ABSTRACT
BACKGROUND: In esophageal cancer (EC), there is a paucity of knowledge regarding the interplay between the tumor immune microenvironment and response to neoadjuvant treatment and, therefore, which factors may influence outcomes. Thus, our goal was to investigate the changes in the immune microenvironment with neoadjuvant treatment in EC by assessing the expression of immune related genes and their association with prognosis. METHODS: We examined the transcriptome of paired pre- and post-neoadjuvant treated EC specimens. Based on these findings, we validated the presence of tumor-infiltrating neutrophils using CD15+ immunohistochemistry in a discovery cohort of patients with residual pathologic disease. We developed a nomogram as a predictor of progression-free survival (PFS) incorporating the variables CD15+ cell count, tumor regression grade, and tumor grade. RESULTS: After neoadjuvant treatment, there was an increase in genes related to myeloid cell differentiation and a poor prognosis associated with high neutrophil (CD15+) counts. Our nomogram incorporating CD15+ cell count was predictive of PFS with a C-index of 0.80 (95% confidence interval [CI] 0.68-0.9) and a concordance probability estimate (CPE) of 0.77 (95% CI 0.69-0.86), which indicates high prognostic ability. The C-index and CPE of the validation cohort were 0.81 (95% CI 0.69-0.91) and 0.78 (95% CI 0.7-0.86), respectively. CONCLUSIONS: Our nomogram incorporating CD15+ cell count can potentially be used to identify patients at high risk of recurrent disease and thus stratify patients who will benefit most from adjuvant treatment.
Subject(s)
Esophageal Neoplasms , Neutrophils , Humans , Neutrophils/pathology , Neoadjuvant Therapy , Esophageal Neoplasms/pathology , Prognosis , Nomograms , Tumor MicroenvironmentABSTRACT
Most NUTM1-rearranged neoplasms (NRNs) have fusions between NUTM1 and BRD (bromodomain-containing) family members and are termed NUT carcinomas (NCs) because they show some squamous differentiation. However, some NRNs are associated with fusions between NUTM1 and members of the MAD (MAX dimerization) gene family of MYC antagonists. Here we describe a small round cell malignancy from the gastro-esophageal junction with a previously unreported fusion between NUTM1 and the MAD family member MXI1. In contrast to NCs, the MXI1-NUTM1 tumor did not show squamous differentiation and did not express MYC, TP63 or SOX2, genes known to be targets of BRD-NUTM1 proteins and critical for NC oncogenesis. Transcriptome analysis showed paradoxical enrichment of MYC target genes in the MXI1-NUTM1 tumor despite the lack of MYC expression. When expressed in vitro MXI1-NUTM1 partially phenocopied MYC, enhancing cell proliferation and cooperating with oncogenic HRAS to produce anchorage-independent cell growth. These data provide evidence that MAD family members, which are normally repressors of MYC activity, can be converted into MYC-like mimics by fusion to NUTM1. The pathological features and novel oncogenic mechanism of the MXI1-NUTM1 tumor show that identification of NUTM1 fusion partners can be important for accurate diagnostic classification of some NRN subtypes, and potentially may guide therapeutic options.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Esophageal Neoplasms/genetics , Esophagogastric Junction/pathology , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Stomach Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Esophageal Neoplasms/metabolism , Esophageal Neoplasms/pathology , Fatal Outcome , Female , Humans , Middle Aged , Oncogene Proteins, Fusion , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , TranscriptomeABSTRACT
Structural alterations of NUTM1 were originally thought to be restricted to poorly differentiated carcinomas with variable squamous differentiation originating in the midline organs of children and adolescents. Termed NUT carcinomas (NCs), they were defined by a t(15;19) chromosomal rearrangement that was found to result in a BRD4-NUTM1 gene fusion. However, the use of DNA and RNA-based next-generation sequencing has recently revealed a multitude of new NUTM1 fusion partners in a diverse array of neoplasms including sarcoma-like tumors, poromas, and acute lymphoblastic leukemias (ALLs) that we propose to call NUTM1-rearranged neoplasms (NRNs). Intriguingly, the nosology of NRNs often correlates with the functional classification of the fusion partner, suggesting different oncogenic mechanisms within each NRN division. Indeed, whereas NCs are characterized by their aggressiveness and intransigence to standard therapeutic measures, the more positive clinical outcomes seen in some sarcoma and ALL NRNs may reflect these mechanistic differences. Here we provide a broad overview of the molecular, nosological, and clinical features in these newly discovered neoplastic entities. We describe how aberrant expression of NUTM1 due to fusion with an N-terminal DNA/chromatin-binding protein can generate a potentially powerful chromatin modifier that can give rise to oncogenic transformation in numerous cellular contexts. We also conclude that classification, clinical behavior, and therapeutic options may be best defined by the NUTM1 fusion partner rather than by tumor morphology or immunohistochemical profile.
Subject(s)
Carcinoma/genetics , Gene Rearrangement , Neoplasm Proteins/genetics , Nuclear Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Sarcoma/genetics , Carcinoma/pathology , Gene Fusion , Humans , Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Sarcoma/pathologyABSTRACT
There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.
Subject(s)
Carcinoma, Acinar Cell/genetics , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins c-raf/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Carcinoma, Acinar Cell/pathology , Databases, Factual , Female , Gene Fusion , Gene Rearrangement , Humans , Male , Middle Aged , Pancreatic Neoplasms/pathology , Young AdultABSTRACT
NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.
Subject(s)
Cell Separation/methods , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/metabolism , Myoblasts, Cardiac/cytology , Myocytes, Cardiac/cytology , Transcription Factors/metabolism , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Biomarkers/analysis , Cell Differentiation , Gene Expression Profiling , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Humans , Myoblasts, Cardiac/metabolism , Myocytes, Cardiac/metabolism , Polymerase Chain Reaction , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Transcription Factors/genetics , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
PURPOSE: The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. EXPERIMENTAL DESIGN: To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. RESULTS: Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. CONCLUSIONS: SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.
Subject(s)
Adaptor Proteins, Signal Transducing , DNA Helicases , Lung Neoplasms , Mutation , Nuclear Proteins , Small Cell Lung Carcinoma , Transcription Factors , YAP-Signaling Proteins , Humans , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , Small Cell Lung Carcinoma/metabolism , Transcription Factors/genetics , DNA Helicases/genetics , Nuclear Proteins/genetics , Cell Line, Tumor , Animals , Adaptor Proteins, Signal Transducing/genetics , YAP-Signaling Proteins/genetics , Mice , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Phosphoproteins/genetics , Biomarkers, Tumor/genetics , Gene Expression ProfilingABSTRACT
BACKGROUND: Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers where a primary tissue of origin (TOO) is uncertain. Most patients with CUP have limited treatment options and poor survival outcomes. Immune checkpoint inhibitors (ICIs) can be efficacious in some patients with CUP, but the optimal predictive biomarkers are unknown. We therefore assessed immune and genomic biomarkers as well as predicted TOO in patients with CUP, including a subset treated with ICIs. METHODS: Patients with CUP were subject to gene-expression profiling (GEP) and DNA panel sequencing. Immune and stromal-related gene expression was explored by NanoString, including genes associated with immunotherapy response (IR) in other solid malignancies. ICI responsive cancer types were assigned based on Food and Drug Administration-approved indications, and either detection of a latent primary tumor or the TOO was suspected based on genomics informed pathology review. Tumor mutation burden (TMB) and gene mutations were also assessed. RESULTS: A total of 219 patients with CUP were included, 215 assessed for TOO in a previous study, with the majority (163) receiving both RNA and DNA tests. Of GEP profiled cases, 33% (59/175) had a high IR gene-expression score. Of the DNA sequenced cases, 16% (32/203) had high TMB (>10 mutations/Mb), including two with mismatch repair deficiency. Low correlation was observed between TMB and an IR score (R=0.26, p<0.001). Among 110 CUPs with a latent primary or suspected TOO, 47% (52/110) belonged to ICI-responsive cancer types. More than half of the CUPs had at least one feature that may predict ICI response (high IR score, high TMB, ICI-responsive cancer type). Among patients with CUP treated with ICIs, 8/28 (29%) responded (2 complete responses and 6 partial responses). Among non-responders, 9 had stable and 11 had progressive disease. All responders had a high IR score (7/8) and/or high TMB (3/8), while most (5/8) belonged to ICI-responsive cancer types. These features were detected at a lower frequency in non-responders and mostly in patients with stable disease. CONCLUSIONS: A significant fraction of CUP tumors had genomic features previously associated with ICI response. High IR score was the most sensitive predictive feature of ICI response, warranting evaluation in a larger patient series.
Subject(s)
Neoplasms, Unknown Primary , United States , Humans , Neoplasms, Unknown Primary/drug therapy , Neoplasms, Unknown Primary/genetics , Mutation , Biomarkers, Tumor/genetics , Immunotherapy , GenomicsABSTRACT
RATIONALE: The transcriptional networks guiding heart development remain poorly understood, despite the identification of several essential cardiac transcription factors. OBJECTIVE: To isolate novel cardiac transcription factors, we performed gene chip analysis and found that Zac1, a zinc finger-type transcription factor, was strongly expressed in the developing heart. This study was designed to investigate the molecular and functional role of Zac1 as a cardiac transcription factor. METHODS AND RESULTS: Zac1 was strongly expressed in the heart from cardiac crescent stages and in the looping heart showed a chamber-restricted pattern. Zac1 stimulated luciferase reporter constructs driven by ANF, BNP, or alphaMHC promoters. Strong functional synergy was seen between Zac1 and Nkx2-5 on the ANF promoter, which carries adjacent Zac1 and Nkx2-5 DNA-binding sites. Zac1 directly associated with the ANF promoter in vitro and in vivo, and Zac1 and Nkx2-5 physically associated through zinc fingers 5 and 6 in Zac1, and the homeodomain in Nkx2-5. Zac1 is a maternally imprinted gene and is the first such gene found to be involved in heart development. Homozygous and paternally derived heterozygous mice carrying an interruption in the Zac1 locus showed decreased levels of chamber and myofilament genes, increased apoptotic cells, partially penetrant lethality and morphological defects including atrial and ventricular septal defects, and thin ventricular walls. CONCLUSIONS: Zac1 plays an essential role in the cardiac gene regulatory network. Our data provide a potential mechanistic link between Zac1 in cardiogenesis and congenital heart disease manifestations associated with genetic or epigenetic defects in an imprinted gene network.
Subject(s)
Cell Cycle Proteins/genetics , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Heart Defects, Congenital/genetics , Heart/embryology , Transcription Factors/genetics , Animals , Apoptosis/genetics , Atrial Natriuretic Factor/genetics , Atrial Natriuretic Factor/metabolism , Binding Sites , COS Cells , Cell Cycle Proteins/metabolism , Chlorocebus aethiops , Gene Expression Profiling/methods , Genes, Tumor Suppressor , Genomic Imprinting , Gestational Age , Heart Defects, Congenital/embryology , Heart Defects, Congenital/metabolism , Heart Defects, Congenital/pathology , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Mutant Strains , Morphogenesis/genetics , Mutation , Natriuretic Peptide, C-Type/genetics , Natriuretic Peptide, C-Type/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Precursors/genetics , Protein Precursors/metabolism , Rats , Transcription Factors/metabolism , Transcriptional Activation , TransfectionABSTRACT
SUMMARY: Adrenocortical carcinoma is a rare disease with poor prognosis whose clinical heterogeneity can at times present a challenge to accurate and timely diagnosis. We present the case of a patient who presented with extensive pulmonary lesions, mediastinal and hilar lymphadenopathy and an adrenal mass in whom the oncological diagnosis was initially uncertain. Through the use of immunohistochemistry, biochemistry and genomic testing, an accurate diagnosis of adrenocortical carcinoma was ultimately made which resulted in more directed treatment being administered. The use of multidisciplinary input and genomics to aid in diagnosis and prognosis of adrenocortical carcinoma is discussed. LEARNING POINTS: Adrenocortical carcinomas can present a diagnostic challenge to clinicians given it is a rare malignancy with significant clinical heterogeneity. Specialist multidisciplinary team input is vital in the diagnosis and management of adrenocortical carcinomas. Hormonal testing is recommended in the diagnostic workup of adrenal masses, even in the absence of overt clinical signs/symptoms of hormone excess. Immunostaining for the highly sensitive and specific steroidogenic factor-1 is vital for accurate diagnosis. Genomics can provide prognostic utility in management of adrenocortical carcinoma.
ABSTRACT
Therapeutically actionable ROS1 rearrangements have been described in 1-3% of non-small cell lung cancer (NSCLC). Screening for ROS1 rearrangements is recommended to be by immunohistochemistry (IHC), followed by confirmation with fluorescence in situ hybridisation (FISH) or sequencing. However, in practise ROS1 IHC presents difficulties due to conflicting scoring systems, multiple clones and expression in tumours that are wild-type for ROS1. We assessed ROS1 IHC in 285 consecutive cases of NSCLC with non-squamous histology over a nearly 2-year period. IHC was scored with ROS1 clone D4D6 (n=270), clone SP384 (n=275) or both clones (n=260). Results were correlated with ROS1 break-apart FISH (n=67), ALK status (n=194), and sequence data of EGFR (n=178) and other drivers, where possible. ROS1 expression was detected in 161/285 cases (56.5%), including 13/14 ROS1 FISH-positive cases. There was no ROS1 expression in one ROS1 FISH-positive case in which sequencing detected an ALK-EML4 fusion, but not a ROS1 fusion. The other 13 ROS1 FISH-positive cases showed moderate to strong staining with both IHC clones. However, one case with a TPM3-ROS1 fusion would have been scored as negative with SP384 and D4D6 clones by some previous criteria. ROS1 expression was also detected in 58/285 cases (20.4%) that had driver mutations in genes other than ROS1. A sensitivity of 100% for detecting a ROS1 rearrangement by FISH was achieved by omitting intensity from the IHC scoring criteria and expression in >0% cells with D4D6 or in ≥50% cells with SP384. Excluding cases with driver events in any MAPK pathway gene (e.g., in ALK, EGFR, KRAS, BRAF, ERBB2 and MET) substantially reduced the number of cases proceeding to ROS1 FISH. Only 15.9% of MAPK-negative NSCLC would proceed to FISH for an IHC threshold of >0% cells with D4D6, with a specificity of 42.4%. For a threshold of ≥50% cells with SP384, only 18.5% of MAPK-negative cases would proceed to FISH, with a specificity of 31.4%. Based on our data we suggest an algorithm for screening for ROS1 rearrangements in NSCLC in which ROS1 FISH is only performed in cases that have been demonstrated to lack activating mutations in any MAPK pathway gene by comprehensive sequencing and ALK IHC, and show staining at any intensity in ≥50% of cells with clone SP384, or >0% cells with D4D6.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Early Detection of Cancer , Gene Rearrangement , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence/methods , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolismABSTRACT
The pulmonary vein is sleeved by myocardium, which is a major source of atrial fibrillation and is involved in congenital sinus venosus defects. Little is known about the cellular origin and mechanism of formation of the pulmonary myocardium. We observed a biphasic process of pulmonary myocardium formation in mice. Firstly, a myocardial cell population forms de novo at the connection of the pulmonary vein and the atrium. Genetic labeling revealed that atrial cells do not contribute to this population, indicating it forms by differentiation of pulmonary mesenchymal cells. Secondly, these pulmonary myocardial cells initiate a phase of rapid proliferation and form the pulmonary myocardial sleeve. Pitx2c-deficient mice do not develop a pulmonary myocardial sleeve because they fail to form the initial pulmonary myocardial cells. Genetic-labeling analyses demonstrated that whereas the systemic venous return derives from Nkx2-5-negative precursors, the pulmonary myocardium derives from Nkx2-5-expressing precursors, indicating a distinct origin of the 2 venous systems. Nkx2-5 and its target gap-junction gene Cx40 are expressed in the atria and in the pulmonary myocardium but not in the systemic venous return, which expresses the essential pacemaker channel Hcn4. When Nkx2-5 protein level was lowered in a hypomorphic model, the pulmonary myocardium switched to a Cx40-negative, Hcn4-positive phenotype resembling that of the systemic venous return. In conclusion, our data suggest a cellular mechanism for pulmonary myocardium formation and highlight the key roles played by Pitx2c and Nkx2-5 in its formation and identity.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/physiology , Pulmonary Veins/embryology , Pulmonary Veins/physiology , Transcription Factors/physiology , Animals , Atrial Fibrillation/physiopathology , Cell Differentiation/physiology , Cell Lineage/physiology , Heart/embryology , Heart/physiology , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Mesoderm/cytology , Mice , Mice, Transgenic , Myocardium/cytology , Phenotype , Pulmonary Veins/cytology , Transcription Factors/genetics , Homeobox Protein PITX2ABSTRACT
The sinoatrial node, which resides at the junction of the right atrium and the superior caval vein, contains specialized myocardial cells that initiate the heart beat. Despite this fundamental role in heart function, the embryonic origin and mechanisms of localized formation of the sinoatrial node have not been defined. Here we show that subsequent to the formation of the Nkx2-5-positive heart tube, cells bordering the inflow tract of the heart tube give rise to the Nkx2-5-negative myocardial cells of the sinoatrial node and the sinus horns. Using genetic models, we show that as the myocardium of the heart tube matures, Nkx2-5 suppresses pacemaker channel gene Hcn4 and T-box transcription factor gene Tbx3, thereby enforcing a progressive confinement of their expression to the forming Nkx2-5-negative sinoatrial node and sinus horns. Thus, Nkx2-5 is essential for establishing a gene expression border between the atrium and sinoatrial node. Tbx3 was found to suppress chamber differentiation, providing an additional mechanism by which the Tbx3-positive sinoatrial node is shielded from differentiating into atrial myocardium. Pitx2c-deficient fetuses form sinoatrial nodes with indistinguishable molecular signatures at both the right and left sinuatrial junction, indicating that Pitx2c functions within the left/right pathway to suppress a default program for sinuatrial node formation on the left. Our molecular pathway provides a mechanism for how pacemaker activity becomes progressively relegated to the most recently added components of the venous pole of the heart and, ultimately, to the junction of the right atrium and superior caval vein.
Subject(s)
Body Patterning/physiology , Gene Expression Regulation, Developmental/physiology , Heart Atria/embryology , Heart Ventricles/embryology , Homeodomain Proteins/physiology , Ion Channels/biosynthesis , Sinoatrial Node/embryology , T-Box Domain Proteins/physiology , Transcription Factors/physiology , Animals , Atrial Natriuretic Factor , Biomarkers , Body Patterning/genetics , Cardiac Myosins/biosynthesis , Cardiac Myosins/genetics , Connexins/biosynthesis , Connexins/genetics , Cyclic Nucleotide-Gated Cation Channels , Gene Expression Regulation, Developmental/genetics , Genes, Reporter , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Imaging, Three-Dimensional , In Situ Hybridization , Ion Channels/genetics , Mice , Mice, Knockout , Mice, Transgenic , Myocardium/metabolism , Myosin Light Chains/biosynthesis , Myosin Light Chains/genetics , Natriuretic Peptide, C-Type/biosynthesis , Natriuretic Peptide, C-Type/genetics , Protein Precursors/biosynthesis , Protein Precursors/genetics , Recombinant Fusion Proteins/physiology , Sinoatrial Node/cytology , T-Box Domain Proteins/biosynthesis , T-Box Domain Proteins/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Troponin I/biosynthesis , Troponin I/genetics , Gap Junction alpha-5 Protein , Homeobox Protein PITX2ABSTRACT
The venous pole of the mammalian heart is a structurally and electrically complex region, yet the lineage and molecular mechanisms underlying its formation have remained largely unexplored. In contrast to classical studies that attribute the origin of the myocardial sinus horns to the embryonic venous pole, we find that the sinus horns form only after heart looping by differentiation of mesenchymal cells of the septum transversum region into myocardium. The myocardial sinus horns and their mesenchymal precursor cells never express Nkx2-5, a transcription factor critical for heart development. In addition, lineage studies show that the sinus horns do not derive from cells previously positive for Nkx2-5. In contrast, the sinus horns express the T-box transcription factor gene Tbx18. Mice deficient for Tbx18 fail to form sinus horns from the pericardial mesenchyme and have defective caval veins, whereas the pulmonary vein and atrial structures are unaffected. Our studies define a novel heart precursor population that contributes exclusively to the myocardium surrounding the sinus horns or systemic venous tributaries of the developing heart, which are a source of congenital malformation and cardiac arrhythmias.
Subject(s)
Cell Differentiation , Coronary Circulation , Heart/embryology , Myocardium/cytology , Stem Cells/cytology , Transcription Factors/deficiency , Transcription Factors/physiology , Animals , Cell Lineage , Embryonic Development/physiology , Homeobox Protein Nkx-2.5 , Homeodomain Proteins , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Mice , Mice, Knockout , Stem Cells/metabolism , T-Box Domain Proteins , Veins/abnormalities , Veins/embryologyABSTRACT
The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes downregulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.
Subject(s)
Homeodomain Proteins/genetics , Spleen/abnormalities , Splenic Diseases/genetics , Transcription Factors/genetics , Adolescent , Amino Acid Sequence , Animals , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p15/metabolism , DNA-Binding Proteins/deficiency , Exome , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/metabolism , Humans , Infant , Male , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation, Missense , Pedigree , Pre-B-Cell Leukemia Transcription Factor 1 , Proto-Oncogene Proteins/deficiency , Transcription Factors/deficiency , Transcription Factors/metabolismABSTRACT
Colony-forming units - fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.