Jamming of nephron-forming niches in the developing mouse kidney creates cyclical mechanical stresses.

Urinary collecting tubules form during kidney embryogenesis through the branching of the ureteric bud epithelium. A travelling mesenchyme niche of nephron progenitor cells caps each branching ureteric bud tip. These 'tip domain' niches pack more closely over developmental time and their number relates to nephron endowment at birth. Yet, how the crowded tissue environment impacts niche number and cell decision-making remains unclear. Here, through experiments and mathematical modelling, we show that niche packing conforms to physical limitations imposed by kidney curvature. We relate packing geometries to rigidity theory to predict a stiffening transition starting at embryonic day 15 in the mouse, validated by micromechanical analysis. Using a method to estimate tip domain 'ages' relative to their most recent branch events, we find that new niches overcome mechanical resistance as they branch and displace neighbours. This creates rhythmic mechanical stress in the niche. These findings expand our understanding of kidney development and inform engineering strategies for synthetic regenerative tissues.

Measurement of adhesion and traction of cells at high yield reveals an energetic ratchet operating during nephron condensation.

Developmental biology-inspired strategies for tissue-building have extraordinary promise for regenerative medicine, spurring 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 multiplexed adhesion and traction of cells at high yield (MATCHY). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and a semiautomated 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 rearranged during transfection (RET) tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We then use MATCHY to create a biophysical atlas of mouse embryonic kidney primary cells and 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 accounts for spatial trends in nephron progenitor cell condensation as they differentiate into early nephron structures, which we validate through agent-based computational simulation. MATCHY offers semiautomated cell biophysical characterization at >10,000-cell throughput, an advance benefiting fundamental studies and new synthetic tissue strategies for regenerative medicine.

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.

Nephron progenitors rhythmically alternate between renewal and differentiation phases that synchronize with kidney branching morphogenesis.

The mammalian kidney achieves massive parallelization of function by exponentially duplicating nephron-forming niches during development. Each niche caps a tip of the ureteric bud epithelium (the future urinary collecting duct tree) as it undergoes branching morphogenesis, while nephron progenitors within niches balance self-renewal and differentiation to early nephron cells. Nephron formation rate approximately matches branching rate over a large fraction of mouse gestation, yet the nature of this apparent pace-maker is unknown. Here we correlate spatial transcriptomics data with branching 'life-cycle' to discover rhythmically alternating signatures of nephron progenitor differentiation and renewal across Wnt, Hippo-Yap, retinoic acid (RA), and other pathways. We then find in human stem-cell derived nephron progenitor organoids that Wnt/ß-catenin-induced differentiation is converted to a renewal signal when it temporally overlaps with YAP activation. Similar experiments using RA activation indicate a role in setting nephron progenitor exit from the naive state, the spatial extent of differentiation, and nephron segment bias. Together the data suggest that nephron progenitor interpretation of consistent Wnt/ß-catenin differentiation signaling in the niche may be modified by rhythmic activity in ancillary pathways to set the pace of nephron formation. This would synchronize nephron formation with ureteric bud branching, which creates new sites for nephron condensation. Our data bring temporal resolution to the renewal vs. differentiation balance in the nephrogenic niche and inform new strategies to achieve self-sustaining nephron formation in synthetic human kidney tissues.

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.

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

Tissue boundaries and interfaces are engines of 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 positioning within tissue constructs remains an engineering challenge. To address this, we augment DNA "velcro" technology for selective patterning of ssDNA-labeled cells on mechanically defined photoactive polyacrylamide hydrogels. Hydrogels bearing photopatterned single-stranded DNA (ssDNA) features for cell capture are then co-functionalized with extracellular matrix (ECM) proteins to support subsequent adhesion of patterned tissues. ECM protein co-functionalization does not alter ssDNA pattern fidelity, cell capture, or hydrogel elastic stiffness. This approach enables mechanobiology studies and measurements of signaling activity at dynamic cell interfaces with precise initial patterning. Combining DNA velcro patterning and ECM functionalization provides independent control of initial cell placement, adhesion, and mechanics, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

The developing murine kidney actively negotiates geometric packing conflicts to avoid defects.

The physiological functions of several organs rely on branched epithelial tubule networks bearing specialized structures for secretion, gas exchange, or filtration. 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 increasing tubule tip numbers through branching and limited organ surface area. Through imaging of whole kidney explants, combined with computational and soft material modeling of tubule families, we identify six possible geometric packing phases, including two defective ones. Experiments in explants show that a radially oriented tension on tubule families is necessary and sufficient for them to switch to a vertical packing arrangement that increases surface tip density while avoiding defects. These results reveal developmental contingencies in response to physical limitations and create a framework for classifying congenital kidney defects.

Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development. The mice exhibited only a mild developmental phenotype; however, with aging, the CD developed a disruption of epithelial integrity and function. Despite intact integrin signaling, Rac1-null CD cells had profound adhesion and polarity abnormalities that were independent of the major downstream Rac1 effector, Pak1. These cells did however have a defect in the WAVE2-Arp2/3 actin nucleation and polymerization apparatus, resulting in actomyosin hyperactivity. The epithelial defects were reversible with direct myosin II inhibition. Furthermore, Rac1 controlled lateral membrane height and overall epithelial morphology by maintaining lateral F-actin and restricting actomyosin. Thus, Rac1 promotes CD epithelial integrity and morphology by restricting actomyosin via Arp2/3-dependent cytoskeletal branching.