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Open Access Publications from the University of California

Scholarly Works (15 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2018)

Zygotic gene activation (ZGA) is the process in the earliest stages of animal development when maternal transcription factors activate the expression of the first zygotic genes. In most metazoans, this process is coupled with specification of the three germ layers. In Xenopus, these two processes are initiated by the function of maternal transcription factors (TFs), which are localized in the egg and asymmetrically distributed to different cells of the embryo during cleavage stages. In this thesis, I uncover the combinatorial function of vegetally-localized TFs that are involved in the activation of the endodermal fate.

By gain- and loss-of-function approaches, I elucidate a novel function of the TF Otx1 in specification of the endoderm. Together with the well-known endodermal TF Vegt, Otx1 activates endodermal genes, while inhibiting mesodermal genes in the presumptive endoderm. By chromatin immunoprecipitation followed by qPCR or sequencing, I show that Vegt and Otx1 co-bind in the chromatin with the ubiquitously expressed Foxh1 prior to ZGA. These maternal TFs initiate the emergence of cis-regulatory regions by docking to clusters of consensus DNA binding motifs. The binding of their assembly pre-marks regions of active cis-regulation, which are subsequently decorated with epigenetic enhancer marks and then bound by zygotically-active endodermal TFs.

In addition to co-regulation between maternal TFs, by curating evidence to generate the Xenopus endoderm gene regulatory network (GRN), I find evidence that maternal TFs co-regulate downstream target genes with zygotic TFs. Analysis of the Xenopus endodermal GRN suggests that feed-forward loops, where a maternal TF activates a primary zygotic gene encoding a TF, and combinatorially these TFs activate a secondary zygotic gene. From ChIP-seq analysis, the chromatin binding of Vegt and the zygotically active Smad2/3 support this finding. While the functional role of this network motif is unknown, computational modeling suggests that this motif could regulate the multiple waves of zygotic transcription during ZGA.

My findings synthesize the roles of maternal TFs in relation to each other, as well as to the roles of zygotic TFs, using cis-regulatory and gene regulatory network perspectives. The use of multiple approaches provides a broader picture of transcriptional regulation and cell specification during this critical process of ZGA.

    Cover page: Combinatorial regulation by maternal transcription factors during activation of the endoderm gene regulatory networkCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2020)
    For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.
      Cover page: Early Xenopus gene regulatory programs, chromatin states, and the role of maternal transcription factors.
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2017)
      Germ layer formation is among the earliest differentiation events in metazoan embryos. In triploblasts, three germ layers are formed, among which the endoderm gives rise to the epithelial lining of the gut tube and associated organs including the liver, pancreas and lungs. In frogs (Xenopus), where early germ layer formation has been studied extensively, the process of endoderm specification involves the interplay of dozens of transcription factors. Here, we review the interactions between these factors, summarized in a transcriptional gene regulatory network (GRN). We highlight regulatory connections conserved between frog, fish, mouse, and human endodermal lineages. Especially prominent is the conserved role and regulatory targets of the Nodal signaling pathway and the T-box transcription factors, Vegt and Eomes. Additionally, we highlight network topologies and motifs, and speculate on their possible roles in development.
        Cover page: A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2019)
        It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.
          Cover page: Morpholinos Do Not Elicit an Innate Immune Response during Early Xenopus Embryogenesis
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2020)
          Although Wnt/β-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide β-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of β-catenin association. We discover that genomic β-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfβ signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease.
            Cover page: Foxh1/Nodal Defines Context-Specific Direct Maternal Wnt/β-Catenin Target Gene Regulation in Early Development
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2019)
            Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA.
              Cover page: Endodermal Maternal Transcription Factors Establish Super-Enhancers during Zygotic Genome ActivationCreative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2017)
              Gene regulatory networks (GRNs) involve highly combinatorial interactions between transcription factors and short sequence motifs in cis-regulatory modules of target genes to control cellular phenotypes. The GRNs specifying most cell types are largely unknown and are the subject of wide interest. A catalog of transcription factors is a valuable tool toward obtaining a deeper understanding of the role of these critical effectors in any biological setting. Here we present a comprehensive catalog of the transcription factors for the diploid frog Xenopus tropicalis. We identify 1235 genes encoding DNA-binding transcription factors, comparable to the numbers found in typical mammalian species. In detail, the repertoire of X. tropicalis transcription factor genes is nearly identical to human and mouse, with the exception of zinc finger family members, and a small number of species/lineage-specific gene duplications and losses relative to the mammalian repertoires. We applied this resource to the identification of transcription factors differentially expressed in the early gastrula stage embryo. We find transcription factor enrichment in Spemann's organizer, the ventral mesoderm, ectoderm and endoderm, and report 218 TFs that show regionalized expression patterns at this stage. Many of these have not been previously reported as expressed in the early embryo, suggesting thus far unappreciated roles for many transcription factors in the GRNs regulating early development. We expect our transcription factor catalog will facilitate myriad studies using Xenopus as a model system to understand basic biology and human disease.
                Cover page: A catalog of Xenopus tropicalis transcription factors and their regional expression in the early gastrula stage embryoCreative Commons 'BY' version 4.0 license
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2017)
                The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-β signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied by Foxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.
                  Cover page: Foxh1 Occupies cis-Regulatory Modules Prior to Dynamic Transcription Factor Interactions Controlling the Mesendoderm Gene Program
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2017)
                  Advances in RNA sequencing technologies have led to the surprising discovery that a vast number of transcripts emanate from regions of the genome that are not part of coding genes. Although some of the smaller ncRNAs such as microRNAs have well-characterized functions, the majority of long ncRNA (lncRNA) functions remain poorly understood. Understanding the significance of lncRNAs is an important challenge facing biology today. A powerful approach to uncovering the function of lncRNAs is to explore temporal and spatial expression profiling. This may be particularly useful for classes of lncRNAs that have developmentally important roles as the expression of such lncRNAs will be expected to be both spatially and temporally regulated during development. Here, we take advantage of our ultra-high frequency (temporal) sampling of Xenopus embryos to analyze gene expression trajectories of lncRNA transcripts over the first 3 days of development. We computationally identify 5689 potential single- and multi-exon lncRNAs. These lncRNAs demonstrate clear dynamic expression patterns. A subset of them displays highly correlative temporal expression profiles with respect to those of the neighboring genes. We also identified spatially localized lncRNAs in the gastrula stage embryo. These results suggest that lncRNAs have regulatory roles during early embryonic development.
                    Cover page: Developmentally regulated long non-coding RNAs in Xenopus tropicalisCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2020)
                    Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN in Xenopus gastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.
                      Cover page: Sox17 and β-catenin co-occupy Wnt-responsive enhancers to govern the endoderm gene regulatory network
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