Lineage Tracing Research Articles

Evidence for a lipofibroblast-to- Cthrc1 + myofibroblast reversible switch during the development and resolution of lung fibrosis in young mice.

Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease (ILD) to which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken center stage as these cells are believed to drive structural remodeling and lung function impairment. While aMYFs are now widely believed to originate from resident fibroblasts, the heterogeneity of aMYFs and ultimate fate during fibrosis resolution remain elusive. We have previously shown that aMYFs dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution. In this study, we combined genetic lineage tracing and single-cell transcriptomics in mice, and data mining of human IPF datasets to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis resolution. Furthermore, organoid cultures were utilized as a functional readout for the alveolar mesenchymal niche activity during various phases of injury and repair in mice. Our data demonstrate that aMYFs consist of four subclusters displaying unique pro-alveologenic versus profibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrogenesis and resolution respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature. Our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

Lineage tracing of Shh+ floor plate cells and dynamics of dorsal-ventral gene expression in the regenerating axolotl spinal cord.

Both development and regeneration depend on signaling centers, which are sources of locally secreted tissue-patterning molecules. As many signaling centers are decommissioned before the end of embryogenesis, a fundamental question is how signaling centers can be re-induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration. The floor plate is an archetypal vertebrate signaling center that secretes Shh ligand and patterns neural progenitor cells during embryogenesis. Unlike mammals, axolotls continue to express floor plate genes (including Shh) and downstream dorsal-ventral patterning genes in their spinal cord throughout life, including at steady state. The parsimonious hypothesis that Shh+ cells give rise to functional floor plate cells for regeneration had not been tested. Using HCR in situ hybridization and mathematical modeling, we first quantified the behaviors of dorsal-ventral spinal cord domains, identifying significant increases in gene expression level and floor plate size during regeneration. Next, we established a transgenic axolotl to specifically label and fate map Shh+ cells in vivo. We found that labeled Shh+ cells gave rise to regeneration floor plate, and not to other neural progenitor domains, after tail amputation. Thus, despite changes in domain size and downstream patterning gene expression, Shh+ cells retain their floor plate identity during regeneration, acting as a stable cellular source for this regeneration signaling center in the axolotl spinal cord.

Loss of PI5P4Kα slows the progression of a Pten mutant basal cell model of prostate cancer.

While early prostate cancer (PCa) depends on the androgen receptor (AR) signaling pathway, which is predominant in luminal cells, there is much to be understood about the contribution of epithelial basal cells in cancer progression. Herein, we observe cell-type specific differences in the importance of the metabolic enzyme phosphatidylinositol 5-phosphate 4-kinase alpha (PI5P4Kα β ; gene name PIP4K2A) in the prostate epithelium. We report the development of a basal-cell-specific genetically engineered mouse model (GEMM) targeting Pip4k2a alone or in combination with the tumor suppressor phosphatase and tensin homolog (Pten). PI5P4Kα is enriched in basal cells, and no major histopathological changes were detectable following gene deletion. Notably, the combined loss of Pip4k2a slowed the development of Pten mutant mouse prostatic intraepithelial neoplasia (mPIN). Through the inclusion of a lineage tracing reporter, we utilize single-cell RNA sequencing to evaluate changes resulting from in vivo downregulation of Pip4k2a and characterize cell populations influenced in the established Probasin-Cre and Cytokeratin 5 (CK5)-Cre driven GEMMs. Transcriptomic pathway analysis points towards the disruption of lipid metabolism as a mechanism for reduced tumor progression. This was functionally supported by shifts of carnitine lipids in LNCaP PCa cells treated with siPIP4K2A. Overall, these data nominate PI5P4Kα as a target for PTEN mutant PCa. Implications: PI5P4Kα is enriched in prostate basal cells and its targeted loss slows the progression of a model of advanced PCa.

8235 Mutations in EMX2, a Homeodomain Transcription Factor, Cause Idiopathic Hypogonadotropic Hypogonadism

Abstract Disclosure: M. Stamou: None. M. Tompkin: None. H. Bow: None. H. Brand: None. L. Plummer: None. M. Talkowski: None. Y. Shen: None. D. Wu: None. R. Balasubramanian: None. S. Wray: None. S.B. Seminara: None. The genetic etiology of infertility remains largely unknown. To identify novel human infertility genes, we applied a de novo rare variant analysis in 142 parent-proband trios with idiopathic hypogonadotropic hypogonadism (IHH), a Mendelian disease caused by Gonadotropin Releasing Hormone (GnRH) deficiency. The discovery pipeline identified 43 rare de novo copy number variants (CNVs-deletions) spanning 263 genes and 213 rare de novo single nucleotide variants (SNVs) in 191 genes. Four genes were found to be affected by both rare de novo CNVs and SNVs among unrelated sporadic IHH probands. A rare variant association testing for protein truncating variants (PTVs) in the IHH cohort (N=1,394) compared to gnomAD controls (N=125,784) revealed that 3/4 genes demonstrated enrichment for PTVs in the IHH cohort compared to controls: the 2 known for IHH genes (ANOS1 and FGFR1) and one novel candidate gene, EMX2. IHH probands with de novo EMX2 variants demonstrated developmental delay and hearing loss. Common EMX2 variants were linked to delayed puberty/infertility, hearing loss and Parkinson’s disease in a phenome-wide association study of the Massachusetts General Brigham Biobank (N=65,253). Emx2 lineage tracing was conducted on Emx2Cre/+; RosatdT/+ and Emx2Cre/-; RosatdT/+ mouse embryos at embryonic day(E) 13.5. The tdT reporter was co-expressed in neuroendocrine GnRH (nGnRH) cells as they migrated from the olfactory pit to the nasal forebrain junction. WT and KO embryos from Emx2+/- matings were immunostained for GnRH across E12.5, E13.5, E14.5 and E16. The total number of nGnRH cells was greater in the Emx2 KO mice compared to WT littermates. However, by E16.5, fewer nGnRH cells were detected in the forebrain of Emx2 KO, compared to WT (WT= 496±28, KO=384±147, p=0.0125). Thus, failure of nGnRH neurons to enter the forebrain could contribute to the IHH pathogenesis. In conclusion, by utilizing a de novo variant analysis and a mouse model, EMX2 was uncovered as a novel gene for human infertility. Presentation: 6/1/2024

8515 Role of Insulin Receptor in Trpv1-positive Adipocyte Progenitor Cellsin Adipocyte Differentiation and Metabolic Function

Abstract Disclosure: C. Sahin: None. H. Camara: None. F. Shamsi: None. M. Lynes: None. Y. Tseng: None. Brown adipose tissue (BAT), a specialized fat that dissipates energy to produce heat, is an important endocrine organ in the regulation of energy balance. Stimulating BAT activation and beiging of white adipose tissue (WAT) are new strategies to combat metabolic diseases, such as obesity and diabetes. Traditionally, thermogenic adipocytes are known to originate from mesenchymal Pdgfrα-expressing adipocyte progenitor cells (APCs). A previous study conducted by our lab identified vascular smooth muscle cells as a novel cellular origin of APCs. These cells are characterized by the expression of transient receptor potential cation channel subfamily V member 1 (Trpv1+). With cold exposure, the Trpv1+ APC-derived adipocytes are increased in BAT and WAT and differentiated into highly thermogenic adipocytes. These findings indicate that the Trpv1+-APCs may play an important role in the development of brown and beige adipocytes in response to cold. This study was designed to explore the function of the Trpv1+ APC-derived adipocytes in thermogenic adipocyte differentiation and metabolic regulation. We generated a mouse model in which insulin receptor (IR), a well-known player in adipocyte differentiation, was deleted in the Trpv1+ APCs to impair their adipogenic potential. Trpv1-Cre::mTmG::IRflox transgenic and IRwt control mice were housed at cold temperature (5°C) for 7 days. Because the lineage tracing mTmG system was included, we were able to trace the fate of Trpv1+ APCs in the presence or absence of IR. We first examined the adipogenic capacity of the IR-deficient Trpv1+ cells in BAT and WAT tissues using whole-mounted adipose tissue imaging. In control mice, approximately 30% of all mature adipocytes were derived from Trpv1+ APCs in adipose tissue in response to cold. With ablation of IR in Trpv1+ APCs, this contribution was significantly reduced in BAT (by 11.7 % in females; 6.1% in males), inguinal WAT (by 16.8% in females; 19.3% males), and perigonadal WAT (by 11.4% in females; 14.8% in males) in transgenic mice compared to control mice. These results showed that IR-deficiency in the Trpv1-expressing cells impaired their adipogenesis in both male and female mice. In contrast, thermogenic genes (e.g., Ucp1, Pparγc1a, Cox7a1) were not significantly altered in BAT and inguinal WAT of the IR-deficient Trpv1+ APCs mice. Despite the changes in the adipocyte populations, we observed that deletion of IR in Trpv1+ APCs has no impact on body weight, food intake, fat mass, glucose tolerance, and insulin sensitivity relative to control mice. Taken together, blocking the contribution of Trpv1+ APCs to the mature adipocyte population results in no metabolic changes in mice, suggesting that the other APCs, such as PdgfRα+ APCs, may potentially compensate for impaired adipogenesis in different adipose tissues. Presentation: 6/1/2024

8235 Mutations in EMX2, a Homeodomain Transcription Factor, Cause Idiopathic Hypogonadotropic Hypogonadism

Abstract Disclosure: M. Stamou: None. M. Tompkin: None. H. Bow: None. H. Brand: None. L. Plummer: None. M. Talkowski: None. Y. Shen: None. D. Wu: None. R. Balasubramanian: None. S. Wray: None. S.B. Seminara: None. The genetic etiology of infertility remains largely unknown. To identify novel human infertility genes, we applied a de novo rare variant analysis in 142 parent-proband trios with idiopathic hypogonadotropic hypogonadism (IHH), a Mendelian disease caused by Gonadotropin Releasing Hormone (GnRH) deficiency. The discovery pipeline identified 43 rare de novo copy number variants (CNVs-deletions) spanning 263 genes and 213 rare de novo single nucleotide variants (SNVs) in 191 genes. Four genes were found to be affected by both rare de novo CNVs and SNVs among unrelated sporadic IHH probands. A rare variant association testing for protein truncating variants (PTVs) in the IHH cohort (N=1,394) compared to gnomAD controls (N=125,784) revealed that 3/4 genes demonstrated enrichment for PTVs in the IHH cohort compared to controls: the 2 known for IHH genes (ANOS1 and FGFR1) and one novel candidate gene, EMX2. IHH probands with de novo EMX2 variants demonstrated developmental delay and hearing loss. Common EMX2 variants were linked to delayed puberty/infertility, hearing loss and Parkinson’s disease in a phenome-wide association study of the Massachusetts General Brigham Biobank (N=65,253). Emx2 lineage tracing was conducted on Emx2Cre/+; RosatdT/+ and Emx2Cre/-; RosatdT/+ mouse embryos at embryonic day(E) 13.5. The tdT reporter was co-expressed in neuroendocrine GnRH (nGnRH) cells as they migrated from the olfactory pit to the nasal forebrain junction. WT and KO embryos from Emx2+/- matings were immunostained for GnRH across E12.5, E13.5, E14.5 and E16. The total number of nGnRH cells was greater in the Emx2 KO mice compared to WT littermates. However, by E16.5, fewer nGnRH cells were detected in the forebrain of Emx2 KO, compared to WT (WT= 496±28, KO=384±147, p=0.0125). Thus, failure of nGnRH neurons to enter the forebrain could contribute to the IHH pathogenesis. In conclusion, by utilizing a de novo variant analysis and a mouse model, EMX2 was uncovered as a novel gene for human infertility. Presentation: 6/1/2024

12439 Investigating The Regulation Of Islet Alpha-cell Function Mediated By The ISL1 And LDB1 Transcriptional Complex

Abstract Disclosure: I. Deeba: None. S. Poole: None. Y. Liu: None. C. Hunter: None. Glucose homeostasis is maintained by the action of insulin-secreting β-cells and glucagon-producing α-cells in the pancreatic islet. Diabetes, traditionally considered a dysglycemia centered on the β-cell, also has clear contributions by dysregulated α-cells. However, little is known of the key transcriptional regulators, including transcription factors (TFs) and interacting co-regulators, driving α-cell development and function. A published pancreas-wide knockout (KO) of the LIM-Homeodomain Islet-1 (ISL1) TF in mice led to a dominant β-cell phenotype, including glucose intolerance and reduced insulin secretion. However, significant reductions of glucagon (Gcg) and Arx mRNA and pancreatic GCG content were also noted, suggesting α-cell impacts. Furthermore, we demonstrated that pancreatic ISL1 activity involves interactions with the LDB1 scaffolding co-regulator. However, nothing is known of the comparative α-cell-specific target genes and functions of these protein partners. To first assess ISL1:LDB1 complexes in α-cells, we performed co-IP and proximity ligation assay (PLA) in mouse α-cell lines and found low-glucose stimulated ISL1 and LDB1 interactions. These observations led us to hypothesize that ISL1 and LDB1 complexes regulate transcription of target genes to drive α-cell function. To assess comparative DNA occupancy, we performed ChIP and observed significant ISL1 and LDB1 binding in conserved Gcg promoter and intronic domains. siRNA-mediated Isl1 or Ldb1 knockdown followed by RNA analysis revealed a similar differential expression of α-cell mRNAs (e.g., Gcg, IL6ra, Sox8, Cox5a, Atp5k, Atp5e), suggesting roles in α-cell function. To compare ISL1 and LDB1 importance in vivo, we developed two novel mouse α-cell ISL1 or LDB1 KO models using Gcg-Cre, termed Isl1Δα and Ldb1Δα, respectively. ISL1 and LDB1 loss was confirmed in α-cells by immunofluorescence. Significant fasting hypoglycemia and hypoglucagonemia were found in postnatal Isl1Δα and Ldb1Δα mice, along with decreased GCG+ cell numbers in Ldb1Δα mice, compared to controls. These findings underscore the crucial roles of these factors in regulating GCG production and secretion. Immunostaining for the MAFB α-cell TF in Ldb1Δα islets revealed potential heterogeneous MAFB expression, marked by MAFB+ cells lacking GCG, or co-expressing INS, each indicating a potential loss of α-cell identity. Our lineage tracing analysis revealed α-cell lineage labeled tomato+ cells in Isl1Δα mice appearing to co-express INS, suggesting transdifferentiation. Currently, inducible α-cell-specific KO mice are in development to investigate the role of ISL1 and LDB1 in adult α-cells. In the future, we will also explore the effects of α-cell-specific ISL1 or LDB1 deficiency during embryogenesis. Overall, our study will benefit efforts in identifying new molecular targets and cell-based therapies to combat diabetes. Presentation: 6/1/2024

Transcription factor KROX20 marks epithelial stem cells for hair follicle formation.

Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), telogen (quiescence), and catagen (regression). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor Krox20 is enriched in an epidermal stem cell population located in the upper/ middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20-lineage cells migrate out of this HF region and contribute to the formation of bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a novel marker for an epidermal stem cell population that is indispensable for hair homeostasis.

Periderm fate and independence of tooth formation are conserved across osteichthyans

BackgroundPrevious studies have reported that periderm (the outer ectodermal layer) in zebrafish partially expands into the mouth and pharyngeal pouches, but does not reach the medial endoderm, where the pharyngeal teeth develop. Instead, periderm-like cells, arising independently from the outer periderm, cover prospective tooth-forming epithelia and are crucial for tooth germ initiation. Here we test the hypothesis that restricted expansion of periderm is a teleost-specific character possibly related to the derived way of early embryonic development. To this end, we performed lineage tracing of the periderm in a non-teleost actinopterygian species possessing pharyngeal teeth, the sterlet sturgeon (Acipenser ruthenus), and a sarcopterygian species lacking pharyngeal teeth, the axolotl (Ambystoma mexicanum).ResultsIn sturgeon, a stratified ectoderm is firmly established at the end of gastrulation, with minimally a basal ectodermal layer and a surface layer that can be homologized to a periderm. Periderm expands to a limited extent into the mouth and remains restricted to the distal parts of the pouches. It does not reach the medial pharyngeal endoderm, where pharyngeal teeth are located. Thus, periderm in sturgeon covers prospective odontogenic epithelium in the jaw region (oral teeth) but not in the pharyngeal region. In axolotl, like in sturgeon, periderm expansion in the oropharynx is restricted to the distal parts of the opening pouches. Oral teeth in axolotl develop long before mouth opening and possible expansion of the periderm into the mouth cavity.ConclusionsRestricted periderm expansion into the oropharynx appears to be an ancestral feature for osteichthyans, as it is found in sturgeon, zebrafish and axolotl. Periderm behavior does not correlate with presence or absence of oral or pharyngeal teeth, whose induction may depend on ‘ectodermalized’ endoderm. It is proposed that periderm assists in lumenization of the pouches to create an open gill slit. Comparison of basal and advanced actinopterygians with sarcopterygians (axolotl) shows that different trajectories of embryonic development converge on similar dynamics of the periderm: a restricted expansion into the mouth and prospective gill slits.

Murine parainfluenza virus persists in lung innate immune cells sustaining chronic lung pathology.

Common respiratory viruses, including the human parainfluenza viruses, threaten human health seasonally and associate with the development of chronic lung diseases. Evidence suggests that these viruses can persist, but the sources of viral products in vivo and their impact on chronic respiratory diseases remain unknown. Using the murine parainfluenza virus Sendai, we demonstrate that viral protein and RNA persist in lung macrophages, type 2 innate lymphoid cells (ILC2s) and dendritic cells long after the infectious virus is cleared. Cells containing persistent viral protein expressed Th2 inflammation-related transcriptomic signatures associated with the development of chronic lung diseases, including asthma. Lineage tracing demonstrated that distinct functional groups of cells contribute to the chronic pathology. Importantly, targeted ablation of infected cells significantly ameliorated chronic lung disease. Overall, we identified persistent infection of innate immune cells as a key factor in the progression from acute to chronic lung disease after infection with parainfluenza virus.

LineageVAE: reconstructing historical cell states and transcriptomes toward unobserved progenitors.

Single-cell RNA sequencing (scRNA-seq) enables comprehensive characterization of the cell state. However, its destructive nature prohibits measuring gene expression changes during dynamic processes such as embryogenesis or cell state divergence due to injury or disease. Although recent studies integrating scRNA-seq with lineage tracing have provided clonal insights between progenitor and mature cells, challenges remain. Because of their experimental nature, observations are sparse, and cells observed in the early state are not the exact progenitors of cells observed at later time points. To overcome these limitations, we developed LineageVAE, a novel computational methodology that utilizes deep learning based on the property that cells sharing barcodes have identical progenitors. LineageVAE is a deep generative model that transforms scRNA-seq observations with identical lineage barcodes into sequential trajectories toward a common progenitor in a latent cell state space. This method enables the reconstruction of unobservable cell state transitions, historical transcriptomes, and regulatory dynamics at a single-cell resolution. Applied to hematopoiesis and reprogrammed fibroblast datasets, LineageVAE demonstrated its ability to restore backward cell state transitions and infer progenitor heterogeneity and transcription factor activity along differentiation trajectories. The LineageVAE model was implemented in Python using the PyTorch deep learning library. The code is available on GitHub at https://github.com/LzrRacer/LineageVAE/.

Cell-cell contact-dependent secretion of large-extracellular vesicles from EFNBhigh cancer cells accelerates peritoneal dissemination.

Large non-apoptotic vesicles released from the plasma membrane protrusions are classified as large-EVs (LEVs). However, the triggers of LEV secretion and their functions in tumors remain unknown. Coculture system of cancer cells, peritoneal mesothelial cells (PMCs), and macrophages (MΦs) was conducted to observe cell-cell contact-mediated LEV secretion. Lineage tracing of PMCs was performed using Wt1CreERT2-tdTnu mice to explore the effects of LEVs on PMCs in vivo, and lymphangiogenesis was assessed by qRT-PCR and flow-cytometry. In peritoneal dissemination, cancer cells expressing Ephrin-B (EFNB) secreted LEVs upon the contact with PMCs expressing ephrin type-B (EphB) receptors, which degraded mesothelial barrier by augmenting mesothelial-mesenchymal transition. LEVs were incorporated in subpleural MΦs, and these MΦs transdifferentiated into lymphatic endothelial cells (LEC) and integrated into the lymphatic vessels. LEC differentiation was also induced in PMCs by interacting with LEV-treated MΦs, which promoted lymphangiogenesis. Mechanistically, activation of RhoA-ROCK pathway through EFNB reverse signaling induced LEV secretion. EFNBs on LEVs activated EphB forward signaling in PMC and MΦs, activating Akt, ERK and TGF-β1 pathway, which were indispensable for causing MMT and LEC differentiation. LEVs accelerated peritoneal dissemination and lymphatic invasions by cancer cells. Blocking of EFNBs on LEVs using EphB-Fc-fusion protein attenuated these events. EFNBhigh cancer cells scattered LEVs when they attached to PMCs, which augmented the local reactions of PMC and MΦ (MMT and lymphangiogenesis) and exaggerated peritoneal dissemination.

Mitochondrial genetic variations in leukemia: a comprehensive overview.

Leukemias are a group of heterogeneous hematological malignancies driven by diverse genetic variations, and the advent of genomic sequencing technologies facilitates the investigation of genetic abnormalities in leukemia. However, these sequencing-based studies mainly focus on nuclear DNAs. Increasing evidence indicates that mitochondrial dysfunction is an important mechanism of leukemia pathogenesis, which is closely related to the mitochondrial genome variations. Here, we provide an overview of current research progress concerning mitochondrial genetic variations in leukemia, encompassing gene mutations and copy number variations. We also summarize currently accessible mitochondrial DNA (mtDNA) sequencing methods. Notably, somatic mtDNA mutations may serve as natural genetic barcodes for lineage tracing and longitudinal assessment of clonal dynamics. Collectively, these findings enhance our understanding of leukemia pathogenesis and foster the identification of novel therapeutic targets and interventions.

P53 mutation biases squamocolumnar junction progenitor cells towards dysplasia rather than metaplasia in Barrett’s oesophagus

BackgroundWhile p53 mutations occur early in Barrett’s oesophagus (BE) progression to oesophageal adenocarcinoma (EAC), their role in gastric cardia stem cells remains unclear.ObjectiveThis study investigates the impact of p53 mutation...

New insights into the endothelial origin of hematopoietic system inspired by "TIF" approaches.

Hematopoietic stem progenitor cells (HSPCs) are derived from a specialized subset of endothelial cells named hemogenic endothelial cells (HECs) via a process of endothelial-to-hematopoietic transition during embryogenesis. Recently, with the usage of multiple single-cell technologies and advanced genetic lineage tracing techniques, namely, "TIF" approaches that combining transcriptome, immunophenotype and function/fate analyses, massive new insights have been achieved regarding the cellular and molecular evolution underlying the emergence of HSPCs from embryonic vascular beds. In this review, we focus on the most recent advances in the enrichment markers, functional characteristics, developmental paths, molecular controls, and the embryonic site-relevance of the key intermediate cell populations bridging embryonic vascular and hematopoietic systems, namely HECs and pre-hematopoietic stem cells, the immediate progenies of some HECs, in mouse and human embryos. Specifically, using expression analyses at both transcriptional and protein levels and especially efficient functional assays, we propose that the onset of Kit expression is at the HEC stage, which has previously been controversial.

Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease

The contribution of progenitor subtypes to generating the billions of neurons produced during human cortical neurogenesis is not well understood. We developed the cortical organoid lineage-tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all excitatory neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic CTNNB1 variant demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play roles in generating the diverse neurons of the human cerebral cortex.

MLKL-mediated endothelial necroptosis drives vascular damage and mortality in systemic inflammatory response syndrome.

The hypersecretion of cytokines triggers life-threatening systemic inflammatory response syndrome (SIRS), leading to multiple organ dysfunction syndrome (MODS) and mortality. Although both coagulopathy and necroptosis have been identified as important factors in the pathogenesis of SIRS, the specific cell types that undergo necroptosis and the interrelationships between coagulopathy and necroptosis remain unclear. In this study, we utilized visualization analysis via intravital microscopy to demonstrate that both anticoagulant heparin and nonanticoagulant heparin (NAH) pretreatment protect mice against TNF-α-induced mortality in SIRS. Moreover, the deletion of Mlkl or Ripk3 resulted in decreased coagulation and reduced mortality in TNF-α-induced SIRS. These findings suggest that necroptosis plays a key role upstream of coagulation in SIRS-related mortality. Furthermore, using a genetic lineage tracing mouse model (Tie2-Cre;Rosa26-tdT), we tracked endothelial cells (ECs) and verified that EC necroptosis is responsible for the vascular damage observed in TNF-α-treated mice. Importantly, Mlkl deletion in vascular ECs in mice had a similar protective effect against lethal SIRS by blocking EC necroptosis to protect the integrity of the endothelium. Collectively, our findings demonstrated that RIPK3-MLKL-dependent necroptosis disrupted vascular integrity, resulting in coagulopathy and multiorgan failure, eventually leading to mortality in SIRS patients. These results highlight the importance of targeting vascular EC necroptosis for the development of effective treatments for SIRS patients.

Tissue resident memory CD4 + T cells are sustained by site-specific levels of self-renewal and replacement from precursors.

Tissue-resident memory T cells (T RM ) protect from repeated infections within organs and barrier sites. The breadth and duration of such protection is defined at minimum by three quantities; the rate at which new T RM are generated from precursors, their rate of self-renewal, and their loss rate through death, egress, or differentiation. Quantifying these processes in isolation is challenging. Here we combine genetic fate mapping tools and mathematical models to untangle these basic homeostatic properties of CD4 + T RM in skin and lamina propria (LP) of healthy adult mice. We show that CD69 + CD4 + T RM in skin reside for ∼ 24 days and self-renew more slowly, such that clones halve in size approximately every 5 weeks; and approximately 2% of cells are replaced daily from precursors. CD69 + CD4 + T RM in LP have shorter residencies ( ∼ 14 days) and are maintained largely by immigration (4-6% per day). We also find evidence that the constitutive replacement of CD69 + CD4 + T RM at both sites derives from circulating effectormemory CD4 + T cells, in skin possibly via a CD69 - intermediate. Our integrated approach maps the ontogeny of CD4 + T RM in skin and LP and exposes their dynamic and distinct behaviours, with continual seeding and erosion potentially impacting the duration of immunity at these sites.

Defining heritability, plasticity, and transition dynamics of cellular phenotypes in somatic evolution.

Single-cell sequencing has characterized cell state heterogeneity across diverse healthy and malignant tissues. However, the plasticity or heritability of these cell states remains largely unknown. To address this, we introduce PATH (phylogenetic analysis of trait heritability), a framework to quantify cell state heritability versus plasticity and infer cell state transition and proliferation dynamics from single-cell lineage tracing data. Applying PATH to a mouse model of pancreatic cancer, we observed heritability at the ends of the epithelial-to-mesenchymal transition spectrum, with higher plasticity at more intermediate states. In primary glioblastoma, we identified bidirectional transitions between stem- and mesenchymal-like cells, which use the astrocyte-like state as an intermediary. Finally, we reconstructed a phylogeny from single-cell whole-genome sequencing in B cell acute lymphoblastic leukemia and delineated the heritability of B cell differentiation states linked with genetic drivers. Altogether, PATH replaces qualitative conceptions of plasticity with quantitative measures, offering a framework to study somatic evolution.

Spatiotemporal Analysis of Mesenchymal Stem Cells Fate Determination by Inflammatory Niche Following Soft Tissue Injury at a Single‐Cell Level

AbstractHeterotopic ossification (HO), often arising in response to traumatic challenges, results from the aberrant osteochondral differentiation of mesenchymal stem cells (MSCs). Nevertheless, the impact of trauma‐induced inflammatory exposure on MSC fate determination remains ambiguous. In this study, the cellular diversity within inflammatory lesions is elucidated, comprising MSCs and several innate and adaptive immune cells. It is observed that quiescent MSCs transition into cycling MSCs, subsequently giving rise to chondrogenic (cMSC) and/or osteogenic (oMSC) lineages within the inflammatory microenvironment following muscle or tendon injuries, as revealed through single‐cell RNA sequencing (scRNA‐seq), spatial transcriptome and lineage tracing analysis. Moreover, these investigations demonstrate that neutrophils and natural killer (NK) cells enhance transition of quiescent MSCs into cycling MSCs, which is also controlled by M1 macrophages, a subpopulation of macrophages can also stimulate cMSC and oMSC production from cycling MSCs. Additionally, M2 macrophages, CD4+ and CD8+ T lymphocytes are found to promote chondrogenesis. Further analysis demonstrates that immune cells promotes the activation of signaling transducers and activators of transcription (STAT) pathway and phosphoinositide 3 (PI3K)/protein kinase B (AKT) pathway in MSC proliferation and osteochondral progenitors’ production, respectively. These findings highlight the dynamics of MSC fate within the inflammatory lesion and unveil the molecular landscape of osteoimmunological interactions, which holds promise for advancing HO treatment.