ROR2 regulates cellular plasticity in pancreatic neoplasia and adenocarcinoma.

Cellular plasticity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) starting from the conversion of normal cells into precancerous lesions to the progression of carcinoma subtypes associated with aggressiveness and therapeutic response. We discovered that normal acinar cell differentiation, maintained by the transcription factor Pdx1, suppresses a broad gastric cell identity that is maintained in metaplasia, neoplasia, and the classical subtype of PDAC in mouse and human. We have identified the receptor tyrosine kinase Ror2 as marker of a gastric metaplasia (SPEM)-like identity in the pancreas. Ablation of Ror2 in a mouse model of pancreatic tumorigenesis promoted a switch to a gastric pit cell identity that largely persisted through progression to the classical subtype of PDAC. In both human and mouse pancreatic cancer, ROR2 activity continued to antagonize the gastric pit cell identity, strongly promoting an epithelial to mesenchymal transition, conferring resistance to KRAS inhibition, and vulnerability to AKT inhibition.

A mucus production programme promotes classical pancreatic ductal adenocarcinoma.

OBJECTIVE: The optimal therapeutic response in cancer patients is highly dependent upon the differentiation state of their tumours. Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer that harbours distinct phenotypic subtypes with preferential sensitivities to standard therapies. This study aimed to investigate intratumour heterogeneity and plasticity of cancer cell states in PDA in order to reveal cell state-specific regulators. DESIGN: We analysed single-cell expression profiling of mouse PDAs, revealing intratumour heterogeneity and cell plasticity and identified pathways activated in the different cell states. We performed comparative analysis of murine and human expression states and confirmed their phenotypic diversity in specimens by immunolabeling. We assessed the function of phenotypic regulators using mouse models of PDA, organoids, cell lines and orthotopically grafted tumour models. RESULTS: Our expression analysis and immunolabeling analysis show that a mucus production programme regulated by the transcription factor SPDEF is highly active in precancerous lesions and the classical subtype of PDA - the most common differentiation state. SPDEF maintains the classical differentiation and supports PDA transformation in vivo. The SPDEF tumour-promoting function is mediated by its target genes AGR2 and ERN2/IRE1ß that regulate mucus production, and inactivation of the SPDEF programme impairs tumour growth and facilitates subtype interconversion from classical towards basal-like differentiation. CONCLUSIONS: Our findings expand our understanding of the transcriptional programmes active in precancerous lesions and PDAs of classical differentiation, determine the regulators of mucus production as specific vulnerabilities in these cell states and reveal phenotype switching as a response mechanism to inactivation of differentiation states determinants.

Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies against self-antigens.

Intratumoral B cell responses are associated with more favorable clinical outcomes in human pancreatic ductal adenocarcinoma (PDAC). However, the antigens driving these B cell responses are largely unknown. We sought to discover these antigens by using single-cell RNA sequencing (scRNA-Seq) and immunoglobulin (Ig) sequencing of tumor-infiltrating immune cells from 7 primary PDAC samples. We identified activated T and B cell responses and evidence of germinal center reactions. Ig sequencing identified plasma cell (PC) clones expressing isotype-switched and hypermutated Igs, suggesting the occurrence of T cell-dependent B cell responses. We assessed the reactivity of 41 recombinant antibodies that represented the products of 235 PCs and 12 B cells toward multiple cell lines and PDAC tissues and observed frequent staining of intracellular self-antigens. Three of these antigens were identified: the filamentous actin (F-actin), the nucleic protein RuvB like AAA ATPase 2 (RUVBL2), and the mitochondrial protein heat shock protein family D (Hsp60) member 1 (HSPD1). Antibody titers against F-actin and HSPD1 were substantially elevated in the plasma of patients with PDAC compared with healthy donors. Thus, PCs in PDAC produce autoantibodies reacting with intracellular self-antigens, which may result from promotion of preexisting, autoreactive B cell responses. These observations indicate the chronic inflammatory microenvironment of PDAC can support the adaptive immune response.

Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma.

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.

Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy.

Cystatin C (CyC), a secreted cysteine protease inhibitor, has unclear biological functions. Many patients exhibit elevated plasma CyC levels, particularly during glucocorticoid (GC) treatment. This study links GCs with CyC's systemic regulation by utilizing genome-wide association and structural equation modeling to determine CyC production genetics in the UK Biobank. Both CyC production and a polygenic score (PGS) capturing predisposition to CyC production were associated with increased all-cause and cancer-specific mortality. We found that the GC receptor directly targets CyC, leading to GC-responsive CyC secretion in macrophages and cancer cells. CyC-knockout tumors displayed significantly reduced growth and diminished recruitment of TREM2+ macrophages, which have been connected to cancer immunotherapy failure. Furthermore, the CyC-production PGS predicted checkpoint immunotherapy failure in 685 patients with metastatic cancer from combined clinical trial cohorts. In conclusion, CyC may act as a GC effector pathway via TREM2+ macrophage recruitment and may be a potential target for combination cancer immunotherapy.

Practical Considerations for Single-Cell Genomics.

The single-cell revolution in the field of genomics is in full bloom, with clever new molecular biology tricks appearing regularly that allow researchers to explore new modalities or scale up their projects to millions of cells and beyond. Techniques abound to measure RNA expression, DNA alterations, protein abundance, chromatin accessibility, and more, all with single-cell resolution and often in combination. Despite such a rapidly changing technology landscape, there are several fundamental principles that are applicable to the majority of experimental workflows to help users avoid pitfalls and exploit the advantages of the chosen platform. In this overview article, we describe a variety of popular single-cell genomics technologies and address some common questions pertaining to study design, sample preparation, quality control, and sequencing strategy. As the majority of relevant publications currently revolve around single-cell RNA-seq, we will prioritize this genomics modality in our discussion. © 2022 Wiley Periodicals LLC.

OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage.

Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.

Oral famotidine versus placebo in non-hospitalised patients with COVID-19: a randomised, double-blind, data-intense, phase 2 clinical trial.

OBJECTIVE: We assessed whether famotidine improved inflammation and symptomatic recovery in outpatients with mild to moderate COVID-19. DESIGN: Randomised, double-blind, placebo-controlled, fully remote, phase 2 clinical trial (NCT04724720) enrolling symptomatic unvaccinated adult outpatients with confirmed COVID-19 between January 2021 and April 2021 from two US centres. Patients self-administered 80 mg famotidine (n=28) or placebo (n=27) orally three times a day for 14 consecutive days. Endpoints were time to (primary) or rate of (secondary) symptom resolution, and resolution of inflammation (exploratory). RESULTS: Of 55 patients in the intention-to-treat group (median age 35 years (IQR: 20); 35 women (64%); 18 African American (33%); 14 Hispanic (26%)), 52 (95%) completed the trial, submitting 1358 electronic symptom surveys. Time to symptom resolution was not statistically improved (p=0.4). Rate of symptom resolution was improved for patients taking famotidine (p<0.0001). Estimated 50% reduction of overall baseline symptom scores were achieved at 8.2 days (95% CI: 7 to 9.8 days) for famotidine and 11.4 days (95% CI: 10.3 to 12.6 days) for placebo treated patients. Differences were independent of patient sex, race or ethnicity. Five self-limiting adverse events occurred (famotidine, n=2 (40%); placebo, n=3 (60%)). On day 7, fewer patients on famotidine had detectable interferon alpha plasma levels (p=0.04). Plasma immunoglobulin type G levels to SARS-CoV-2 nucleocapsid core protein were similar between both arms. CONCLUSIONS: Famotidine was safe and well tolerated in outpatients with mild to moderate COVID-19. Famotidine led to earlier resolution of symptoms and inflammation without reducing anti-SARS-CoV-2 immunity. Additional randomised trials are required.

Patient-Derived Triple-Negative Breast Cancer Organoids Provide Robust Model Systems That Recapitulate Tumor Intrinsic Characteristics.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with poor patient outcomes, highlighting the unmet clinical need for targeted therapies and better model systems. Here, we developed and comprehensively characterized a diverse biobank of normal and breast cancer patient-derived organoids (PDO) with a focus on TNBCs. PDOs recapitulated patient tumor intrinsic properties and a subset of PDOs can be propagated for long-term culture (LT-TNBC). Single cell profiling of PDOs identified cell types and gene candidates affiliated with different aspects of cancer progression. The LT-TNBC organoids exhibit signatures of aggressive MYC-driven, basal-like breast cancers and are largely comprised of luminal progenitor (LP)-like cells. The TNBC LP-like cells are distinct from normal LPs and exhibit hyperactivation of NOTCH and MYC signaling. Overall, this study validates TNBC PDOs as robust models for understanding breast cancer biology and progression, paving the way for personalized medicine and tailored treatment options. SIGNIFICANCE: A comprehensive analysis of patient-derived organoids of TNBC provides insights into cellular heterogeneity and mechanisms of tumorigenesis at the single-cell level.

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment. EXPERIMENTAL DESIGN: We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC. RESULTS: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression. CONCLUSIONS: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

ZBED2 is an antagonist of interferon regulatory factor 1 and modifies cell identity in pancreatic cancer.

Lineage plasticity is a prominent feature of pancreatic ductal adenocarcinoma (PDA) cells, which can occur via deregulation of lineage-specifying transcription factors. Here, we show that the zinc finger protein ZBED2 is aberrantly expressed in PDA and alters tumor cell identity in this disease. Unexpectedly, our epigenomic experiments reveal that ZBED2 is a sequence-specific transcriptional repressor of IFN-stimulated genes, which occurs through antagonism of IFN regulatory factor 1 (IRF1)-mediated transcriptional activation at cooccupied promoter elements. Consequently, ZBED2 attenuates the transcriptional output and growth arrest phenotypes downstream of IFN signaling in multiple PDA cell line models. We also found that ZBED2 is preferentially expressed in the squamous molecular subtype of human PDA, in association with inferior patient survival outcomes. Consistent with this observation, we show that ZBED2 can repress the pancreatic progenitor transcriptional program, enhance motility, and promote invasion in PDA cells. Collectively, our findings suggest that high ZBED2 expression is acquired during PDA progression to suppress the IFN response pathway and to promote lineage plasticity in this disease.

Squamous trans-differentiation of pancreatic cancer cells promotes stromal inflammation.

A highly aggressive subset of pancreatic ductal adenocarcinomas undergo trans-differentiation into the squamous lineage during disease progression. Here, we investigated whether squamous trans-differentiation of human and mouse pancreatic cancer cells can influence the phenotype of non-neoplastic cells in the tumor microenvironment. Conditioned media experiments revealed that squamous pancreatic cancer cells secrete factors that recruit neutrophils and convert pancreatic stellate cells into cancer-associated fibroblasts (CAFs) that express inflammatory cytokines at high levels. We use gain- and loss-of-function approaches to show that squamous-subtype pancreatic tumor models become enriched with neutrophils and inflammatory CAFs in a p63-dependent manner. These effects occur, at least in part, through p63-mediated activation of enhancers at pro-inflammatory cytokine loci, which includes IL1A and CXCL1 as key targets. Taken together, our findings reveal enhanced tissue inflammation as a consequence of squamous trans-differentiation in pancreatic cancer, thus highlighting an instructive role of tumor cell lineage in reprogramming the stromal microenvironment.

Bayesian Inference of Allelic Inclusion Rates in the Human T Cell Receptor Repertoire.

A small population of αß T cells is characterized by the expression of more than one unique T cell receptor (TCR); this outcome is the result of "allelic inclusion," that is, inclusion of both α- or ß-chain alleles during V(D)J recombination. Limitations in single-cell sequencing technology, however, have precluded comprehensive enumeration of these dual receptor T cells. Here, we develop and experimentally validate a fully Bayesian inference model capable of reliably estimating the true rate of α and ß TCR allelic inclusion across two different emulsion-barcoding single-cell sequencing platforms. We provide a database composed of over 51,000 previously unpublished allelic inclusion TCR sequence sets drawn from eight healthy individuals and show that allelic inclusion contributes a distinct and functionally important set of sequences to the human TCR repertoire. This database and a Python implementation of our statistical inference model are freely available at our Github repository (https://github.com/JasonACarter/Allelic_inclusion).

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.

Single T Cell Sequencing Demonstrates the Functional Role of αß TCR Pairing in Cell Lineage and Antigen Specificity.

Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and ß chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the ß chain alone. To determine the amount of useful information about TCR repertoire function encoded within αß pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and ß chains are only weakly associated with lineage, αß pairings appear to synergistically drive TCR-MHC interactions. Vαß gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αß TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and ß chains that each recognize different known antigens, suggesting that αß pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αß pairing and highlight the utility of single-cell sequencing in immunogenomics.

Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population "antigen-presenting CAFs" and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II-expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors.See related commentary by Belle and DeNardo, p. 1001.This article is highlighted in the In This Issue feature, p. 983.

IL1-Induced JAK/STAT Signaling Is Antagonized by TGFß to Shape CAF Heterogeneity in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is poorly responsive to therapies and histologically contains a paucity of neoplastic cells embedded within a dense desmoplastic stroma. Within the stroma, cancer-associated fibroblasts (CAF) secrete tropic factors and extracellular matrix components, and have been implicated in PDAC progression and chemotherapy resistance. We recently identified two distinct CAF subtypes characterized by either myofibroblastic or inflammatory phenotypes; however, the mechanisms underlying their diversity and their roles in PDAC remain unknown. Here, we use organoid and mouse models to identify TGFß and IL1 as tumor-secreted ligands that promote CAF heterogeneity. We show that IL1 induces LIF expression and downstream JAK/STAT activation to generate inflammatory CAFs and demonstrate that TGFß antagonizes this process by downregulating IL1R1 expression and promoting differentiation into myofibroblasts. Our results provide a mechanism through which distinct fibroblast niches are established in the PDAC microenvironment and illuminate strategies to selectively target CAFs that support tumor growth. SIGNIFICANCE: Understanding the mechanisms that determine CAF heterogeneity in PDAC is a prerequisite for the rational development of approaches that selectively target tumor-promoting CAFs. Here, we identify an IL1-induced signaling cascade that leads to JAK/STAT activation and promotes an inflammatory CAF state, suggesting multiple strategies to target these cells in vivo. See related commentary by Ling and Chiao, p. 173. This article is highlighted in the In This Issue feature, p. 151.

Regulation of Ribosome Biogenesis and Protein Synthesis Controls Germline Stem Cell Differentiation.

Complex regulatory networks regulate stem cell behavior and contributions to tissue growth, repair, and homeostasis. A full understanding of the networks controlling stem cell self-renewal and differentiation, however, has not yet been realized. To systematically dissect these networks and identify their components, we performed an unbiased, transcriptome-wide in vivo RNAi screen in female Drosophila germline stem cells (GSCs). Based on characterized cellular defects, we classified 646 identified genes into phenotypic and functional groups and unveiled a comprehensive set of networks regulating GSC maintenance, survival, and differentiation. This analysis revealed an unexpected role for ribosomal assembly factors in controlling stem cell cytokinesis. Moreover, our data show that the transition from self-renewal to differentiation relies on enhanced ribosome biogenesis accompanied by increased protein synthesis. Collectively, these results detail the extensive genetic networks that control stem cell homeostasis and highlight the intricate regulation of protein synthesis during differentiation.

Panoramix enforces piRNA-dependent cotranscriptional silencing.

The Piwi-interacting RNA (piRNA) pathway is a small RNA-based innate immune system that defends germ cell genomes against transposons. In Drosophila ovaries, the nuclear Piwi protein is required for transcriptional silencing of transposons, though the precise mechanisms by which this occurs are unknown. Here we show that the CG9754 protein is a component of Piwi complexes that functions downstream of Piwi and its binding partner, Asterix, in transcriptional silencing. Enforced tethering of CG9754 to nascent messenger RNA transcripts causes cotranscriptional silencing of the source locus and the deposition of repressive chromatin marks. We have named CG9754 "Panoramix," and we propose that this protein could act as an adaptor, scaffolding interactions between the piRNA pathway and the general silencing machinery that it recruits to enforce transcriptional repression.

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation.

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation.