Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (23 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2013)

Sperm and oocytes are terminally differentiated, sex-specific germ cells, which, upon fertilization will generate a new embryo and leads to species propagation by sexual reproduction. Though fated only to generate eggs or sperm, germ cells have the unique property to imbue zygotes with totipotent capacity, which facilitates the formation of all tissues the embryo will need to survive to adulthood. These characteristics are facilitated by germ cells' ability to pass on genetic information to the next generation, as well as their capacity to initiate genome-wide reorganization and removal of epigenetic information inherited by germ cells during embryogenesis. It is hypothesized that remodeling of this epigenetic information is essential to drive proper embryo development. While these events are not well understood, it is known that the events that underlie these unique properties are initiated in early development, shortly after the germ line is established as a pool of primordial germ cells (PGCs). Efforts to unravel these mechanisms that underlie totipotency in germ cells have been limited due to the inability to isolate, study, and manipulate PGCs. To overcome this obstacle, we hypothesized that PGCs cells could be differentiated from pluripotent embryonic stem cells, and that these cells would serve as a surrogate cell type for the study of PGC biology.

Establishing a new model of lineage differentiation from embryonic stem cells required the development of assays and criteria to rigorously test identity, developmental staging, and epigenetic progression to determine if an in vitro model is able to recapitulate features of endogenous PGCs. To accomplish this, we developed a scalable and transgene-free method to differentiate immature PGCs in vitro using the cell surface markers SSEA1 and cKit that are developmentally and epigenetically reminiscent of immature PGCs. We applied existing assays to validate PGC identity, and devised a new stringent assay based on genetic deletion of a known PGC determinant. We developed a single-cell gene expression methodology to compare gene expression signatures of in vitro derived PGCs and endogenous PGCs, and identified novel criteria to define PGC identity from early endogenous PGCs and in vitro-generated PGCs.

We next used in vitro PGC differentiation to investigate genome-wide DNA demethylation, one of the first epigenetic reprogramming events undertaken by early PGCs. By combining the scalability of this differentiation system with next generation methylation sequencing techniques, we generated the first DNA methylation maps of in vitro derived PGCs, and determined with sequence-specific information that DNA demethylation is genome-wide and likely to involve loss of DNA methylation as a consequence of cell division. We also investigated potentiators of active DNA methylation loss, including the Tet proteins, and their roles in early PGC development.

Finally, we applied single cell gene expression technology to define developmental progression of human PGCs isolated from the gonads of fetuses from elective terminations. We identified a common progenitor stage of PGC development in the human fetal gonad. Furthermore, we adapted our single cell gene expression approaches to interrogate differentiation strategies in the generation of the common human PGC progenitor in vitro.

Together, we have developed a differentiation system to ask questions about epigenetic progression in early germ cells, and have utilized single cell gene expression technology and genomics to characterize seminal events in the epigenetic reprogramming of human and mouse germ cells.

    Cover page: Primordial Germ Cell Differentiation in Vitro: A Model for Understanding Epigenetic Reprogramming and Genome-Wide DNA Demethylation in Mouse Primordial Germ Cells
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2011)
    Graph coloring and linear scan are two appealing techniques for register allocation as the underlying formalism are extremely clean and simple. This paper advocates a decoupled approach that first lowers the register pressure by spilling variables, and then performs live ranges splitting/coalescing /coloring in a separate phase; this enables the design of simpler, cleaner, and more efficient register allocators. This paper gives a new and more general approach to deal with register constraints. This approach called repairing does not require pre live range splitting and does not introduce additional spill code. It ignores register constraints during coloring/coalescing, and repairs violated constraints afterwards. We applied this method to both graph based and scan based allocators into a decoupled approach. Here, the Iterated Register Coalescer (IRC) and a scan algorithm that uses Static Single Assignment (SSA) properties, the treescan. Moreover, this paper provides a survey on existing and new techniques of bias coloring during scan approaches. Our experimental evaluation shows for the graph based approach, that we reduced the number of vertices (edges) in the interference graph by 26% (33%) without compromising the quality of the generated code. The treescan algorithm improved the compile time of the allocation process by 6:97x over IRC while providing comparable results for the quality of the generated code. Copyright © 2011 ACM.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2015)
      Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.
        Cover page: The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2011)
        Pluripotent stem cells (PSCs) hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into every cell type in the body. One major barrier to the use of PSCs is their potential risk for tumor initiation following differentiation and transplantation in vivo. In the current study we sought to evaluate the role of the tumor suppressor Pten in murine PSC neoplastic progression. Using eight functional assays that have previously been used to indicate PSC adaptation or transformation, Pten null embryonic stem cells (ESCs) failed to rate as significant in five of them. Instead, our data demonstrate that the loss of Pten causes the emergence of a small number of aggressive, teratoma-initiating embryonic carcinoma cells (ECCs) during differentiation in vitro, while the remaining 90-95% of differentiated cells are non-tumorigenic. Furthermore, our data show that the mechanism by which Pten null ECCs emerge in vitro and cause tumors in vivo is through increased survival and self-renewal, due to failed repression of the transcription factor Nanog.
          Cover page: Loss of Pten Causes Tumor Initiation Following Differentiation of Murine Pluripotent Stem Cells Due to Failed Repression of Nanog
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2014)
          Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT OMIM 312750). Alternative inclusion of MECP2/Mecp2 exon 1 with exons 3 and 4 encodes MeCP2-e1 or MeCP2-e2 protein isoforms with unique amino termini. While most MECP2 mutations are located in exons 3 and 4 thus affecting both isoforms, MECP2 exon 1 mutations but not exon 2 mutations have been identified in RTT patients, suggesting that MeCP2-e1 deficiency is sufficient to cause RTT. As expected, genetic deletion of Mecp2 exons 3 and/or 4 recapitulates RTT-like neurologic defects in mice. However, Mecp2 exon 2 knockout mice have normal neurologic function. Here, a naturally occurring MECP2 exon 1 mutation is recapitulated in a mouse model by genetic engineering. A point mutation in the translational start codon of Mecp2 exon 1, transmitted through the germline, ablates MeCP2-e1 translation while preserving MeCP2-e2 production in mouse brain. The resulting MeCP2-e1 deficient mice developed forelimb stereotypy, hindlimb clasping, excessive grooming and hypo-activity prior to death between 7 and 31 weeks. MeCP2-e1 deficient mice also exhibited abnormal anxiety, sociability and ambulation. Despite MeCP2-e1 and MeCP2-e2 sharing, 96% amino acid identity, differences were identified. A fraction of phosphorylated MeCP2-e1 differed from the bulk of MeCP2 in subnuclear localization and co-factor interaction. Furthermore, MeCP2-e1 exhibited enhanced stability compared with MeCP2-e2 in neurons. Therefore, MeCP2-e1 deficient mice implicate MeCP2-e1 as the sole contributor to RTT with non-redundant functions.
            Cover page: Mice with an isoform-ablating Mecp2 exon 1 mutation recapitulate the neurologic deficits of Rett syndrome
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2022)
            Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.
              Cover page: Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2015)

              Background

              Cohen Syndrome (COH1) is a rare autosomal recessive disorder, principally identified by ocular, neural and muscular deficits. We identified three large consanguineous Pakistani families with intellectual disability and in some cases with autistic traits.

              Methods

              Clinical assessments were performed in order to allow comparison of clinical features with other VPS13B mutations. Homozygosity mapping followed by whole exome sequencing and Sanger sequencing strategies were used to identify disease-related mutations.

              Results

              We identified two novel homozygous deletion mutations in VPS13B, firstly a 1 bp deletion, NM_017890.4:c.6879delT; p.Phe2293Leufs*24, and secondly a deletion of exons 37-40, which co-segregate with affected status. In addition to COH1-related traits, autistic features were reported in a number of family members, contrasting with the "friendly" demeanour often associated with COH1. The c.6879delT mutation is present in two families from different regions of the country, but both from the Baloch sub-ethnic group, and with a shared haplotype, indicating a founder effect among the Baloch population.

              Conclusion

              We suspect that the c.6879delT mutation may be a common cause of COH1 and similar phenotypes among the Baloch population. Additionally, most of the individuals with the c.6879delT mutation in these two families also present with autistic like traits, and suggests that this variant may lead to a distinct autistic-like COH1 subgroup.
                Cover page: Novel VPS13B Mutations in Three Large Pakistani Cohen Syndrome Families Suggests a Baloch Variant with Autistic-Like Features
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2021)
                Bipolar disorder (BD) is a serious mental illness with substantial common variant heritability. However, the role of rare coding variation in BD is not well established. We examined the protein-coding (exonic) sequences of 3,987 unrelated individuals with BD and 5,322 controls of predominantly European ancestry across four cohorts from the Bipolar Sequencing Consortium (BSC). We assessed the burden of rare, protein-altering, single nucleotide variants classified as pathogenic or likely pathogenic (P-LP) both exome-wide and within several groups of genes with phenotypic or biologic plausibility in BD. While we observed an increased burden of rare coding P-LP variants within 165 genes identified as BD GWAS regions in 3,987 BD cases (meta-analysis OR = 1.9, 95% CI = 1.3-2.8, one-sided p = 6.0 × 10-4), this enrichment did not replicate in an additional 9,929 BD cases and 14,018 controls (OR = 0.9, one-side p = 0.70). Although BD shares common variant heritability with schizophrenia, in the BSC sample we did not observe a significant enrichment of P-LP variants in SCZ GWAS genes, in two classes of neuronal synaptic genes (RBFOX2 and FMRP) associated with SCZ or in loss-of-function intolerant genes. In this study, the largest analysis of exonic variation in BD, individuals with BD do not carry a replicable enrichment of rare P-LP variants across the exome or in any of several groups of genes with biologic plausibility. Moreover, despite a strong shared susceptibility between BD and SCZ through common genetic variation, we do not observe an association between BD risk and rare P-LP coding variants in genes known to modulate risk for SCZ.
                  Cover page: Investigating rare pathogenic/likely pathogenic exonic variation in bipolar disorder.
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2012)
                  Autism spectrum disorder (ASD) is a highly heritable disorder of complex and heterogeneous aetiology. It is primarily characterized by altered cognitive ability including impaired language and communication skills and fundamental deficits in social reciprocity. Despite some notable successes in neuropsychiatric genetics, overall, the high heritability of ASD (~90%) remains poorly explained by common genetic risk variants. However, recent studies suggest that rare genomic variation, in particular copy number variation, may account for a significant proportion of the genetic basis of ASD. We present a large scale analysis to identify candidate genes which may contain low-frequency recessive variation contributing to ASD while taking into account the potential contribution of population differences to the genetic heterogeneity of ASD. Our strategy, homozygous haplotype (HH) mapping, aims to detect homozygous segments of identical haplotype structure that are shared at a higher frequency amongst ASD patients compared to parental controls. The analysis was performed on 1,402 Autism Genome Project trios genotyped for 1 million single nucleotide polymorphisms (SNPs). We identified 25 known and 1,218 novel ASD candidate genes in the discovery analysis including CADM2, ABHD14A, CHRFAM7A, GRIK2, GRM3, EPHA3, FGF10, KCND2, PDZK1, IMMP2L and FOXP2. Furthermore, 10 of the previously reported ASD genes and 300 of the novel candidates identified in the discovery analysis were replicated in an independent sample of 1,182 trios. Our results demonstrate that regions of HH are significantly enriched for previously reported ASD candidate genes and the observed association is independent of gene size (odds ratio 2.10). Our findings highlight the applicability of HH mapping in complex disorders such as ASD and offer an alternative approach to the analysis of genome-wide association data.
                    Cover page: A novel approach of homozygous haplotype sharing identifies candidate genes in autism spectrum disorder
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2007)
                    Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental conditions. The genetic architecture of ASDs is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASDs by using Affymetrix 10K SNP arrays and 1,181 [corrected] families with at least two affected individuals, performing the largest linkage scan to date while also analyzing copy number variation in these families. Linkage and copy number variation analyses implicate chromosome 11p12-p13 and neurexins, respectively, among other candidate loci. Neurexins team with previously implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for contributing to ASDs.
                      Cover page: Mapping autism risk loci using genetic linkage and chromosomal rearrangementsCreative Commons 'BY' version 4.0 license
                      Top