Biology subjects

Macara, I. G.

Publications and source records attributed to Macara, I. G..

8 recordsLinked to original sources

bioRxiv2024

Actomyosin Contractility is a Potent Suppressor of Mesoderm Induction by Human Pluripotent Stem Cells

The activation of WNT signaling in human pluripotent stem cells (hPSCs) drives efficient conversion to lateral mesoderm, which can be further differentiated into cardiomyocytes. Stabilization of the WNT effector {beta}-catenin promotes expression of mesoderm-specifying genes such as TBXT (which encodes Brachyury) and drives an epithelial-mesenchymal transition (EMT). Mechanical forces are essential for the self-organization and development of vertebrate embryos but the role of forces, especially actomyosin contractility, in mesoderm specification has remained controversial. We discovered that, unexpectedly, increasing actomyosin contractility by expression of constitutively active Rho kinase or MLC kinase, efficiently blocked any induction of mesoderm by WNT signaling, and cells failed to undergo EMT. Conversely, the suppression of contractility by inhibitors and genetic approaches significantly accelerated differentiation: Brachyury induction was enhanced, and EMT initiated 24hrs earlier than in control settings. These data were initially puzzling because we observed that WNT signaling was sufficient by itself to promote contractility of hiPSC colonies. Notably, however, we showed that contractility must be inhibited prior to WNT activation to efficiently promote mesoderm specification, suggesting that reduced tension primes the pluripotent state, not the induced state, to accelerate differentiation along this trajectory. Mechanistically, we found that loss of contractility decreased junctional {beta}-catenin and promoted active {beta}-catenin levels in the cytoplasm and nucleus. Increased contractility had opposite effects, highlighting actomyosin contractility at the pluripotent state as a key regulator of WNT signaling responsiveness through effects on adherens junctions.

cell biology↗

bioRxiv2024

Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries

Current methods for single-cell RNA profiling of pooled CRISPR screens are limited, either by indirect capture of single guide RNAs (sgRNAs) or by custom modification of plasmid libraries. Here, we present a direct sgRNA capture platform called Native sgRNA Capture and sequencing (NSC-seq) that enables single-cell CRISPR screens using common knockout plasmid libraries, facilitating genotype-phenotype mapping at multiple scales in vitro and in vivo. Additionally, we characterize sgRNA expression in three whole-genome knockout libraries, revealing a substantial subset of truncated (isoform) spacer reads. We provide this dataset as a reference of expressed sgRNA isoforms that may potentially have compromised CRISPR gene editing efficacy and precision.

genomics↗

bioRxiv2023

Temporal recording of mammalian development and precancer

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

developmental biology↗

bioRxiv2022

A Junction-Dependent Mechanism Drives Mammary Cell Intercalation for Ductal Elongation

Mammary glands contain branched networks of ducts and alveoli that function to produce milk for offspring. While the murine luminal epithelium is organized as a cellular monolayer, it originates from multilayered structures called terminal end buds (TEB). The TEBs generate ducts of monolayered epithelial cells as they invade the fat pad, but little is known about underlying mechanisms. While apoptosis provides a plausible mechanism for cavitation of the ductal lumen, it does not account for elongation of ducts behind the TEBs. Our spatial calculations suggest that most cells in TEBs need to intercalate into the outermost luminal layer and that this migration of cells is the primary driver of cavitation and ductal elongation. To study the progression of multilayered to monolayered epithelium, we developed a quantitative cell culture assay that determines the efficiency of intercalation into an epithelial monolayer. Using this tool, we verified that loss of adherens junctions prevents stable integration of cells into monolayers, consistent with previous data in cultured cells and in primary tissue. Interestingly, tight junction (TJ) proteins also play a key role in this integration process. Although loss of the ZO-1 TJ protein in intercalating cells suppresses intercalation, loss of ZO-1 in the monolayer has the reverse effect, promoting intercalation - even though ZO-1 is not necessary for establishment of TJs. ZO-1-positive puncta form between cells and the monolayer, which then resolves into a new intercellular boundary as intercalation proceeds. ZO-1 loss also reduces engraftment when cells are transplanted into the mammary gland via intraductal injection. We further show that intercalation is dependent on dynamic cytoskeletal rearrangements in both the existing monolayer and intercalating cells. These data identify luminal cell rearrangements necessary for mammary gland development and suggest a molecular mechanism for integration of cells into an existing monolayer.

cell biology↗

bioRxiv2021

A Self-Limiting Circuit Regulates Mammary Cap Cell Plasticity Through TGF-beta Signaling

The organization and maintenance of complex tissues requires emergent properties driven by self-organizing and self-limiting cell-cell interactions. We examined these interactions in the murine mammary gland. Luminal and myoepithelial subpopulations of the postnatal mammary gland arise from unipotent progenitors, but the destiny of cap cells, which enclose terminal end buds (TEB) in pubertal mice, remains controversial. Using a transgenic strain (Tg11.5kb-GFP) that specifically marks cap cells, we found ~50% of these cells undergo divisions perpendicular to the TEB surface, suggesting they might contribute to the underlying luminal cell population. To address their stemness potential we developed a lineage tracing mouse driven from the s-SHIP (11.5 kb) promoter. Induction of tdTomato (tdTom) from this promoter in vivo demonstrated that all cap cell progeny are myoepithelial, with no conversion to luminal lineage. Organoid cultures also exhibited unipotency. However, isolated cap cells cultured as mammospheres generated mixed luminal/myoepithelial spheres. Moreover, ablation of luminal cells in vivo using diphtheria toxin triggered repopulation by progeny of tdTom+ cap cells. A signaling inhibitor screen identified the TGF{beta} pathway as a potential regulator of multipotency. TGF{beta}R inhibitors or gene deletion blocked conversion to the luminal lineage, consistent with an autocrine loop in which cap cells secrete TGF{beta} to activate the receptor and promote luminal transdifferentiation. Ductal tree regeneration in vivo from isolated cap cells was much more efficient when they were pre-treated with inhibitor, consistent with more cells retaining cap cell potential prior to transplantation. Notably, in vitro transdifferentiation of cap cells was blocked by co-culture with luminal cells. Overall, these data reveal a self-limiting cell circuit through which mammary luminal cells suppress cap cell conversion to the luminal lineage.

developmental biology↗

bioRxiv2021

Apoptotic Find-me Signals are an Essential Driver of Stem Cell Conversion To The Cardiac Lineage

Pluripotent stem cells can be driven by manipulation of Wnt signaling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic mesoderm lineage and ultimately into functional cardiomyocytes 1-4. Strikingly, we observed that induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) undergo widespread apoptosis upon Wnt activation, followed by a synchronous epithelial-mesenchymal transition (EMT). The EMT requires induction of transcription factors SNAI1/SNAI2 downstream of MESP1 expression, and double knock-out of SNAI1/2, or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis chemically or by ablation of pro-apoptotic genes also completely prevents the EMT, suppressing even the earliest events in mesoderm conversion, including EOMES, TBX6, and MESP1 induction. Conditioned medium from WNT-activated WT iPSCs overcomes the block to EMT by cells incapable of apoptosis (Apop-), suggesting the involvement of soluble factors from apoptotic cells in mesoderm conversion. Treatment with a purinergic P2Y receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signaling. ATP was sufficient to induce a partial EMT in Apop- cells treated with WNT activator. We conclude that nucleotides, in addition to acting as chemo-attractants for clearance of apoptotic cells can, unexpectedly, function as essential paracrine signals in mesoderm specification.

cell biology↗

bioRxiv2020

Genome-Wide CRISPR Screen Identifies Non-Canonical NF-κB Signaling as a Potent Regulator of Density-dependent Proliferation

Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on FUCCI, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-{kappa}B signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates non-canonical NF-{kappa}B signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin kinase inhibitors, by blocking entry into quiescence.

cell biology↗

bioRxiv2019

Non-autonomous induction of epithelial lineage infidelity and hyperplasia by DNA damage

Several epithelial tissues contain stem cell reserves to replenish cells lost during normal homeostasis or upon injury. However, how epithelial tissues respond to distinct types of damage, and how stem cell plasticity and proliferation are regulated in these contexts, remain poorly understood. Here, we reveal that genotoxic agents, but not mechanical damage, induce hyperplasia and lineage infidelity in three related epithelial tissues: the mammary gland, interfollicular epidermis and hair follicle. Furthermore, DNA damage also promotes stromal proliferation. In the mammary gland, we find that DNA damage activates multipotency within the myoepithelial population and hyper-proliferation of their luminal progeny, resulting in tissue disorganization. Additionally, in epidermal and hair follicle epithelia, DNA damage induces basal cell hyperplasia with the formation of abnormal, multi-layered K14+/K10+ cells. This behavior does not involve apoptosis or immunity, and is epithelial cell non-autonomous; stromal fibroblasts are both necessary and sufficient to induce the epithelial response. Thus, genotoxic agents that are used chemotherapeutically to promote cancer cell death can have the opposite effect on wild-type epithelial tissue, paradoxically promoting hyperplasia and inducing both stemness and lineage infidelity.

cell biology↗