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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2015)

In this thesis, I describe the transcriptional regulation of neural crest throughout neurulation of Xenopus embryos. This work focuses on the global expression dynamics of genes involved in the specification of the ectodermal territories, i.e., neural plate, pre-placodal, neural border, and epidermis. In particular, I study the function of tfap2e, a member of the tfap2 family of transcription factors that is expressed in the neural crest of Xenopus.

In the first chapter, I provide an overview of the morphogenetic movements and signaling pathways that contribute to adoption of the ectodermal fates in Xenopus embryos, and in particular, to the process of specification of the neural crest. Furthermore, I describe what is currently known about the role of Tfap2 transcription factors in vertebrate neural crest development.

In the second chapter, I characterize the functional role of tfap2e in Xenopus development. I identified tfap2e in a screen for genes that are expressed exclusively in the neural crest. Through gain and loss of function analyses, I concluded that tfap2e is required for the early processes of neural crest specification, and it is also necessary and sufficient to promote neural crest delamination, a mesenchymal property essential for neural crest cell behaviors. To better understand why tfap2e is important for these processes, I developed a transcriptomic analysis strategy to identify genes whose expression is either positively or negatively correlated with Tfap2e activity. These genes are likely to be regulated directly or indirectly by Tfap2e. The results of this study contribute to a further understanding of the roles of Tfap2 factors within the neural crest module of the gene regulatory network.

The third chapter summarizes the results of collaborative work between the Harland and Eisen labs, at U.C. Berkeley, and the Monsoro-Burq lab at the Curie Institute in Orsay, France. I describe the expression dynamics of genes known to specify the different ectodermal fates in Xenopus embryos. In addition, I describe how we obtained and analyzed the expression profiles of manually dissected ectodermal tissues throughout neurulation. The dissection dataset was utilized as a reference to evaluate how tfap2e knockdown altered the fate of the neural plate border. In agreement with the analyses from the second chapter, we conclude that loss of tfap2e delays neural crest specification, and consequently, neural plate border cells seem to adopt an expression profile that is similar to the anterior placodes.

In the fourth and final chapter, I describe the syntenic relationships between the different tfap2 clusters. The analysis provides insights into the genomic rearrangements that have occurred during the evolution of vertebrates, which may have contributed to alterations in gene expression patterns after the divergence between the common ancestor of vertebrates and tunicates. Finally, I summarize the results of chromatin immunoprecipitation to identify putative cis-regulatory elements of genes that are downstream of Tfap2e. This protein may have both activator and repressor functions that help maintain the neural crest fate over other neural plate border fates.

The work described here has contributed to our understanding of how the development the neural crest is regulated, both through focusing on a single transcription factor, tfap2e, and more broadly by characterizing gene expression dynamics in this unique cell population and important vertebrate innovation.

    Cover page: Transcriptional regulation of the developing neural crest in Xenopus
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2014)
    A forward genetic screen of N-ethyl-N-nitrosourea mutagenized Xenopus tropicalis has identified an inner ear mutant named eclipse (ecl). Mutants developed enlarged otic vesicles and various defects of otoconia development; they also showed abnormal circular and inverted swimming patterns. Positional cloning identified specificity protein 8 (sp8), which was previously found to regulate limb and brain development. Two different loss-of-function approaches using transcription activator-like effector nucleases and morpholino oligonucleotides confirmed that the ecl mutant phenotype is caused by down-regulation of sp8. Depletion of sp8 resulted in otic dysmorphogenesis, such as uncompartmentalized and enlarged otic vesicles, epithelial dilation with abnormal sensory end organs. When overexpressed, sp8 was sufficient to induce ectopic otic vesicles possessing sensory hair cells, neurofilament innervation in a thickened sensory epithelium, and otoconia, all of which are found in the endogenous otic vesicle. We propose that sp8 is an important factor for initiation and elaboration of inner ear development.
      Cover page: Sp8 regulates inner ear development
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2015)
      Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.
        Cover page: Cell-fate determination by ubiquitin-dependent regulation of translation
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2022)
        Cephalopods are known for their large nervous systems, complex behaviors and morphological innovations. To investigate the genomic underpinnings of these features, we assembled the chromosomes of the Boston market squid, Doryteuthis (Loligo) pealeii, and the California two-spot octopus, Octopus bimaculoides, and compared them with those of the Hawaiian bobtail squid, Euprymna scolopes. The genomes of the soft-bodied (coleoid) cephalopods are highly rearranged relative to other extant molluscs, indicating an intense, early burst of genome restructuring. The coleoid genomes feature multi-megabase, tandem arrays of genes associated with brain development and cephalopod-specific innovations. We find that a known coleoid hallmark, extensive A-to-I mRNA editing, displays two fundamentally distinct patterns: one exclusive to the nervous system and concentrated in genic sequences, the other widespread and directed toward repetitive elements. We conclude that coleoid novelty is mediated in part by substantial genome reorganization, gene family expansion, and tissue-dependent mRNA editing.
          Cover page: Genome and transcriptome mechanisms driving cephalopod evolution
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2017)
          During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.
            Cover page: A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates
            • Article
            • Peer Reviewed
            UCLA Previously Published Works (2017)
            The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes.
              Cover page: Evolution of substrate specificity in a retained enzyme driven by gene loss
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2024)
              Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.
                Cover page: Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs
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