Lineage Tracing Research Articles

Uncovering the role of oncogenic HER2 variants as drivers of pancreatic cancer.

772 Background: The therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC) to conventional cytotoxic therapy underscores the need for advances in targeted molecular therapeutics. Previous studies have reported approximately 2% of PDAC patients with HER2 gene amplification. While anti- HER2 cancer therapy has yielded success in other disease sites, it has had minimal reported benefit in PDAC. Multiple HER2 isoforms exist that are generated either by loss of exon 16 ( d16 HER2 ), or through N-terminal truncations ( p95 HER2 ). These isoforms are implicated in differential tumor behavior and response to anti-HER2 therapy in the breast but have not been investigated in PDAC. Herein, we hypothesize that these isoforms may underlie the observed resistance to HER2 therapy in PDAC. Methods: To test for the presence of HER2 structural variants within human PDAC, a tissue microarray was constructed of fifty-one primary tumor samples and two metastatic samples. Multiplex immunohistochemistry (mIHC) was performed for staining of the extracellular N-terminal and intracellular C-terminal HER2 domains. We then utilized our previously described HER2 + cancer rainbow (Crainbow) mouse model in which human wild-type HER2 ( WT HER2 ), d16 HER2 , and p95 HER2 were expressed in the same mouse along with fluorescent protein reporters. HER2 Crainbow mice were crossed with PDX1-Cre recombinase to initiate recombination and expression of each fluorescently labelled HER2 isoform during pancreas development. PDX1-Cre / HER2 Crainbow mice were followed for up to one year and sacrificed to lineage trace each isoform and register HER2 lineages with histopathology. Results: In our human data, four of fifty-three patient samples (three primary and one metastatic) had moderate-to-strong HER2 staining. All samples identified on mIHC as HER2 positive stained primarily for the p95 variant of HER2 , with absence of the N-terminal domain and preservation of the intracellular domain. In our transgenic mice, 65% of the mouse cohort developed pre-malignant low grade pancreatic intraepithelial neoplasia (PanIN) by eight weeks. At one year, 58.3% of mice demonstrated advanced high grade PanIN, and 25% developed invasive disease. Lineage tracing revealed that while early low grade PanIN began as polyclonal lesions, comprised of WT- , d16- , and p95 HER2 expressing cells, advanced PanIN and invasive disease were unanimously monoclonal with one dominant isoform arising. WT HER2 was represented abundantly in low grade disease, but high grade and invasive disease were predominantly driven by d16- and p95-HER2. Conclusions: Our data implicates the oncogenic d16- and p95 HER2 variants, rather than WT HER2 , as the primary drivers of HER2+ PDAC tumorigenesis. In breast p95 HER2 has been described as a known cause of Trastuzumab resistance. Therefore, future efforts will be aimed at characterizing the mechanisms driving therapeutic resistance in p95-HER2 PDAC tumors.

Spermidine alleviates diabetic periodontitis by reversing human periodontal ligament stem cell senescence via mitophagy.

Type 2 Diabetes Mellitus (T2DM) exacerbates periodontal disease lesions, and human periodontal ligament stem cells (PDLSCs) depletion may be the key to periodontal healing impair by T2DM. This study aims to explore the mechanism of PDLSCs depletion in diabetes periodontitis (DP). Firstly, we observed aggravated periodontal destruction in the DP animal model, accompanied by oxidative damage and accumulation of senescent cells. In the high-glucose inflammatory environment in vitro, we revealed that PDLSCs underwent senescence, oxidative stress, mitochondrial dysfunction, and activation of cGAS-STING signaling pathway triggered by mitochondrial DNA. Lineage tracing confirmed that SPD recruited Tdtomato-Gli1+ PDLSCs to the damaged area and alleviated periodontal destruction in DP models. Evidence in vitro further showed that SPD inhibited PDLSCs senescence and oxidative stress, enhanced mitochondrial function, reduced membrane permeability transition pore opening, and reduced DNA leakage, which blocked the STING activation. Mechanistically, SPD reduced STING-TBK1 phosphorylation by scavenging mitochondrial-derived dsDNA in a mitophagy-dependent manner, its therapeutic effect was abolished by incorporation of cGAMP, a STING activator. In summary, our study reveals the mechanism of PDLSCs depletion due to excessive oxidative damage in the DP environment. Local injection of SPD reactivates mitophagy, recruits Gli1+ stem cells by inhibiting STING activation for periodontal regeneration.

Promoting epithelial regeneration in chemically induced acute lung injury through Sox9-positive alveolar type 2 epithelial cells

BackgroundChemical-induced acute lung injury is characterized by impaired epithelial regenerative capacity, leading to acute pulmonary edema. Numerous studies have investigated the therapeutic potential of endogenous stem cells with particular emphasis on alveolar type 2 epithelial (AEC2) cells owing to their involvement in lung cell renewal. Sox9, a transcription factor known for its role in maintaining stem cell properties and guiding cell differentiation, marks a subset of AEC2 cells believed to contribute to epithelial repair. However, the role of Sox9+AEC2 cells in the distal lung alveolar cells and the potential roles in chemically induced acute lung injury have never been explored.MethodsIn this study, we generated Sox9flox/flox;SftpcCre−ERT2 mice and examined the effects of Sox9+AEC2 cells on the pathophysiology of epithelial damage during chemical-induced acute lung injury. Subsequently, Sox9-CreERT2 Ai9 mice were used for lineage tracing to elucidate the repair mechanisms.ResultsOur findings revealed that Sox9+AEC2 cells endowed with stem cell properties induced cell proliferation during lung injury, predominantly in the damaged alveolar region. This process is accompanied by the regulation of inflammatory responses and orderly differentiation, thereby promoting epithelial regeneration.ConclusionThese results provide compelling in vivo genetic evidence supporting the characterization of Sox9+AEC2 cells as bona fide lung epithelial stem cells, demonstrating their multipotency and self-renewal capabilities during lung repair and regeneration. The identification of Sox9+AEC2 cells as crucial contributors to the promotion of epithelial repair underscores their potential as therapeutic targets in chemical-induced acute lung injury.

DOP073 Gli1+ Mesenchymal Cells Drive Intestinal Fibrostenosis by Secreting SMOC2: A Biomarker and Therapeutic Target

Abstract Background Intestinal fibrostenosis in Crohn’s disease (CD) represents a significant clinical challenge, driven by the absence of reliable early biomarkers, limited therapeutic options, and high postoperative recurrence rates, all of which contribute to poor patient outcomes. This study aims to elucidate the role of Gli1+ mesenchymal cells in the pathogenesis of intestinal fibrostenosis and to explore their potential as a novel therapeutic target. Methods CD patients who underwent surgery for intestinal obstruction due to fibrosis were included in this study, and intestinal tissues were collected from both fibrotic and normal sites. Dextrose sodium sulfate (DSS)-induced and 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced chronic colitis mouse models and their normal controls were also included. Single-cell RNA sequencing was performed on both human and murine tissues. Gli1-CreERT2; R26-tdTomato mice were generated for lineage tracing. Cell sorting combined with Smart-seq was used to isolate Gli1+ mesenchymal cells, revealing their pathogenic determinants. Results Single-cell RNA sequencing data and immunofluorescence staining from surgical patients revealed an enrichment of Gli1+ mesenchymal cells at fibrotic sites. Lineage tracing of Gli1+ mesenchymal cells demonstrated their proliferation and acquisition of a myofibroblast phenotype following chronic colitis. In vivo ablation of Gli1+ mesenchymal cells attenuated the severity of intestinal fibrosis. Compared to Gli1- cells, Gli1+ mesenchymal cells expressed a distinct gene expression profile, including markers of extracellular matrix (ECM)-related genes and Smoc2 (SPARC Related Modular Calcium Binding 2, encodes an extracellular matrix protein). Elevated Smoc2 expression was confirmed in both human and murine tissues, and Smoc2 knockdown further supported its role in promoting intestinal fibrosis. Mechanistically, Gli1+ mesenchymal cells establish a pro-fibrotic niche through the endocrine and exocrine functions of SMOC2, promoting ECM deposition and the activation of other fibroblasts. Additionally, a predictive and diagnostic model was established by assessing SMOC2 levels in tissue and plasma samples from CD patients with intestinal fibrosis, highlighting its potential as a biomarker for postoperative recurrence and as a therapeutic target. Conclusion Our results demonstrate that Gli1+ mesenchymal cells are key drivers of intestinal fibrostenosis, forming a pro-fibrotic niche through their unique secretion of the SMOC2 protein at fibrotic sites. SMOC2 could therefore be used as a predictive biomarker for postoperative recurrence and as a potential therapeutic target.

DOP112 Inflammation-associated monocytes express ACOD1 to curtail inflammatory behaviour in IBD and experimental colitis

Abstract Background ACOD1 is the ubiquitous source of the anti-inflammatory metabolite itaconate. We hypothesised monocyte ACOD1 expression is an inhibitory pathway that limits intestinal inflammation. Methods We performed single-cell RNA sequencing of colonoscopic biopsies from >120 Crohn’s disease (CD) patients and healthy controls, with complementary analysis of DSS treated C57BL/6J (WT) mice, validated using flow cytometry in independent cohorts. Acod1–/– mice received 2% DSS in acute and recovery models, complemented by competitive bone marrow chimeric mice, anti-IFN / itaconate-derivate treatment and transgenic cell depletion models. Results We profile the monocyte / macrophage compartment in human IBD using single-cell RNA sequencing and identify a discrete population of intestinal CXCL9/10+ cells that accumulate in IBD, which we term inflammation associated monocytes (IAMs). These can be identified by the expression of CD319, CD274 and CCRL2, and demonstrate specific expression of IBD susceptibility genes, including NOD2. By performing cross-species analysis in mice, we show an analogous population of IAMs accumulate during chemically induced colitis. Using transgenic fate mapping approaches, we show these cells likely derive from a discrete MDP-precursor in the bone marrow and are locally imprinted to produce heightened IL-1β and TNF in mouse and humans. Importantly, we show that co-incident with a hyper-inflammatory phenotype, these same cells uniquely and specifically express aconitate decarboxylase (ACOD1) in response to local Toll-like receptor (TLR) and interferon (IFN) receptor signalling, to limit unrestricted cytokine production. Conclusion Thus, the intestinal environment after injury instructs both inflammation and recovery specifically within a transitioning population of monocytes that are absent in health. Reduced itaconate production from IAMs in CD may predispose to chronic inflammation and disease progression.

Early precursor-derived pituitary gland tissue-resident macrophages play a pivotal role in modulating hormonal balance.

The pituitary gland is the central endocrine regulatory organ producing and releasing hormones that coordinate major body functions. The physical location of the pituitary gland at the base of the brain, though outside the protective blood-brain barrier, leads to an unexplored special immune environment. Using single-cell transcriptomics, fate mapping, and imaging, we characterize pituitary-resident macrophages (pitMØs), revealing their heterogeneity and spatial specialization. Microglia-like macrophages (ml-MACs) are enriched in the posterior pituitary, while other pitMØs in the anterior pituitary exhibit close interactions with hormone-secreting cells. Importantly, all pitMØs originate from early yolk sac progenitors and maintain themselves through self-renewal, independent of bone marrow-derived monocytes. Macrophage depletion experiments unveil the role of macrophages in regulating intrapituitary hormonal balance through extracellular ATP-mediated intercellular signaling. Altogether, these findings provide information on pituitary gland macrophages and advance our understanding of immune-endocrine system crosstalk.

FlashTag-mediated Labeling for Intraventricular Macrophages in the Embryonic Brain.

The fate mapping technique is essential for understanding how cells differentiate and organize into complex structures. Various methods are used in fate mapping, including dye injections, genetic labeling (e.g., Cre-lox recombination systems), and molecular markers to label cells and track their progeny. One such method, the FlashTag system, was originally developed to label neural progenitors. This technique involves injecting carboxyfluorescein diacetate succinimidyl ester (CFSE) into the lateral ventricles of mouse embryos, relying on the direct uptake of dye by cells. The injection of CFSE into the lateral ventricle allows for the pulse labeling of mitotic (M-phase) neural progenitors in the ventricular zone and their progeny throughout the brain. This approach enables us to trace the future locations and differentiation paths of neural progenitors. In our previous study, we adapted this method to selectively label central nervous system-associated macrophages (CAMs) in the lateral ventricle by using a lower concentration of CFSE compared to the original protocol. Microglia, the brain's immune cells, which play pivotal roles in both physiological and pathological contexts, begin colonizing the brain around embryonic day (E) 9.5 in mice, with their population expanding as development progresses. The modified FlashTag technique allowed us to trace the fate of intraventricular CAMs, revealing that certain populations of microglia are derived from these cells. The optimized approach offers deeper insights into the developmental trajectories of microglia. This protocol outlines the modified FlashTag method for labeling intraventricular CAMs, detailing the CFSE injection procedure, evaluation of CFSE dilution, and preparation of tissue for immunohistochemistry. Key features • This protocol builds upon the method developed by Govindan et al. and extends its application to intraventricular CAMs. • This protocol allows for the cell fate tracking of intraventricular CAMs within 24 h. • This protocol requires the technique of intraventricular injection of CFSE into embryonic brains.

Polycystin-1 regulates tendon-derived mesenchymal stem cells fate and matrix organization in heterotopic ossification

Mechanical stress modulates bone formation and organization of the extracellular matrix (ECM), the interaction of which affects heterotopic ossification (HO). However, the mechanically sensitive cell populations in HO and the underlying mechanism remain elusive. Here, we show that the mechanical protein Polysyctin-1 (PC1, Pkd1) regulates CTSK lineage tendon-derived mesenchymal stem cell (TDMSC) fate and ECM organization, thus affecting HO progression. First, we revealed that CTSK lineage TDMSCs are the major source of osteoblasts and fibroblasts in HO and are responsive to mechanical cues via single-cell RNA sequencing analysis and experiments with a lineage tracing mouse model. Moreover, we showed that PC1 mediates the mechanosignal transduction of CTSK lineage TDMSCs to regulate osteogenic and fibrogenic differentiation and alters the ECM architecture by facilitating TAZ nuclear translocation. Conditional gene depletion of Pkd1 or Taz in CTSK lineage cells and pharmaceutical intervention in the PC1-TAZ axis disrupt osteogenesis, fibrogenesis and ECM organization, and consequently attenuate HO progression. These findings suggest that mechanically sensitive CTSK-lineage TDMSCs contribute to heterotopic ossification through PC1-TAZ signaling axis mediated cell fate determination and ECM organization.

CD11c-Expressing Microglia Are Transient, Driven by Interactions With Apoptotic Cells.

Microglia, the parenchymal macrophage of the central nervous system, serve crucial remodeling functions throughout development. Microglia are transcriptionally heterogenous, suggesting that distinct microglial states confer discrete roles. Currently, little is known about how dynamic these states are, the cues that promote them, or how they impact microglial function. In the developing retina, we previously found a significant proportion of microglia express CD11c (Integrin αX, Itgax, subunit of complement receptor 4) which has also been reported in other developmental and disease contexts. Here, we sought to understand the regulation and function of CD11c+ microglia. We found that CD11c+ microglia track with prominent waves of neuronal apoptosis in postnatal retina. Using genetic fate mapping, we provide evidence that microglia transition out of the CD11c state to return to homeostasis. We show that CD11c+ microglia have elevated lysosomal content and contribute to the clearance of apoptotic neurons, and found that acquisition of CD11c expression is partially dependent upon the TAM receptor AXL. Using selective ablation, we found CD11c+ microglia are not uniquely critical for phagocytic clearance of apoptotic cells. Together, our data suggest that CD11c+ microglia are a transient state induced by developmental apoptosis rather than a specialized subset mediating phagocytic elimination.

High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations.

In vivo lineage tracing holds great potential to reveal fundamental principles of tissue development and homeostasis. However, current lineage tracing in humans relies on extremely rare somatic mutations, which has limited temporal resolution and lineage accuracy. Here, we developed a generic lineage-tracing tool based on frequent epimutations on DNA methylation, enabled by our computational method MethylTree. Using single-cell genome-wide DNA methylation datasets with known lineage and phenotypic labels, MethylTree reconstructed lineage histories at nearly 100% accuracy across different cell types, developmental stages, and species. We demonstrated the epimutation-based single-cell multi-omic lineage tracing in mouse and human blood, where MethylTree recapitulated the differentiation hierarchy in hematopoiesis. Applying MethylTree to human embryos, we revealed early fate commitment at the four-cell stage. In native mouse blood, we identified ~250 clones of hematopoietic stem cells. MethylTree opens the door for high-resolution, noninvasive and multi-omic lineage tracing in humans and beyond.

Early and delayed STAT1-dependent responses drive local trained immunity of macrophages in the spleen.

Trained immunity (TI) is the process wherein innate immune cells gain functional memory upon exposure to specific ligands or pathogens, leading to augmented inflammatory responses and pathogen clearance upon secondary exposure. While the differentiation of hematopoietic stem cells (HSCs) and reprogramming of bone marrow (BM) progenitors are well-established mechanisms underpinning durable TI protection, remodeling of the cellular architecture within the tissue during TI remains underexplored. Here, we study the effects of peritoneal Bacillus Calmette-Guérin (BCG) administration to find TI-mediated protection in the spleen against a subsequent heterologous infection by the Gram-negative pathogen Salmonella Typhimurium (S.Tm). Utilizing single cell RNA-sequencing and flow cytometry, we discerned STAT1-regulated genes in TI-associated resident and recruited splenic myeloid populations. The temporal dynamics of TI were further elucidated, revealing both early and delayed myeloid subsets with time-dependent, cell-type-specific STAT1 signatures. Using lineage tracing, we find that tissue-resident red pulp macrophages (RPM), initially depleted by BCG exposure, are restored from both tissue-trained, self-renewing macrophages and from bone marrow-derived progenitors, fostering long lasting local defense. Early inhibition of STAT1 activation, using specific JAK-STAT inhibitors, reduces both RPM loss and recruitment of trained monocytes. Our study suggests a temporal window soon after BCG vaccination, in which STAT1-dependent activation of long-lived resident cells in the tissue mediates localized protection.

MethylTree: exploring epimutations for accurate and non-invasive lineage tracing.

MethylTree: exploring epimutations for accurate and non-invasive lineage tracing.

Single cell derived multicellular meristem: insights into male-to-hermaphrodite conversion and de novo meristem formation in ceratopteris.

Land plants alternate between asexual sporophytes and sexual gametophytes. Unlike seed plants, ferns develop free-living gametophytes. Gametophytes of the model fern Ceratopteris exhibit two sex types: hermaphrodites with pluripotent meristems and males lacking meristems. In the absence of the pheromone antheridiogen, males convert to hermaphrodites by forming de novo meristems, though the mechanisms remain unclear. Using long-term time-lapse imaging and computational analyses, we captured male-to-hermaphrodite conversion at single-cell resolution and reconstructed the lineage and division atlas of newly formed meristems. Lineage tracing revealed that the de novo-formed meristem originates from a single non-antheridium cell, the meristem progenitor cell (MPC). During conversion, the MPC lineage showed increased mitotic activity, with marginal cells proliferating faster than inner cells. A mathematical model suggests that stochastic variation in cell division, combined with strong inhibitory signals from dividing marginal cells, is sufficient to explain gametophyte dynamics. Experimental disruption of division timing agreed with the model, showing precise cell cycle progression is essential for MPC establishment and sex-type conversion. These findings reveal cellular mechanisms governing sex conversion and de novo meristem formation in land plants.

Studying the Global Progress and Regulatory Landscape of Stem Cell Therapy Research

Objective: Stem cell therapy has emerged as a pioneering front in the biomedical domain, characterized by its rapid strides in innovation and progress. Such advancements hold profound implications for public health. Notably, Europe, the United States, Japan, and China have all crafted comprehensive regulatory frameworks to expedite the fruition of stem cell therapy. This article meticulously scrutinizes and evaluates the progression of clinical trials in cell therapy across major global jurisdictions. It delineates the trajectory of regulatory evolution in Europe, the United States, Japan, and China, and systematically collates the pivotal normative guidelines. This article is crafted with the intent to serve as a reference for regulatory agents, enterprises, and professionals in the field. Methods: Stem cell therapy, a paragon of innovative medical technology, heralds a new era of treatment possibilities for hitherto intractable diseases. It is more than just a benchmark of technological advancement; it represents a revolutionary shift in the pharmaceutical landscape. Since Dr.Thomas was awarded the Nobel Prize for his research on bone marrow transplantation in 1990, he has gradually moved from the laboratory to the clinical application stage, bringing new hope for treatment to patients. Stem cell therapy is a type of cell therapy. There are various types of cell therapy, which can be classified into stem cell therapy and immune cell therapy based on their cell sources. They can also be classified into autologous, allogeneic, and heterologous cells based on their donor sources. As a cutting-edge application technology in life science research, cell therapy has shown great therapeutic potential in various disease fields such as malignant tumors, genetic diseases, and chronic degenerative diseases. Multiple cell therapy products have been successfully launched and demonstrated excellent efficacy, among which stem cell therapy stands out due to its long application history and active research trend. Bone marrow/hematopoietic stem cell therapy is the earliest stem cell therapy, mainly used for bone marrow/hematopoietic stem cell transplantation to treat hematological malignancies such as leukemia. Human derived stem cells and their derived therapeutic products, as important regenerative medicine products, have great potential in cell replacement, tissue repair, disease treatment, and other fields. Nearly 70% of the stem cells used in clinical studies are hematopoietic stem cells and mesenchymal stem cells derived from bone marrow, peripheral blood, and umbilical cord. Technical methods include purification, in vitro culture and amplification, drug treatment, or gene modification. Immune cell therapy is a method of treating diseases using immune cells, which was originally mainly used to treat malignant tumors. According to the specificity of immune cell therapy, it can usually be divided into specific immune cell therapy and non-specific immune cell therapy. Specific immune cell therapy includes chimeric antigen receptor T-cell (CAR-T) therapy, T-cell receptor engineered T cell therapy, chimeric antigen receptor natural killer cell therapy (CAR-NK), and DC-CIK immunotherapy. Nonspecific immune cell therapy mainly includes lymphokine activated killer cell therapy and cytokine induced killer cell therapy. In addition, extracellular vesicles, as cell derivatives, have the advantage of non-living properties that are easier to characterize, store, package, and transport than living cells, and their research has shown explosive growth. Extracellular vesicle is a general term for lipid bilayer membrane-bound vesicles released by cells into the extracellular space, with a diameter range of 50-2,000 nanometers. Exosomes are extracellular vesicles with a diameter of 30-150 nanometers. At present, the Food and Drug Administration (FDA) has approved at least 8 extracellular vesicle products to enter phase II/III clinical trials. In recent years, the development of organoid technology, cell lineage tracing technology, single-cell spatial omics-sequencing technology, single-molecule technology, micro proteomics and proximity labeling technology has greatly promoted the progress of stem cell research and laid a good technical reserve for achieving functional organ reconstruction. Results: In the realm of emerging technologies, industry development often precedes regulatory frameworks. The swift progression of innovative stem cell therapy products not only propels medical innovation but also challenges existing regulatory technologies and institutions. Regulatory agencies must, therefore, continually innovate their strategies and technologies. Conclusion: Countries worldwide have crafted regulatory policies and technical evaluation systems tailored to their specific contexts. The variety of stem cell therapy types and applications defined by national regulations reflects this diversity. This article delves into the clinical research status of stem cell therapy through statistical analysis and dissects the regulatory frameworks governing it across nations, offering a valuable resource for stakeholders in this dynamic field.

Spatiotemporal and genetic cell lineage tracing of endodermal organogenesis at single-cell resolution.

During early mammalian development, the endoderm germ layer forms the foundation of the respiratory and digestive systems through complex patterning. This intricate process, guided by a series of cell fate decisions, remains only partially understood. Our study introduces innovative genetic tracing codes for 14 distinct endodermal regions using novel mouse strains. By integrating high-throughput and high-precision single-cell RNA sequencing with sophisticated imaging, we detailed the spatiotemporal and genetic lineage differentiation of the endoderm at single-cell resolution. We discovered an unexpected multipotentiality within early endodermal regions, allowing differentiation into various organ primordia. This research illuminates the complex and underestimated phenomenon where endodermal organs develop from multiple origins, prompting a reevaluation of traditional differentiation models. Our findings advance understanding in developmental biology and have significant implications for regenerative medicine and the development of advanced organoid models, providing insights into the intricate mechanisms that guide organogenesis.

Endothelial Cpt1a Inhibits Neonatal Hyperoxia-Induced Pulmonary Vascular Remodeling by Repressing Endothelial-Mesenchymal Transition.

Pulmonary hypertension (PH) increases the mortality of preterm infants with bronchopulmonary dysplasia (BPD). There are no curative therapies for this disease. Lung endothelial carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme of the carnitine shuttle system, is reduced in a rodent model of BPD. It is unknown whether endothelial Cpt1a reduction causes pulmonary vascular (PV) remodeling. The latter can be the result of endothelial-mesenchymal transition (EndoMT). Here, endothelial cell (EC)-specific Cpt1a KO and WT mice (<12h old) are exposed to hyperoxia (70% O2) for 14 days and allow them to recover in normoxia until postnatal day 28. Hyperoxia causes PH, which is aggravated in EC-specific Cpt1a KO mice. Upregulating endothelial Cpt1a expression inhibits hyperoxia-induced PV remodeling. Hyperoxia causes lung EndoMT, detected by immunofluorescence, scRNA-sequencing, and EC lineage tracing, which is further increased in EC-specific Cpt1a KO mice. Blocking EndoMT inhibits hyperoxia-induced PV remodeling. Male mice under the same high oxygen conditions develop a higher degree of PH than females, which is associated with reduced endothelial Cpt1a expression. Conclusively, neonatal hyperoxia causes PH by decreasing endothelial Cpt1a expression and upregulating EndoMT. This provides a valuable strategy for developing targeted therapies by upregulating endothelial Cpt1a levels or inhibiting EndoMT to treat BPD-associated PH.

Neural plate pre-patterning enables specification of intermediate neural progenitors in the spinal cord.

Dorsal-ventral patterning of neural progenitors in the posterior neural tube, which gives rise to the spinal cord, has served as a model system to understand how extracellular signals organize developing tissues. While previous work has shown that signaling gradients diversify progenitor fates at the dorsal and ventral ends of the tissue, the basis of fate specification in intermediate regions has remained unclear. Here we use zebrafish to investigate the neural plate, which precedes neural tube formation, and show that its pre-patterning by a distinct signaling environment enables intermediate fate specification. Systematic spatial analysis of transcription factor (TF) expression and signaling activity using a reference-based mapping approach shows that the neural plate is partitioned into a striking complexity of TF co-expression states that, in part, correspond to the activity of gastrulation signals such as FGF and Wnt that persist through axis extension. Using in toto analysis of cellular movement combined with fate mapping, we find that dbx1b -expressing intermediate progenitors (p0) originate from a neural-plate specific state characterized by transient co-expression of the TFs pax3a , olig4 and her3 . Finally, we show that this state is defined by Wnt signaling in the posterior neural plate and that ectopic Wnt activation within pax3a/olig4 + cells is sufficient to promote dbx1b expression. Our data broadly support a model in which neural progenitor specification occurs through the sequential use of multiple signals to progressively diversify the neural tissue as it develops. This has implications for in vitro differentiation of spinal cord cell types and for understanding signal-based patterning in other developmental contexts.

Resolving forebrain developmental organisation by analysis of differential growth patterns.

The forebrain is the most complex region of the vertebrate CNS, and its developmental organisation is controversial. We fate-mapped the embryonic chick forebrain using lipophilic dyes and Cre-recombination lineage tracing, and built a 4D model of brain growth. We reveal modular patterns of anisotropic growth, ascribed to progenitor regions through multiplex HCR. Morphogenesis is dominated by directional growth towards the eye, more isometric expansion of the prethalamus and dorsal telencephalon, and anterior movement of ventral cells into the hypothalamus. Fate conversion experiments in chick and comparative gene expression analysis in chick and mouse support placement of the hypothalamus ventral to structures extending from the telencephalon up to and including the zona limitans intrathalamica (ZLI), with the dorsoventral axis becoming distorted at the base of the ZLI. Our findings challenge the widely accepted prosomere model of forebrain organisation, and we propose an alternative 'tripartite hypothalamus' model.

Irx1 mechanisms for oral epithelial basal stem cell plasticity during reepithelialization after injury.

The oral mucosa undergoes daily insults, and stem cells in the epithelial basal cell layer regenerate gingiva tissue to maintain oral health. The Iroquois Homeobox 1 (IRX1) protein is expressed in the stem cell niches in human/mouse oral epithelium and mesenchyme under homeostasis. We found that Irx1+/- heterozygous (Het) mice have delayed wound closure, delayed morphological changes of regenerated epithelium, and defective keratinocyte proliferation and differentiation during wound healing. RNA-Seq analyses between WT and Irx1+/- mice at 3 days postinjury (dpi) found impaired epithelial migration and decreased keratinocyte-related genes upon injury. IRX1-expressing cells are found in the gingival epithelial basal cell layer, a stem cell niche for gingival maintenance. IRX1-expressing cells are also found in cell niches in the underlying stroma. IRX1 activates SOX9 in the transient amplifying layer to increase cell proliferation, and EGF signaling is activated to induce cell migration. Krt14CreERT lineage tracing experiments reveal defects in the stratification of the Irx1+/- HET mouse oral epithelium. IRX1 is primed at the base of the gingiva in the basal cell layer of the oral epithelium, facilitating rapid and scarless wound healing through activating SOX9 and the EGF signaling pathway.

Oxidative Phosphorylation is a Metabolic Vulnerability of Endocrine Therapy-Tolerant Persister Cells in ER+ Breast Cancer.

Despite adjuvant treatment with endocrine therapies, estrogen receptor-positive (ER+) breast cancers recur in a significant proportion of patients. Recurrences are attributable to clinically undetectable endocrine-tolerant persister cancer cells that retain tumor-forming potential. Therefore, strategies targeting such persister cells may prevent recurrent disease. Using CRISPR-Cas9 genome-wide knockout screening in ER+ breast cancer cells, we identified a survival mechanism involving metabolic reprogramming with reliance upon mitochondrial respiration in endocrine-tolerant persister cells. Quantitative proteomic profiling showed reduced levels of glycolytic proteins in persisters. Metabolic tracing of glucose revealed an energy-depleted state in persisters where oxidative phosphorylation was required to generate ATP. A phase II clinical trial was conducted to evaluate changes in mitochondrial markers in primary ER+/HER2- breast tumors induced by neoadjuvant endocrine therapy (NCT04568616). In an analysis of tumor specimens from 32 patients, tumors exhibiting residual cell proliferation after aromatase inhibitor-induced estrogen deprivation with letrozole showed increased mitochondrial content. Genetic profiling and barcode lineage tracing showed that endocrine-tolerant persistence occurred stochastically without genetic predisposition. Pharmacological inhibition of mitochondrial complex I suppressed the tumor-forming potential of persisters in mice and synergized with the anti-estrogen fulvestrant to induce regression of patient-derived xenografts. These findings indicate that mitochondrial metabolism is essential in endocrine-tolerant persister ER+ breast cancer cells and warrant the development of treatment strategies to leverage this vulnerability for treating breast cancer.