Doctor of Philosophy (Ph.D.)

Biology, Developmental and Stem Cell Biology Program
Duke University
2012

Bachelor of Science (B.S.)

Biology
University of Virginia
2004

Developmental Biology

Biology

Specialty Areas: Cell biology, Model system genetics (Drosophila melanogaster)

Biotechnology (BIOL/BIOG 462)

Topics in Cell and Molecular Biology (BIOL 500)

Genetics (BIOL 200)

Honors Biology

John W Graham, Jr. Faculty Research Award, Clarkson University, 2024

Fellows Award for Research Excellence, NIH, 2017

Poster Prize, NINDS Departmental Retreat, NIH, 2016

Company of Biologists/EMBO conference award, 2015

Biochemical Society Travel Grant, 2014

American Society for Cell Biology Travel Grant, 2014

Sigma Xi Grant-in-Aid, 2010

Grant-in-Aid, Department of Biology, Duke University, 2010

Conference Travel Scholarship, Duke University, 2009

Quantifying bristle cell organization in Drosophila melanogaster using spatial clustering features

Quantifying bristle cell organization in Drosophila melanogaster using spatial clustering features

Developing tissues reproduce nearly identical patterns from animal to animal, despite the process being stochastic at the cell level. A major challenge for researchers is the ability to quantify and classify complex cell and tissue spot patterns across wild type and perturbed conditions. Here, we use the organization of small sensory bristles on the fruit fly thorax as a model system to address this problem. A well-known and easily observable distinguishing feature of spot patterns is density. Beyond this, it is unclear how to quantitatively distinguish between patterns in a reproducible way. Our work evaluates the utility of the spatial clustering of spot patterns in quantifying their organization. We propose four clustering features, obtained using the density-based spatial clustering of applications with noise (DBSCAN) algorithm. Together with pattern density we assess how these features can quantify and distinguish between bristle patterns in a variety of wild-type and mutant flies to confirm known perturbations and discover new ones. Our results show that a combination of spot pattern density and the variance between the size of clusters is sufficient to distinguish between 70% of patterns from wild-type and mutant tissues.

Integrative Computational and Experimental Analysis of Curly Su Mutations in Drosophila melanogaster

Integrative Computational and Experimental Analysis of Curly Su Mutations in Drosophila melanogaster

The Curly Su (dMPO) protein, a homolog of the human myeloperoxidase (hMPO), is critical for wing development in Drosophila melanogaster. Like human peroxidases, dMPO is involved in various cellular and physiological processes, producing significant quantities of reactive oxygen species that contribute to both development and immunity in the fruit fly. Given the significant sequence and structural similarities between dMPO and hMPO, dMPO serves an ideal model for studying peroxidase functions and related pathologies. We performed saturated computational mutagenesis on dMPO, analyzing the effects of 11,191 missense mutations on its stability. Notably, the G378W mutation exhibited the greatest destabilizing effect, while the W621R, potentially pathogenic, also reduced dMPO stability. To investigate these effects in vivo, we used genome editing to generate the transgenic Drosophila with G378W, W621R, and deletion of residues 305-687. Remarkably, G378W mutants displayed significant alterations in wing morphology and reduced lifespan. RNA-seq analysis of transgenic and wild-type flies revealed differentially expressed genes (DEGs), as interpreted through gene ontology analysis. Our integrated computational and genetic approach identified dMPO mutations that disrupt protein stability and alter gene expression. These findings offer new insight into how single-point mutations can lead to systemic biological changes.

Bridging single cells to organs

Bridging single cells to organs: Mesoscale modules as fundamental units of tissue function

Recent studies at molecular and genomic scales have enriched our understanding of life’s most fundamental building block: the cell. However, bridging the gap between single-cell phenotypes and the emergent functions of tissues and organs remains a formidable challenge. Here, we suggest that the conceptual span from cells to tissues and organs is so large as to warrant intermediate stepping stones. Drawing inspiration from “network motifs”—discrete units of cell-level function that emerge from the interactions of a handful of genes or enzymes—we argue that similarly identifiable units of tissue-level function, which we term “mesoscale modules,” emerge from coordinated “interactions” among relatively small numbers of cells and their extracellular milieu. We outline several such modules and propose that a concerted effort to study them will deepen our foundational understanding of tissue and organ functions. By developing these mesoscale insights, we anticipate a more tractable and mechanistic approach to complex human conditions rooted in tissue- and organ-scale dysregulation, including developmental defects, cancer, cardiovascular disease, immune-related disorders, infectious disease, and aging.

Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition

Myosin XV is a negative regulator of signaling filopodia during long-range lateral inhibition

The self-organization of cells during development is essential for the formation of healthy tissues, and requires the coordination of cell activities at local scales. Cytonemes, or signaling filopodia, are dynamic actin-based cellular protrusions that allow cells to engage in contact mediated signaling at a distance. While signaling filopodia have been shown to support several signaling paradigms during development, less is understood about how these protrusions are regulated. We investigated the role of the plus-end directed, unconventional MyTH4-FERM myosins in regulating signaling filopodia during sensory bristle patterning on the dorsal thorax of the fruit fly Drosophila melanogaster. We found that Myosin XV is required for regulating signaling filopodia dynamics and, as a consequence, lateral inhibition more broadly throughout the patterning epithelium. We found that Myosin XV is required for limiting the length and number of signaling filopodia generated by bristle precursor cells. Cells with additional and longer signaling filopodia due to loss of Myosin XV are not signaling competent, due to altered levels of Delta ligand and Notch receptor along their lengths. We conclude that Myosin XV acts to negatively regulate signaling filopodia, as well as promote the ability of signaling filopodia to engage in long-range Notch signaling. Since Myosin XV is present across several vertebrate and invertebrate systems, this may have significance for other long-range signaling mechanisms.

Scabrous modulates Notch response via signaling filopodia during bristle pattern formation in Drosophila

Scabrous modulates Notch response via signaling filopodia during bristle pattern formation in Drosophila

During development, cells in tissues must be patterned correctly in order to support tissue function and shape. The sensory bristles of the peripheral nervous system on the thorax of Drosophila melanogaster self-organizes from a unpatterned epithelial tissue to a regular spot pattern during pupal stages. Wild type patterning requires Notch-mediated lateral inhibition. Scabrous is a protein that can bind to and modify Notch receptor activity. Scabrous can be secreted, but it is also known to be localized to basal signaling filopodia, or cytonemes, that play a role in long-range Notch signaling. Here we show that Scabrous is primarily distributed basally, within the range of signaling filopodia extension. We show that filamentous actin dynamics are required for the distribution of Scabrous protein during sensory bristle patterning stages. We show that the Notch response of epithelial cells is sensitive to the level of Scabrous protein being expressed by the sensory bristle precursor cell. Our findings at the cell-level suggest a model for how epithelial cells engaged in lateral inhibition at a distance are sensitive local levels of Scabrous protein.

Modeling collective cell behavior in cancer: perspectives from an interdisciplinary conversation

Modeling collective cell behavior in cancer: perspectives from an interdisciplinary conversation

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

Talking to your neighbors across scales

Talking to your neighbors across scales: long-distance Notch signaling during patterning

Tissue patterning is a critical part of animal development. Here we review the role that length- and timescales play in shaping patterns during development, focusing on the mechanisms by which Notch-mediated lateral inhibition signaling generates periodic tissue patterns. Because Notch ligands and receptors are membrane bound, the signaling that underlies lateral inhibition depends on direct cell-cell contacts. Nevertheless, there are many biological examples where effective Notch signaling occurs over distances larger than adjacent cells. Here, we summarize the theoretical and experimental evidence for mechanisms that modify the scale of Notch-mediated lateral inhibition. We focus on how cell protrusions, in addition to other cell behaviors like proliferation and neighbor exchange, allow for Notch signaling to both extend lateral inhibition beyond nearest neighbors and impact the timescale of patterning. Using recent examples, we examine how dynamic cell behaviors like the formation of protrusions affect the timing of Notch-mediated lateral inhibition as well as the density of the final tissue pattern. We suggest that mechanisms that affect the length and timescale of Notch signaling may have key implications for the evolution of patterns. This review highlights the role of cell behaviors in controlling the temporal and spatial dynamics of pattern formation across scales.

Phosphorylation and proteolytic cleavage of Notch in canonical and non-canonical Notch signaling

Phosphorylation and proteolytic cleavage of Notch in canonical and non-canonical Notch signaling

The Notch signaling pathway seems deceptively simple, with its key feature being a direct connection between extracellular signal and transcriptional output without the need for an extended chain of protein intermediaries as required by so many other signaling paradigms. However, this apparent simplicity hides considerable complexity. Consistent with its central role in many aspects of development, Notch signaling has an extensive collection of mechanisms that it employs alongside of its core transcriptional machinery. These so-called noncanonical Notch pathways diversify the potential outputs of Notch, and allow it to coordinate regulation of many aspects of the biology of cells. Here we will review noncanonical Notch signaling with special attention to the role of posttranslational modifications of Notch. We will also consider the importance of coordinating the activity of gene expression with regulation of cell morphology in biological processes, including axon guidance and other morphological events during embryogenesis.

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle

The ability of cells to divide along their longest axis has been proposed to play an important role in maintaining epithelial tissue homeostasis in many systems. Because the division plane is largely set by the position of the anaphase spindle, it is important to understand how spindles become oriented. While several molecules have been identified that play key roles in spindle orientation across systems, most notably Mud/NuMA and cortical dynein, the precise mechanism by which spindles detect and align with the long cell axis remain poorly understood. Here, in exploring the dynamics of spindle orientation in mechanically distinct regions of the fly notum, we find that the ability of cells to properly reorient their divisions depends on local tissue tension. Thus, spindles reorient to align with the long cell axis in regions where isotropic tension is elevated, but fail to do so in elongated cells within the crowded midline, where tension is low, or in regions that have been mechanically isolated from the rest of the tissue via laser ablation. Importantly, these differences in spindle behavior outside and inside the midline can be recapitulated by corresponding changes in tension induced by perturbations that alter nonmuscle myosin II activity. These data lead us to propose that isotropic tension within an epithelium provides cells with a mechanically stable substrate upon which localized cortical motor complexes can act on astral microtubules to orient the spindle.

A role for actomyosin contractility in Notch signaling

A role for actomyosin contractility in Notch signaling

Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling.

A new mechanism for spatial pattern formation via lateral and protrusion-mediated lateral signalling

A new mechanism for spatial pattern formation via lateral and protrusion-mediated lateral signalling

Tissue organization and patterning are critical during development when genetically identical cells take on different fates. Lateral signalling plays an important role in this process by helping to generate self-organized spatial patterns in an otherwise uniform collection of cells. Recent data suggest that lateral signalling can be mediated both by junctional contacts between neighbouring cells and via cellular protrusions that allow non-neighbouring cells to interact with one another at a distance. However, it remains unclear precisely how signalling mediated by these distinct types of cell–cell contact can physically contribute to the generation of complex patterns without the assistance of diffusible morphogens or pre-patterns. To explore this question, in this work we develop a model of lateral signalling based on a single receptor/ligand pair as exemplified by Notch and Delta.

Coordinated control of Notch-Delta signalling and cell division aids lateral inhibition mediated tissue patterning

Coordinated control of Notch-Delta signalling and cell division aids lateral inhibition mediated tissue patterning

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.

An absolute interval scale of order for point patterns

An absolute interval scale of order for point patterns

Human observers readily make judgements about the degree of order in planar arrangements of points (point patterns). Here, based on pairwise ranking of 20 point patterns by degree of order, we have been able to show that judgements of order are highly consistent across individuals and the dimension of order has an interval scale structure spanning roughly 10 just-notable-differences (jnd) between disorder and order. We describe a geometric algorithm that estimates order to an accuracy of half a jnd by quantifying the variability of the size and shape of spaces between points. The algorithm is 70% more accurate than the best available measures. By anchoring the output of the algorithm so that Poisson point processes score on average 0, perfect lattices score 10 and unit steps correspond closely to jnds, we construct an absolute interval scale of order. We demonstrate its utility in biology by using this scale to quantify order during the development of the pattern of bristles on the dorsal thorax of the fruit fly.

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

Ion channels contribute to the regulation of cell sheet forces during Drosophila dorsal closure

We demonstrate that ion channels contribute to the regulation of dorsal closure in Drosophila, a model system for cell sheet morphogenesis. We find that Ca2+ is sufficient to cause cell contraction in dorsal closure tissues, as UV-mediated release of caged Ca2+ leads to cell contraction. Furthermore, endogenous Ca2+ fluxes correlate with cell contraction in the amnioserosa during closure, whereas the chelation of Ca2+ slows closure. Microinjection of high concentrations of the peptide GsMTx4, which is a specific modulator of mechanically gated ion channel function, causes increases in cytoplasmic free Ca2+and actomyosin contractility and, in the long term, blocks closure in a dose-dependent manner. We identify two channel subunits, ripped pocket and dtrpA1 (TrpA1), that play a role in closure and other morphogenetic events. Blocking channels leads to defects in force generation via failure of actomyosin structures, and impairs the ability of tissues to regulate forces in response to laser microsurgery. Our results point to a key role for ion channels in closure, and suggest a mechanism for the coordination of force-producing cell behaviors across the embryo.