Activation of WNT signaling in human pluripotent stem cells efficiently drives lateral mesoderm specification and subsequent cardiomyocyte differentiation. Stabilization of the WNT effector β-catenin induces mesodermal genes such as TBXT (Brachyury) and triggers an epithelial-mesenchymal transition (EMT). Although mechanical forces are essential for embryonic development, the role of actomyosin contractility during human mesoderm specification remains unclear. We show that increasing contractility through constitutively active Rho kinase or myosin light-chain kinase unexpectedly blocks β-catenin-dependent mesoderm induction and prevents EMT. In contrast, pharmacological or genetic suppression of contractility enhances Brachyury expression and advances EMT onset by 24 h. While β-catenin signaling alone promotes colony-level contractility, we find that contractility must be reduced prior to WNT activation to promote mesoderm specification, indicating a sensitization effect at the pluripotent state. Mechanistically, reduced tension decreases junctional β-catenin and increases nuclear active β-catenin, identifying actomyosin contractility as a key regulator of lineage commitment following WNT pathway activation.

Individual epithelial cells that acutely express oncogenes such as Ras or Src are extruded apically from monolayers of wildtype cells. Multiple signaling networks have been implicated but the extrusion mechanism is still not fully understood. We examined extrusion of mammary epithelial cells caused by acute induction of oncogenic Ras(Q61L). As reported by others, Ras-dependent extrusion requires downstream activation of ERK but not AKT. Unexpectedly, however, extrusion was completely blocked by Erlotinib, which inhibits Epidermal growth factor receptor (EGFR) activity, or by deletion of the receptor. In pancreatic and lung cancers, EGFR is required for full activation of Ras and consequent ERK activation. However, inhibition or deletion of EGFR had no impact in our system on Ras(Q61L)-GTP levels or ERK phosphorylation. Importantly, receptor function was cell autonomous, because EGFR expression was not required in surrounding WT cells but was essential in the Ras(Q61L) cells, yet did not act through the canonical ERK signaling pathway. Deletion of Ras exchange factors Sos1/2 did not block cell extrusion. Moreover, expression of a constitutively active MEK mutant, instead of Ras, was sufficient to drive extrusion, and EGFR inhibition or knockout in these cells blocked extrusion, with no change in phospho-ERK levels. Notably, acute expression of Ras triggered internalization of E-cadherin, which was partially blocked by inhibition of EGFR. Knockout of E-cadherin was alone sufficient to promote extrusion. Together, these data demonstrate an unanticipated requirement for noncanonical EGFR signaling in cancer cell extrusion, which may act in part through the promotion of E-cadherin endocytosis.

Temporal ordering of cellular events offers fundamental insights into biological phenomena. Although this is traditionally achieved through continuous direct observations1,2, an alternative solution leverages irreversible genetic changes, such as naturally occurring mutations, to create indelible marks that enables retrospective temporal ordering3-5. Using a multipurpose, single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, with incorporation of cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during mouse embryonic development, unconventional developmental relationships between cell types and new epithelial progenitor states by their unique genetic histories. Analysis of mouse adenomas, coupled to multiomic and single-cell profiling of human precancers, with clonal analysis of 418 human polyps, demonstrated the occurrence of polyclonal initiation in 15-30% of colonic precancers, showing their origins from multiple normal founders. Our study presents a multimodal framework that lays the foundation for in vivo recording, integrating synthetic or natural indelible genetic changes with single-cell analyses, to explore the origins and timing of development and tumorigenesis in mammalian systems.

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.### Competing Interest StatementJ.C.R. is on the scientific advisory board of Sitryx Therapeutics. All other authors declare no competing interests.

The activation of WNT signaling in human pluripotent stem cells drives efficient conversion to lateral mesoderm, which can be further differentiated into cardiomyocytes. Stabilization of the WNT effector β-catenin promotes expression of mesoderm-specifying genes such as Brachyury (Bra/T, encoded by TBXT) and Eomesodermin and drives an epithelial-mesenchymal transition (EMT) that occurs at 49-52 hrs post WNT activation. Interestingly, WNT activation also triggers an early contraction of stem cell colonies, with increased myosin light chain (MLC) phosphorylation, resulting to cellular morphological changes. Small molecule inhibitors and genetic approaches were applied to determine if contractility is related to mesoderm differentiation. Contrary to expectation, suppressing contractility accelerated differentiation; Bra/T induction was enhanced, and EMT initiated as early as 24 hrs post WNT activation. Conversely, expression of constitutively active Rho kinase or myosin light chain kinase, to increase contractility, efficiently blocked any induction of mesoderm, including EMT. Mechanistically, reduced contractility promoted active β-catenin levels in the cytoplasm and nucleus. We propose that the early contraction of human pluripotent stem cell colonies in response to WNT activation initially delays mesoderm induction by restraining accumulation of active β-catenin, suggesting a role for contractility as a temporal gate. ### Competing Interest Statement The authors have declared no competing interest.

Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. Here we used a synthetic biology approach to investigate whether a size barrier at the Golgi apparatus might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3, Ace2 and Muc1 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, increasing the size of their cytoplasmic domains caused partial mislocalization to the basolateral cortex and significantly delayed Golgi departure. Moreover, N-glycosylation of ‘large’ Crb3 was delayed, and ‘small’ Crb3 segregated into spatially distinct Golgi regions. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the endoplasmic reticulum and Golgi, Pals1 disassociated before Crb3 departure. Notably, a non-dissociable mutant Pals1 hampered the exit of Crb3. We conclude that, unexpectedly, a size filter at the Golgi facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for normal Crb3 sorting. de Caestecker and Macara study apical sorting of proteins with varying cytoplasmic tail length in epithelial cells. They propose that a size filter at the Golgi facilitates apical sorting of proteins with small cytoplasmic domains.

Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. We investigated whether a size barrier at the trans-Golgi network (TGN) might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3 and Ace2 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum (ER) until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, departure from the Golgi could be significantly delayed simply by increasing cytoplasmic bulk. Moreover, large and small Crb3 segregated into spatially distinct Golgi regions as detected by super resolution imaging. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the ER and Golgi, we found that Pals1 disassociates prior to Crb3 departure. Notably, a non-dissociable mutant Pals1 hampers the exit of Crb3. We conclude that an unexpected mechanism involving a size filter at the TGN facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for the normal kinetics of Crb3 sorting.

The luminal epithelium of the mammary gland is organized into monolayers; however, it originates from multilayered terminal end buds (TEBs) during development. Although apoptosis provides a plausible mechanism for cavitation of the ductal lumen, it doesn’t account for ductal elongation behind TEBs. Spatial calculations in mice suggest that most TEB cells integrate into the outermost luminal layer to generate elongation. We developed a quantitative cell culture assay that models intercalation into epithelial monolayers. We found that tight junction proteins play a key role in this process. ZO-1 puncta form at the new cellular interface and resolve into a new boundary as intercalation proceeds. Deleting ZO-1 suppresses intercalation both in culture and in cells transplanted into mammary glands via intraductal injection. Cytoskeletal rearrangements at the interface are critical for intercalation. These data identify luminal cell rearrangements necessary for mammary development and suggest a mechanism for integration of cells into an existing monolayer.

Pluripotent stem cells can be driven by manipulation of Wnt signalling 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. Strikingly, we observed that initiation of differentiation in induced pluripotent stem cells (iPSCs) and embryonic stem cells triggers widespread apoptosis, followed by a synchronous epithelial-mesenchymal transition (EMT). Apoptosis is caused by the absence of bFGF in the differentiation medium. EMT requires induction of the transcription factors SNAI1 and SNAI2 downstream of MESP1 expression, and double knockout of SNAI1 and SNAI2 or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis, either chemically or by ablation of pro-apoptotic genes, also completely prevents EMT, suppressing even the earliest events in mesoderm conversion, including T/BRA, TBX6 and MESP1 induction. Conditioned medium from WNT-activated wild-type iPSCs overcomes the block to EMT by cells incapable of apoptosis, suggesting involvement of soluble factors from apoptotic cells in mesoderm conversion. Knockout of the PANX1 channel blocked EMT, whereas treatment with a purinergic P2-receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signalling. ATP and/or UTP was sufficient to induce a partial EMT in apoptosis-incapable cells treated with WNT activator. Notably, knockout of the ATP/UTP-specific P2Y2 receptor blocked EMT and mesoderm induction. We conclude that in addition to acting as chemo-attractants for clearance of apoptotic cells, nucleotides can function as essential paracrine signals that, with WNT signalling, create a logical AND gate for mesoderm specification.

Mitotic spindle orientation is a conserved, dynamic, and highly complex process that plays a key role in dictating the cleavage plane, fate

SUMMARYThe 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 TGFβ pathway as a potential regulator of multipotency. TGFβR inhibitors or gene deletion blocked conversion to the luminal lineage, consistent with an autocrine loop in which cap cells secrete TGFβ 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.

ABSTRACTThe myoepithelial cell compartment of the murine postnatal mammary gland is generated from basal cap cells in the terminal end bud and maintained by self-renewal. Transdifferentiation to the luminal lineage does not normally occur but can be induced by DNA damage, luminal cell death or transplantation into a recipient mammary fat pad. Myoepithelial cells cultivated in vitro can also transdifferentiate towards the luminal lineage. Little is known about the molecular mechanisms and gene regulatory networks underlying this plasticity. Using a transgenic mouse (Tg11.5kb-GFP) that marks cap cells with GFP, we discovered that mature myoepithelial cells placed in culture begin to express GFP within ∼24 hrs and later express the Keratin 8 (K8) luminal marker. Cell tracking showed that most K8+ cells arose from GFP+ cells, suggesting that myoepithelial cells de-differentiate towards a progenitor state before changing lineage. Differential gene expression analysis, comparing pure GFP+ cap cells with mature myoepithelial cells, identified multiple transcription factors that iRegulon predicted might regulate the myoepithelial to cap cell transition. Knockout of one of these genes, Regulatory Factor 3 (Rfx3), significantly reduced the population of GFP+ cells and increased differentiation to the K8+ luminal lineage. Rfx3 knockout also reduced mammosphere growth and mammary gland regeneration efficiency in a transplantation assay, but had no effect on proliferation in vitro. Together, these data support a key role for Rfx3 in the stabilization of the mammary basal cell lineages.

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.

Most human cancers arise from epithelial tissues, which are apical-basally polarized and possess intercellular adhesive junctions. Epithelial cells grow to characteristic densities, often from proliferative progenitors, which arrest as they mature. Homeostatic mechanisms can maintain this characteristic density if it is exceeded (crowding) or is too low (e.g. in response to wounding). During tumor initiation and progression this homeostatic mechanism is lost. Some aspects of cell polarity are also lost, although many carcinomas retain intercellular junctions and even apical domains. In other cases, and particularly in recurrent tumors, however, the cells become predominantly mesenchymal. A major question, still only incompletely answered, is whether the proteins that determine cell polarity function as tumor suppressors or tumor promoters. Here we discuss recent advances in understanding the role of polarity proteins and homeostasis in cancer.

DNA crosslinking agents are commonly used in cancer chemotherapy; however, responses of normal tissues to these agents have not been widely investigated. We reveal in mouse interfollicular epidermal, mammary and hair follicle epithelia that genotoxicity does not promote apoptosis but paradoxically induces hyperplasia and fate specification defects in quiescent stem cells. DNA damage to skin causes epithelial and dermal hyperplasia, tissue expansion, and proliferation-independent formation of abnormal K14/K10 dual-positive suprabasal cells. Unexpectedly, this behavior is epithelial cell non-autonomous and independent of an intact immune system. Instead, dermal fibroblasts are both necessary and sufficient to induce the epithelial response, which is mediated by activation of a fibroblast-specific NLRP3 inflammasome and subsequent IL-1β production. Thus, genotoxic agents that are used chemotherapeutically to promote cancer cell death can have the opposite effect on wild-type epithelia by inducing, via a non-autonomous IL-1β-driven mechanism, both hyperplasia and stem cell lineage defects.

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 fluorescence ubiquitination cell cycle indicator, 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-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB 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-dependent kinase inhibitors, by blocking entry into quiescence.

The exocyst, an octameric protein complex, is an essential component of the membrane transport machinery required for tethering and fusion of vesicles at the plasma membrane. We report pathogenic variants in an exocyst subunit, EXOC2 (Sec5). Affected individuals have severe developmental delay, dysmorphism, and brain abnormalities; variability associated with epilepsy; and poor motor skills. Family 1 had two offspring with a homozygous truncating variant in EXOC2 that leads to nonsense-mediated decay of EXOC2 transcript, a severe reduction in exocytosis and vesicle fusion, and undetectable levels of EXOC2 protein. The patient from Family 2 had a milder clinical phenotype and reduced exocytosis. Cells from both patients showed defective Arl13b localization to the primary cilium. The discovery of mutations that partially disable exocyst function provides valuable insight into this essential protein complex in neural development. Since EXOC2 and other exocyst complex subunits are critical to neuronal function, our findings suggest that EXOC2 variants are the cause of the patients' neurological disorders.

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.

The original version of this Article contained errors in the author affiliations. Affiliation 2 incorrectly read ‘Department of Biochemistry and Molecular Genetics and Breast Surgery, Ehime University Graduate School of Medicine, 7910295, Japan’ and affiliation 3 incorrectly read ‘Department of Hepato-Biliary-Pancreatic and Breast Surgery, Ehime University Graduate School of Medicine, Matsuyama 7910295, Japan.’ These errors have now been corrected in both the PDF and HTML versions of the Article.