Biology subjects

Hughes, A. J.

Publications and source records attributed to Hughes, A. J..

16 recordsLinked to original sources

bioRxiv2026

Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

The mammalian kidney relies on a branched network of collecting ducts for fluid transport and homeostasis. Replicating this network in vitro would parallelize function in synthetic replacement kidneys, yet current organoids have limited branching capacity. Here, we establish a developmentally-informed strategy to control organoid budding through optogenetic control of a receptor tyrosine kinase, RET. We first show pharmacological manipulation of RET signaling controls the extent of branching in mouse embryonic kidneys and human stem cell-derived kidney organoids. Next, we develop an optogenetic RET receptor (optoRET) that signals in a ligand-independent manner via blue light-mediated clustering. Epithelial cells expressing optoRET reproduce stereotyped RET signaling, scattering, and symmetry breaking in response to blue light. Human kidney organoids undergo budding with controllable orientation in response to spatially patterned optoRET stimulation. Our results establish ligand-free optogenetic control of branching and inspire new synthetic biology strategies for epithelial organoid design. HighlightsGDNF-RET controls branching and tip cell state in mouse and human kidney tissues. OptoRET reproduces endogenous RET signaling and morphogenesis in cell lines. OptoRET enables ligand-free budding in human renal epithelial organoids. Spatially patterned optoRET stimulation controls budding orientation.

bioengineering↗

bioRxiv2025

Single-Cell Spatial Mapping of Human Kidney Development Reveals the Critical Role of the Local Microenvironment in Cell Fate Decisions

Cell-cell interactions play a pivotal role in organ development, yet these communications have previously been studied one interaction at a time in model organisms, leaving a gap in our understanding of the cellular interplay in human development. To address this, we investigated human kidney development using single-cell RNA sequencing and spatial transcriptomics, analyzing over 500,000 cells. By mapping gene expression and differentiation trajectories in histologic space, we define the spatial organization of kidney development. Our analysis revealed newfound plasticity, showing that nephron progenitor cells undergo an early fate decision between renal corpuscle and tubular lineages. However, this choice is later reversed with some mature tubule cells transitioning back to a renal corpuscle fate. Further, through a genome-wide, spatially-aware cell-cell interaction analysis, we identified specific ligands and neighboring cell signals that create biologically meaningful cellular neighborhoods and mediate cell fate choices, offering a blueprint to understand the coordination of human development at scale.

genomics↗

bioRxiv2024

Engineering kidney developmental trajectory using culture boundary conditions

Kidney explant cultures are traditionally carried out at air-liquid interfaces, which disrupts 3D tissue structure and limits interpretation of developmental data. To overcome this limitation, we developed a 3D culture technique using hydrogel embedding to capture morphogenesis in real time. We show that 3D culture better approximates in vivo-like niche spacing and dynamic tubule tip rearrangement, as well as in vivo-like presentation of branching defects under perturbations to glial cell-derived neurotrophic factor (GDNF)-REarranged during Transfection (RET) tyrosine kinase signaling. We find that the concentration of the embedding matrix influences the number of nephrons per ureteric bud (UB) tip and the spacing between tips. To isolate the effect of specific material properties on explant development, we introduce engineered acrylated hyaluronic acid hydrogels that allow independent tuning of stiffness and adhesion. We find that sufficient stiffness and adhesion are both required to maintain kidney shape. Matrix stiffness has a "Goldilocks effect" on the nephron per UB tip balance centered at [~]2 kPa, while higher matrix adhesion increases nephron per UB tip ratio. Our technique captures large-scale, in vivo-like tissue morphogenesis in 3D, providing a platform suited to contrasting normal and congenital disease contexts. Moreover, understanding the impact of boundary condition mechanics on kidney development benefits fundamental renal research and advances the engineering of next-generation kidney replacement tissues.

developmental biology↗

bioRxiv2024

HUHgle: An Interactive Substrate Design Tool for Covalent Protein-ssDNA Labeling Using HUH-tags

HUH-tags have emerged as versatile fusion partners that mediate sequence specific protein-ssDNA bioconjugation through a simple and efficient reaction. Here we present HUHgle, a python-based interactive tool for the visualization, design, and optimization of substrates for HUH-tag mediated covalent labeling of proteins of interest with ssDNA substrates of interest. HUHgle streamlines design processes by integrating an intuitive plotting interface with a search function capable of predicting and displaying protein-ssDNA bioconjugate formation efficiency and specificity in proposed HUH-tag/ssDNA sequence combinations. Validation demonstrates that HUHgle accurately predicts product formation of HUH-tag mediated bioconjugation for single- and orthogonal-labeling reactions. In order to maximize the accessibility and utility of HUHgle, we have implemented it as a user-friendly Google Colab notebook which facilitates broad use of this tool, regardless of coding expertise. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/585203v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1a1bbforg.highwire.dtl.DTLVardef@13d218org.highwire.dtl.DTLVardef@146478corg.highwire.dtl.DTLVardef@1034a6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Reduced Filaggrin expression induces dysregulated intracellular signalling in atopic eczema

Atopic eczema (AE) is the most common inflammatory dermatosis, affecting up to 20% of children. Loss of function mutations in the Filaggrin (FLG) gene are the most strongly implicated genetic risk factor for AE, but little is known about the signalling pathways altered in response to loss of FLG. To explore the downstream effects of loss of FLG on the cellular environment, we combined RNAseq analysis of siRNA knockdown of normal human keratinocytes and analysis of tape strip (TS) samples from AE patients who were clinically phenotyped and genotyped for FLG mutation status. RNA-seq analysis revealed an increase in BMP signalling following FLG KD, which we validated in vivo using TS samples and biopsies. Recombinant BMP2 or BMP6 increased FLG and suprabasal keratin expression in vitro. Phosphoproteomic analysis identified 237 significantly differentially phosphorylated proteins following FLG KD. Kinase enrichment analysis identified downregulation of ERK1/2 and AKT1 signalling which was confirmed in AE biopsies. cFOS was downregulated following FLG KD and correlated with FLG expression in repository datasets. cFOS was downregulated following BMP6 treatment, implying that cFOS may be an important link between FLG and BMP signalling. Finally, proteomic analysis of TS samples identified altered desmosomal expression and phosphorylation following either loss of FLG or increased BMP signalling. Therefore, we have identified a SMAD1/Filaggrin/AKT axis as a potential therapeutic avenue in AE.

cell biology↗

bioRxiv2024

Measurement of adhesion and traction of cells at high yield (MATCHY) reveals an energetic ratchet driving nephron condensation

Engineering of embryonic strategies for tissue-building has extraordinary promise for regenerative medicine. This has led to a resurgence in interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here we present MATCHY (multiplexed adhesion and traction of cells at high yield). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and an automated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on a priori knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for RET tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We go on to create a biophysical atlas of primary cells dissociated from the mouse embryonic kidney and use MATCHY to identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an energetic ratchet that explains spatial nephron progenitor cell condensation from the niche as they differentiate, which we validate through agent-based computational simulation. MATCHY offers automated cell biophysical characterization at >104-cell throughput, a highly enabling advance for fundamental studies and new synthetic tissue design strategies for regenerative medicine.

bioengineering↗

bioRxiv2023

Nephron progenitors rhythmically alternate between renewal and differentiation in synchrony with kidney branching morphogenesis

The developing mammalian kidney exponentially duplicates nephron-forming stem cell niches at the tips of the urinary collecting duct tree to achieve massively parallel function. Nephron formation rate has a clinically meaningful effect on person-to-person variability in nephron endowment1-5, while exerting in vitro control could enable sustained waves of nephrogenesis in organoid-derived synthetic kidney tissues6,7. However, how the kidney arrives at an appropriate number and ratio of nephrons to collecting ducts is unclear. Here we show that nephron formation is rhythmic and synchronized with branching of the ureteric bud tree (the future urinary collecting ducts). We correlate human and mouse spatial transcriptomics data with the branching life-cycle to uncover rhythmically alternating signatures of nephron progenitor differentiation and renewal. The nephron progenitor rhythm parallels rhythmic nuclear elongation and other hallmarks of mechanical tension in surrounding stromal cells that we attribute to branching-induced deformation. Stroma-specific knockdown of actomyosin activity leads to a striking loss of synchronization between nephron formation and ureteric bud branching without blocking either. These results suggest that the stroma acts as a mechanically entrained pacemaker for nephron formation. Together, our findings uncover a feedback mechanism for clock-like coordination of organ composition during exponential growth.

developmental biology↗

bioRxiv2023

Rho/ROCK activity tunes cell compartment segregation and differentiation in nephron-forming niches

Controlling the time and place of nephron formation in vitro would improve nephron density and connectivity in next-generation kidney replacement tissues. Recent developments in kidney organoid technology have paved the way to achieving self-sustaining nephrogenic niches in vitro. The physical and geometric structure of the niche are key control parameters in tissue engineering approaches. However, their relationship to nephron differentiation is unclear. Here we investigate the relationship between niche geometry, cell compartment mixing, and nephron differentiation by targeting the Rho/ROCK pathway, a master regulator of the actin cytoskeleton. We find that the ROCK inhibitor Y-27632 increases mixing between nephron progenitor and stromal compartments in native mouse embryonic kidney niches, and also increases nephrogenesis. Similar increases are also seen in reductionist mouse primary cell and human induced pluripotent stem cell (iPSC)-derived organoids perturbed by Y-27632, dependent on the presence of stromal cells. Our data indicate that niche organization is a determinant of nephron formation rate, bringing renewed focus to the spatial context of cell-cell interactions in kidney tissue engineering efforts.

bioengineering↗

bioRxiv2023

Highly-parallel production of designer organoids by mosaic patterning of progenitors

Human organoids are a promising approach for disease modeling and regenerative medicine. However, organoid variability and limited control over morphological outcomes remain significant challenges. Here we extend a DNA velcro cell patterning approach, precisely controlling the number and ratio of human stem cell-derived progenitors contributing to nephron and mosaic nephron/ureteric bud organoids within arrays of microwells. We demonstrate long-term control over organoid size and morphology, decoupled from geometric constraints.

bioengineering↗

bioRxiv2023

Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 103 molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents -- not previously accessible for in-gel use -- for further signal amplification and detection of low abundance targets.

bioengineering↗

bioRxiv2022

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

Replicating organizational principles that establish fine-scale tissue structure is critical to our capacity for building functional replacement tissues. Tissue boundaries such as epithelial-mesenchymal interfaces are engines for morphogenesis in vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environment in vitro, fine-scale spatial control of cell composition within tissue constructs remains an engineering challenge. To address this, we augment DNA "velcro" technology for selective patterning of ssDNA-labeled cells with long-term culture on mechanically defined polyacrylamide hydrogels. We co-functionalize photoactive benzophenone-containing polyacrylamide gels (BP-PA gels) with spatially precise ssDNA features that confer temporary cell adhesion and with extracellular matrix (ECM) proteins that confer long-term adhesion. We find that co-functionalization does not compromise ssDNA patterning fidelity or cell capture, nor hydrogel mechanical properties or mechanosensitive fibroblast spreading, enabling mechanobiology studies of precise cell interfaces. We then co-pattern colonies of fibroblasts and epithelial cells to study interface formation and extracellular signal-related kinase (ERK) activity at cellular contacts. Combining DNA velcro and ECM functionalization approaches provides independent control of initial cell placement, adhesion, and mechanical environment, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

bioengineering↗

bioRxiv2022

Tubule jamming in the developing kidney creates cyclical mechanical stresses instructive to nephron formation

The kidney develops through branching of progressively crowded ureteric bud (UB) tubules at the organ surface. The elongating tubule tips are surrounded by traveling cap mesenchyme niches consisting of nephron progenitors and separated by stromal boundaries. Dynamic interactions between these tissues coordinate a balance between UB tip branching, elongation, and nephron induction that sets nephron numbers for life, impacting the likelihood of adult disease. Such a crowded tissue environment could place geometric limits on the number of niches that can be formed while maintaining mechanical integrity of the tissue. Since space is at a premium, crowding could also force a given niche to prioritize between nephron formation or UB branching differently depending on its spatial context. Here we study the geometric and mechanical consequences of tubule tip crowding at the embryonic kidney surface. Organ curvature reduces and tubule tip domain niches pack more closely over developmental time. These together create a semi-crystalline geometry of tips at the kidney surface and a rigidity transition to more solid-like tissue properties at later developmental stages. To infer mechanical dynamics over the branching timescale, we define a new method to infer tip domain ages relative to their most recent branch events from fixed kidneys. We find that new tip domains overcome mechanical resistance as they branch and displace close-packed neighbors, transiently increasing mechanical stress in the niche. Ongoing efforts to understand geometric and mechanical effects on niche regulation will clarify variation in kidney tissue composition and advance engineering control strategies for synthetic regenerative tissues.

bioengineering↗

bioRxiv2021

The developing kidney actively negotiates geometric packing conflicts to avoid defects

The physiological functions of several organs rely on branched tubular networks, but little is known about conflicts in development between building enough tubules for adequate function and geometric constraints imposed by organ size. We show that the mouse embryonic kidney epithelium negotiates a physical packing conflict between tubule tip duplication and limited area at the organ surface. Imaging, computational, and soft material modeling of tubule families identifies six geometric packing phases, including two defective ones. Experiments in kidney explants show that a retrograde tension on tubule families is necessary and sufficient for them to avoid defects by switching to a vertical orientation that increases packing density. These results reveal developmental contingencies in response to physical limitations, and create a framework for classifying kidney defects. One-Sentence SummaryEpithelial branching in the kidney causes a geometric packing conflict that is resolved through internally generated tensions

developmental biology↗

bioRxiv2021

Systematically quantifying morphological features reveals constraints on organoid phenotypes

Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoids morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoids morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby Canine Kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

systems biology↗

bioRxiv2019

Kinomorphs: Shape-shifting tissues for developmental engineering

Current methods for building tissues usually start with a non-biological blueprint, or rely on self-organization, which does not extend to organ-scales. This has limited the construction of large tissues that simultaneously encode fine-scale cell organization. Here we bridge scales by mimicking developmental dynamics using \"kinomorphs\", tissue scaffolds that undergo globally programmed shape and density changes to trigger local self-organization of cells in many locations at once. In this first report, we focus on mimicking the extracellular matrix (ECM) compaction and division into leaflets that occurs in kidney collecting duct development. We start by creating single-cell resolution cell patterns in ECM-mimetic hydrogels that are >10x larger than previously described, by leveraging photo-lithographic technology. These patterns are designed to mimic the branch geometry of the embryonic kidney collecting duct tree. We then predict the shape dynamics of kinomorphs driven by cell contractility-based compaction of the ECM using kinematic origami simulations. We show that these dynamics spur centimeter-scale assembly of structurally mature ~50 m-diameter epithelial tubules that are locally self-organized, but globally programmed. Our approach prescribes tubule network geometry at ~5x smaller length-scales than currently possible using 3D printing, and at local cell densities comparable to in vivo tissues. Kinomorphs could be used to scaffold and \"plumb\" arrays of organoids in the future, by guiding the morphogenesis of epithelial networks. Such hybrid globally programmed/locally self-organized tissues address a major gap in our ability to recapitulate organ-scale tissue structure.\n\nSignificance StatementEngineers are attempting to build tissues that mimic human diseases outside of the body. Although stem cells can be coaxed to form small organoids with a diversity of cell types, they do not properly organize over large distances by themselves. We report a strategy to mimic developmental processes using dynamic materials that attempt to guide a cellular \"blueprint\" towards a more complex tissue endpoint. We call these materials kinomorphs, combining the Greek kino (propel, drive) and morfi (form, shape), since they seek to shepherd both the shape and developmental trajectory of cell collectives within them. Kinomorphs could pave the way towards organ-scale synthetic tissues built through a hybrid of engineering and self-organization strategies.

bioengineering↗

bioRxiv2017

Tissue Folding by Mechanical Compaction of the Mesenchyme

Many tissues fold during development into complex shapes. Engineering 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 a mechanical compaction of the ECM during mesenchymal condensation can drive tissue folding along programmed trajectories. The process requires cell contractility, generates strains at nearby tissue interfaces, and causes specific 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 finite element 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 engineered patterns of mesenchymal condensates. These studies provide insight into the active mechanical properties of the embryonic mesenchyme and establish entirely new strategies for more robustly directing tissue morphogenesis ex vivo, without genetic engineering.

bioengineering↗