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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2023)

Animal bodies develop through complex developmental pathways in which cells are programmed for particular fates and functions. Transcriptional regulation has been shown to be central to this process, but we know little about how transcriptional regulatory programs evolved along the animal stem lineage. Can we trace animal developmental programs to their unicellular, pre-animal roots? Which mechanistic aspects of transcriptional regulation are unique to animals and which are more deeply conserved? My doctoral research explored these questions through bioinformatic and genetic approaches in choanoflagellates, the closest living relatives of animals. Through a better understanding of transcription factors and cell type specification in these organisms, I strove to help us triangulate the transcriptional regulatory capacity of the common ancestor of animals and choanoflagellates, which lived hundreds of millions of years in the past.

Chapter 1 reviews how transcriptional regulation has evolved along the animal stem lineage. It has been frequently proposed that animal origins required the evolution of increasingly “complex” transcriptional regulation. I break this idea of complexity into specific mechanisms and trace what is known about the evolution of these mechanisms in animals and their closest living relatives.

In Chapter 2, I present an example of how functional interrogation of choanoflagellate transcriptional networks can help us better understand the ancient roles of specific transcription factors as well as the regulatory architecture of cell differentiation. I explored the function of the RFX family of transcription factors in choanoflagellates, identifying a particular sub-family (cRFXa) that has a functionally conserved role in regulating dozens of genes required for ciliogenesis. By generating genome-edited mutant strains, I show that cRFXa is essential for proper ciliogenesis in the model choanoflagellate Salpingoeca rosetta, and that this defect is coupled with the loss of full expression of dozens of highly conserved ciliary genes. Coupled with existing data from animals, this work shows that the RFX/ciliogenesis regulatory module dates before the divergence of animals and choanoflagellates. It also helps us to understand the regulatory changes that might have been required for the differentiation of ciliated and non-ciliated cells early in animal evolution.

Finally, in the Appendix I present work from early in my dissertation on a novel virus I helped to discover in Entomophthora muscae, a behavior-manipulating fungal pathogen of dipteran flies, including Drosophila melanogaster. We identified this virus through sequence analysis, including small RNA sequencing signatures generated by host Dicer processing, as well as through electron microscopy to directly visualize viral capsids in both cell-free extract and within fungal cells themselves.

    Cover page: Choanoflagellate transcriptional regulation and the origin of animal cell types
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2021)
    Amoeboid cell types are fundamental to animal biology and broadly distributed across animal diversity, but their evolutionary origin is unclear. The closest living relatives of animals, the choanoflagellates, display a polarized cell architecture (with an apical flagellum encircled by microvilli) that resembles that of epithelial cells and suggests homology, but this architecture differs strikingly from the deformable phenotype of animal amoeboid cells, which instead evoke more distantly related eukaryotes, such as diverse amoebae. Here, we show that choanoflagellates subjected to confinement become amoeboid by retracting their flagella and activating myosin-based motility. This switch allows escape from confinement and is conserved across choanoflagellate diversity. The conservation of the amoeboid cell phenotype across animals and choanoflagellates, together with the conserved role of myosin, is consistent with homology of amoeboid motility in both lineages. We hypothesize that the differentiation between animal epithelial and crawling cells might have evolved from a stress-induced switch between flagellate and amoeboid forms in their single-celled ancestors.
      Cover page: A flagellate-to-amoeboid switch in the closest living relatives of animals.
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2023)
      Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey.1,2,3 Regulated ciliogenesis was likely critical for early animal evolution,2,4,5,6 and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals7,8,9 but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.10,11 By comparing the genomes of animals with those of their closest living relatives, the choanoflagellates, we found that the genome of their last common ancestor encoded at least three RFX paralogs and a FoxJ1 homolog. Disruption of the RFX homolog cRFXa in the model choanoflagellate Salpingoeca rosetta resulted in delayed cell proliferation and aberrant ciliogenesis, marked by the collapse and resorption of nascent cilia. In cRFXa mutants, ciliogenesis genes and foxJ1 were significantly downregulated. Moreover, the promoters of S. rosetta ciliary genes are enriched for DNA motifs matching those bound by the cRFXa protein in vitro. These findings suggest that an ancestral cRFXa homolog coordinated ciliogenesis in the progenitors of animals and choanoflagellates and that the selective deployment of the RFX regulatory module may have been necessary to differentiate ciliated from non-ciliated cell types during early animal evolution.
        Cover page: An RFX transcription factor regulates ciliogenesis in the closest living relatives of animals.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2023)
        The construction of three-dimensional (3D) microvascular networks with defined structures remains challenging. Emerging bioprinting strategies provide a means of patterning endothelial cells (ECs) into the geometry of 3D microvascular networks, but the microenvironmental cues necessary to promote their self-organization into cohesive and perfusable microvessels are not well known. To this end, we reconstituted microvessel formation in vitro by patterning thin lines of closely packed ECs fully embedded within a 3D extracellular matrix (ECM) and observed how different microenvironmental parameters influenced EC behaviors and their self-organization into microvessels. We found that the inclusion of fibrillar matrices, such as collagen I, into the ECM positively influenced cell condensation into extended geometries such as cords. We also identified the presence of a high-molecular-weight protein(s) in fetal bovine serum that negatively influenced EC condensation. This component destabilized cord structure by promoting cell protrusions and destabilizing cell-cell adhesions. Endothelial cords cultured in the presence of fibrillar collagen and in the absence of this protein activity were able to polarize, lumenize, incorporate mural cells, and support fluid flow. These optimized conditions allowed for the construction of branched and perfusable microvascular networks directly from patterned cells in as little as 3 days. These findings reveal important design principles for future microvascular engineering efforts based on bioprinting and micropatterning techniques. Impact statement Bioprinting is a potential strategy to achieve microvascularization in engineered tissues. However, the controlled self-organization of patterned endothelial cells into perfusable microvasculature remains challenging. We used DNA Programmed Assembly of Cells to create cell-dense, capillary-sized cords of endothelial cells with complete control over their structure. We optimized the matrix and media conditions to promote self-organization and maturation of these endothelial cords into stable and perfusable microvascular networks.
          Cover page: Programming the Self-Organization of Endothelial Cells into Perfusable Microvasculature
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2018)
          Many tissues fold into complex shapes during development. Controlling this process in vitro would represent an important advance for tissue engineering. We use embryonic tissue explants, finite element modeling, and 3D cell-patterning techniques to show that mechanical compaction of the extracellular matrix during mesenchymal condensation is sufficient to drive tissue folding along programmed trajectories. The process requires cell contractility, generates strains at tissue interfaces, and causes patterns of collagen alignment around and between condensates. Aligned collagen fibers support elevated tensions that promote the folding of interfaces along paths that can be predicted by modeling. We demonstrate the robustness and versatility of this strategy for sculpting tissue interfaces by directing the morphogenesis of a variety of folded tissue forms from patterns of mesenchymal condensates. These studies provide insight into the active mechanical properties of the embryonic mesenchyme and establish engineering strategies for more robustly directing tissue morphogenesis ex vivo.
            Cover page: Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme
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