppress the expression of myogenin after birth. MRF4 is also required during muscle regeneration after injury.^ To determine the degree of genetic redundancy between MRF4-myogenin; and MRF4-MyoD, I crossed the MRF4-null mice with MyoD- and myogenin-null mice respectively. There are no additional muscle phenotypes in double-null progeny from a MRF4 and myogenin cross, suggesting that the existence of residual fibers in myogenin-null mice is not due to the presence of MRF4. MRF4 expression also cannot account for the ability of myogenin-null myoblasts to differentiate in vitro. However, the combination of the MRF4-null mutation with the myogenin-null mutation results in a novel rib phenotype. This result suggests that MRF4 modifies the myogenin-null rib phenotype, and MRF4 and myogenin play redundant roles in rib development.^ MRF4 also shares dosage effects with MyoD during mouse development. (MyoD+/$-$;MRF4$-$/$-$)mice are fertile and viable, while (MyoD$-$/$-$;MRF4+/$-$) mice die between birth and two weeks after birth, and have a small skeletal structure. The double homozygous mice for MRF4 and MyoD mutations are embryonic lethal and die at around E10.5. These results suggest that MRF4 and MyoD share overlapping functions during mouse embryogenesis. ^

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

A conditional Mdm2 allele shows the importance of Mdm2 in heart development

Over 50% of sporadic tumors in humans have a p53 mutation highlighting its importance as a tumor suppressor. Considering additional mutations in other genes involved in p53 pathways, every tumor probably has mutant p53 or impaired p53-mediated functions. In response to a variety of cellular and genotoxic stresses, p53, mainly through its transcriptional activity, induces pathways involved in apoptosis and growth arrest. In these circumstances and under normal situations, p53 must be tightly regulated. Mdm2 is an important regulator of p53. Mdm2 inhibits p53 function by binding and blocking its transactivation domain. In addition, Mdm2 helps target p53 for degradation through its E3 ligase activity. Mdm2 null mice are embryonic lethal due to apoptosis in the blastocysts. However, a p53 null background rescues this lethality demonstrating the importance of the p53-Mdm2 interaction, particularly during development. The lethality of the Mdm2 null mouse prior to implantation limits the ability to investigate the role of Mdm2 in regulating p53 in a temporal and tissue specific manner. Does p53 need to be regulated in all tissues throughout the life of a mouse? Does Mdm2 always have to regulate it? To address these questions, we created a conditional Mdm2 allele. The conditional allele, Mdm2FM, in the presence of Cre recombinase results in the deletion of exons 5 and 6 of Mdm2 (most of the p53 binding domain) and represents a null allele. ^ The Mdm2FM allele was crossed with a heart muscle specific Cre expressing mouse (α-myosin heavy chain promoter driven Cre) to ask whether Mdm2 acts as a negative regulator of p53 in the heart. The heart is the most prominent organ early in embryogenesis and is shaped by cell death and proliferation. p53 does not appear to be active in the heart in response to some types of stress, so it remained to be determined if it has to be regulated in normal heart development. Loss of Mdm2 in the heart results in heart defects as early as E9.5. Loss of Mdm2 results in stabilized p53 and apoptosis. This apoptosis leads to a thinning of the myocardial wall particularly in the ventricles and abnormal ventricular structure. Eventually the abnormal heart fails resulting in lethality by E13.5. The embryonic lethality is rescued in a p53 null background. Thus, Mdm2 is important in regulating p53 in the development of the heart. ^

Abnormal expression of E2F1 and E2F4 results in hyperproliferation of the ependyma and lethal hydrocephalus

The proliferative role of E2F has been under investigation for several years. However, while it is known that E2F1 and E2F4 play a part in development and differentiation, research has not been centered on determining the exact functions these E2Fs play in brain development, given there high expression levels throughout embryogenesis. A GFAP-E2F1 mouse model directing human E2F1 transgene expression to glial cells, such as ependymal cells, was used in the present study in combination with an E2F4 mutant mouse model. Interestingly, 20% of tgE2F1; E2F4 null mice developed a phenotype consisting of domed head, hunched posture, seizures, tremors, hyperactivity or hypeactivity, dysnea, and low body weight. These mice died during the first three weeks of severe hydrocephalus. Similarly, tgE2F1; E2F4 heterozygous mice also develop severe hydrocephalus, although this occurs at 6 weeks at a 2% frequency. Pathological examination of the brains of those animals uncovered enlarged cerebral ventricles with marked thinning of the cerebral cortices, confirming the diagnosis of three-ventricle hydrocephalus, and the absence of tumors. Careful examination of the aqueduct shows an excess of proliferating cells that may cause a blockage of CSF. Of significance, 44% of ependymal cells in hydrocephalic tgE2F1;E2F4-/- mouse brains were positive for BrdU incorporation. Studies determining the molecular rationale for the hydrocephalic phenotype suggest proliferative ependymal cells may not be exclusively related to dysregulated cell cycle in conjuction with E2F activity. Due in part to the deficiency of E2F4 in this mouse model, we find that differentiation of these ependymal cells is not complete and instead undergoes maturation arrest. This suggestion is confirmed by the expression of genes found in neural stem cells or precursor cell populations, in the ependymal cell region of tgE2F1; E2F4-/-. Therefore, from this study, we conclude that dysregulated E2F1 expression in combination with deficient E2F4 expression results in an undifferentiated ependymal cell population that is hyperproliferative in the ventricular system causing an impediment of CSF circulation. It is further concluded that normal E2F1 and E2F4 expression in brain development is crucial for the proper formation and function of the ventricular system.^