RESUMO
Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers.
Assuntos
Proteômica , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , RNA Polimerases Dirigidas por DNA , Sequenciamento de Cromatina por Imunoprecipitação , Fatores de TranscriçãoRESUMO
The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.
Assuntos
Proteínas Morfogenéticas Ósseas , Músculo Esquelético , Animais , Humanos , Camundongos , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Genes Homeobox , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteína Smad6/metabolismoRESUMO
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.
Assuntos
Rabdomiossarcoma , Fator de Ligação a CCAAT/genética , Diferenciação Celular/genética , Aberrações Cromossômicas , Rabdomiossarcoma/genética , Fatores de TranscriçãoRESUMO
Larvacean tunicates feature a spectacular innovation not seen in other animals - the trunk oikoplastic epithelium (OE). This epithelium produces a house, a large and complex extracellular structure used for filtering and concentrating food particles. Previously we identified several homeobox transcription factor genes expressed during early OE patterning. Among these are two Pax3/7 copies that we named pax37A and pax37B. The vertebrate homologs, PAX3 and PAX7 are involved in developmental processes related to neural crest and muscles. In the ascidian tunicate Ciona intestinalis, Pax3/7 plays a role in the development of cells deriving from the neural plate border, including trunk epidermal sensory neurons and tail nerve cord neurons, as well as in the neural tube closure. Here we have investigated the roles of Oikopleura dioica pax37A and pax37B in the development of the OE, by using CRISPR-Cas9 mutant lines and analyzing scRNA-seq data from wild-type animals. We found that pax37B but not pax37A is essential for the differentiation of cell fields that produce the food concentrating filter of the house: the anterior Fol, giant Fol and Nasse cells. Trajectory analysis supported a neuroepithelial-like or a preplacodal ectoderm transcriptional signature in these cells. We propose that the highly specialized secretory epithelial cells of the Fol region either maintained or evolved neuroepithelial features. This is supported by a fragmented gene regulatory network involved in their development that also operates in ascidian epidermal neurons.
RESUMO
Alveolar rhabdomyosarcoma (ARMS) with FOXO1 gene rearrangements is an aggressive pediatric rhabdomyosarcoma subtype that is prognostically distinct from embryonal rhabdomyosarcoma and fusion-negative ARMS. Herein, we report two cases of ARMS with PAX3::MAML3 fusions. The tumors arose in an infant and an adolescent as stage IV metastatic disease (by Children's Oncology Group staging system). Histologically, both cases were small round blue cell tumors arranged in vague nests and solid sheets that were diffusely positive for desmin and myogenin. By methylation profiling and unsupervised clustering analysis, the tumors clustered with ARMS with classic FOXO1 rearrangements and ARMS with variant PAX3::NCOA1/INO80D fusions, but not with biphenotypic sinonasal sarcoma (BSNS) with PAX3::MAML3/NCOA2/FOXO1/YAP1 fusions, nor with other small round blue cell tumors, including embryonal rhabdomyosarcoma. The differentially methylated genes between ARMS and BSNS were highly enriched in genes involved in myogenesis, and 21% of these genes overlap with target genes of the PAX3::FOXO1 fusion transcription factor. On follow-up after initiation of vincristine/actinomycin/cyclophosphamide chemotherapy, the tumors showed partial and complete clinical response, consistent with typical upfront chemotherapy responsiveness of ARMS with the classic FOXO1 rearrangement. We conclude that PAX3::MAML3 is a novel variant fusion of ARMS, which displays a methylation signature distinct from BSNS despite sharing similar PAX3 fusions. These findings highlight the utility of methylation profiling in classifying ARMS with non-canonical fusions.
RESUMO
This chapter discusses the role of cardiac neural crest cells in the formation of the septum that divides the cardiac arterial pole into separate systemic and pulmonary arteries. Further, cardiac neural crest cells directly support the normal development and patterning of derivatives of the caudal pharyngeal arches, including the great arteries, thymus, thyroid, and parathyroids. Recently, cardiac neural crest cells have also been shown to indirectly influence the development of the secondary heart field, another derivative of the caudal pharynx, by modulating signaling in the pharynx. The contribution and function of the cardiac neural crest cells has been learned in avian models; most of the genes associated with cardiac neural crest function have been identified using mouse models. Together these studies show that the neural crest cells may not only critical for normal cardiovascular development but also may be involved secondarily because they represent a major component in the complex tissue interactions in the caudal pharynx and outflow tract. Cardiac neural crest cells span from the caudal pharynx into the outflow tract, and therefore may be susceptible to any perturbation in or by other cells in these regions. Thus, understanding congenital cardiac outflow malformations in human sequences of malformations resulting from genetic and/or environmental insults necessarily requires better understanding the role of cardiac neural crest cells in cardiac development.
Assuntos
Crista Neural , Crista Neural/embriologia , Crista Neural/citologia , Crista Neural/metabolismo , Animais , Humanos , Coração/embriologia , CamundongosRESUMO
Both Warrensburg (WS) and Marfan syndrome (MFS) can impair the vision. Here, we recruited a Chinese family consisting of two WS affected individuals (II:1 and III:3) and five MFS affected individuals( I:1, II:2, III:1, III:2, and III:5) as well as one suspected MFS individual (II:4). Using whole exome sequencing (WES) and subsequent PCR-Sanger sequencing, we identified one novel heterozygous variant NM_000438 (PAX3) c.208 T > C, (p.Cys70Arg) from individuals with WS and one previous reported variant NM_000138 (FBN1) c.2740 T > A, (p.Cys914Ser) from individuals with MFS and co-segregated with the diseases. Real-time PCR and Western blot assay showed that, compared to their wild-type, both mRNAs and proteins of PAX3 and FBN1 mutants reduced in HKE293T cells. Together, our study identified two disease-causing variants in a same Chinese family with WS and MFS, and confirmed their damaged effects on their genes' expression. Therefore, those findings expand the mutation spectrum of PAX3 and provide a new perspective for the potential therapy.
Assuntos
Síndrome de Marfan , Humanos , Síndrome de Marfan/genética , Sequenciamento do Exoma , População do Leste Asiático , Mutação , Heterozigoto , Linhagem , Fator de Transcrição PAX3/genética , Fibrilina-1/genéticaRESUMO
Reexpressed PAX3 transcription factor is believed to be responsible for the differentiation defects observed in neuroblastoma. Although the importance of PAX3 in neuronal differentiation is documented how it is involved in the defective differentiation remains unexplored particularly with its isoforms. Here, first we have analyzed PAX3 expression, its functional status, and its correlation with the neuronal marker expression in SH-SY5Y and its parental SK-N-SH cells. We have found that SH-SY5Y cells which expressed more PAX3 showed increased expression of neuronal marker genes (TUBB, MAP2, NEFL, NEUROG2, SYP) and reported PAX3 target genes (MET, TGFA, and NCAM1) than the SK-N-SH cells that had low PAX3 level. Retinoic acid treatment is unable to induce neuronal differentiation in cells (SK-N-SH) with low PAX3 level/activity. Moreover, ectopic expression of PAX3 in SK-N-SH cells neither induces neuronal marker genes nor its target genes. PAX3 isoform expression analysis revealed the expression of PAX3b isoform that contains only paired domain in SK-N-SH cells, whereas in SH-SY5Y cells, we could also observe PAX3c isoform that contains all functional domains. Further, PAX3b depletion in SK-N-SH cells is not induced PAX3 target genes, and the cells remain poorly differentiated. Interestingly, ectopic PAX3 expression in PAX3b-depleted SK-N-SH cells enhanced neuronal outgrowth along with neuronal marker gene induction. Collectively, these results showed that the PAX3b isoform may be responsible for the differentiation defect observed in SK-N-SH cells and restoration of functional PAX3 in the absence of PAX3b can induce neurogenesis in these cells.
Assuntos
Diferenciação Celular , Neuroblastoma , Fator de Transcrição PAX3 , Humanos , Linhagem Celular Tumoral , Neuroblastoma/genética , Neuroblastoma/metabolismo , Fator de Transcrição PAX3/genética , Isoformas de Proteínas/genética , Tretinoína/farmacologiaRESUMO
Biphenotypic sinonasal sarcoma (BSNS), previously known as low-grade sinonasal sarcoma, is a rare tumour of the sinonasal tract, first described in 2012. It involves both myogenic and neural differentiation and is characterized by PAX3 rearrangement. MAML3 is the most frequent fusion partner of PAX3; however, its partner remains unidentified in a subset of cases. These tumours have significant local recurrence rates but lack metastatic potential. Here, we report a case of BSNS with PAX3/FOXO1 fusion and discuss its clinicopathological features and differential diagnosis.
Assuntos
Neoplasias dos Seios Paranasais , Sarcoma , Neoplasias de Tecidos Moles , Humanos , Biomarcadores Tumorais , Neoplasias dos Seios Paranasais/diagnóstico , Neoplasias dos Seios Paranasais/patologia , Diagnóstico Diferencial , Proteína Forkhead Box O1 , Fator de Transcrição PAX3RESUMO
BACKGROUND: Anomalies of the FOXO1 gene in alveolar rhabdomyosarcoma are associated with a worse clinical prognosis, which determines the high value of studying the status of this gene when choosing a therapy strategy. The «gold standard¼ for determining FOXO1 gene rearrangements is currently the fluorescent in situ hybridization (FISH) technique. OBJECTIVE: Study of the relationship between canonical FOXO1 translocation and immunohistochemical expression of new surrogate markers in alveolar rhabdomyosarcoma to determine their predictive value. MATERIAL AND METHODS: 139 cases of rhabdomyosarcoma were retrospectively studied. The study used tissue matrix technology (TMA). On sections obtained from TMA blocks, the FISH technique was implemented using the locus-specific probe MetaSystems XL FOXO1 Break Apart (Metasystems, Germany). Immunohistochemical studies were performed on similar sections from TMA blocks with OLIG2 (Cell Marque Antibodies, clone 211F1.1) and MUC4 (Cell Marque Antibodies, clone 8G7) antibodies. RESULTS: The final expression analysis and statistical processing using a 2x2 contingency table and Fisher's exact test passed 111 cases (76 without FOXO1 rearrangement and 35 with rearrangement). The specificity of OLIG2 and MUC4 expression for FOXO1-rearranged alveolar rhabdomyosarcoma was 85.53% and 80.26%, respectively (p<0.01). CONCLUSION: The present study confirms the high predictive value of the expression of surrogate markers OLIG2 and MUC4 in determining the genetic status of alveolar rhabdomyosarcoma, which makes it possible to predict with high specificity the detection of the FOXO1 gene rearrangement.
Assuntos
Rabdomiossarcoma Alveolar , Humanos , Rabdomiossarcoma Alveolar/diagnóstico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Hibridização in Situ Fluorescente/métodos , Proteína Forkhead Box O1/genética , Estudos Retrospectivos , Biomarcadores , Translocação Genética/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
In our review we have completed current knowledge on myotomal myogenesis in model and non-model vertebrate species (fishes, amphibians, reptiles, birds and mammals) at morphological and molecular levels. Data obtained from these studies reveal distinct similarities and differences between amniote and anamniote species. Based on the available data, we decided to present evolutionary implications in vertebrate trunk muscle development. Despite the fact that in all vertebrates muscle fibres are multinucleated, the pathways leading to them vary between vertebrate taxa. In fishes during early myogenesis myoblasts differentiate into multinucleated lamellae or multinucleate myotubes. In amphibians, myoblasts fuse to form multinucleated myotubes or, bypassing fusion, directly differentiate into mononucleated myotubes. Furthermore, mononucleated myotubes were also observed during primary myogenesis in amniotes. The mononucleated state of myogenic cells could be considered as an old phylogenetic, plesiomorphic feature, whereas direct multinuclearity of myotubes has a synapomorphic character. On the other hand, the explanation of this phenomenon could also be linked to the environmental conditions in which animals develop. The similarities observed in vertebrate myogenesis might result from a conservative myogenic programme governed by the Pax3/Pax7 and myogenic regulatory factor (MRF) network, whereas differences in anamniotes and amniotes are established by spatiotemporal pattern expression of MRFs during muscle differentiation and/or environmental conditions.
Assuntos
Diferenciação Celular , Músculo Esquelético/citologia , Vertebrados , Animais , Músculo Esquelético/metabolismoRESUMO
Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.
Assuntos
Crista Neural/inervação , Fator de Transcrição PAX3/genética , Bexiga Urinária/inervação , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Gânglios , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Sistema Nervoso/embriologia , Crista Neural/fisiologia , Defeitos do Tubo Neural/genética , Neurogênese , Fator de Transcrição PAX3/fisiologia , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição SOXE , Região Sacrococcígea/inervação , Disrafismo Espinal/complicações , Disrafismo Espinal/genética , Bexiga Urinária/embriologiaRESUMO
BACKGROUND: The PAX3 (paired box gene 3) gene is highly expressed in several cancer types. However, its underlying mechanism of action in skin cutaneous melanoma (SKCM) remains unknown. METHODS: In this study, we used the GEPIA database and western blotting to analyze the expression of PAX3. We performed the Kaplan-Meier survival analysis to evaluate the prognostic value of PAX3 in SKCM. Next, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed to evaluate the function of PAX3-related co-expressed genes. Additionally, the function and potential mechanism of action of PAX3 in SKCM were studied through functional experiments. Western blotting was used to detect the changes in the levels of epithelial-mesenchymal transition (EMT)-related and MET (c-MET tyrosine kinase) proteins following PAX3 knockdown. Finally, we assessed the correlation between PAX3 expression and the infiltration of CD4+/CD8+ T cells using the TISIDB database. RESULTS: We found that PAX3 was overexpressed in the SKCM tissues and that these levels were indicative of a poor prognosis of SKCM. The KEGG pathway enrichment analysis showed that PAX3-related co-expressed genes were mainly associated with the oncogenic pathways. Knocking down PAX3 significantly inhibited the proliferation, invasion, and migration of SK-MEL-28 cells. The PAX3 expression was related significantly to the immune infiltration level of CD4+/CD8+ T cells. CONCLUSIONS: Our findings demonstrated that PAX3 knockdown could reverse the EMT of tumor cells, inhibit the growth, and progression of SKCM cells. Therefore, PAX3 may have implications as a potential therapeutic target and promising prognostic biomarker for SKCM.
Assuntos
Melanoma , Neoplasias Cutâneas , Biomarcadores , Linfócitos T CD8-Positivos , Regulação para Baixo/genética , Humanos , Melanoma/patologia , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Neoplasias Cutâneas/metabolismo , Melanoma Maligno CutâneoRESUMO
Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSCs) allows the generation of myogenic progenitors endowed with enhanced regenerative capacity. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro-generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared with fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple reinjuries, and contribute to long-term regeneration. Upon engraftment, the transcriptome of reisolated Pax3/Pax7-induced PSC-derived myogenic progenitors changes toward a postnatal molecular signature, particularly in genes involved in extracellular matrix remodeling. These findings demonstrate that Pax3/Pax7-induced myogenic progenitors remodel their molecular signature and functionally mature upon in vivo exposure to the adult muscle environment.
Assuntos
Desenvolvimento Muscular/fisiologia , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX7/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Camundongos , Desenvolvimento Muscular/genética , Músculo Esquelético , Mioblastos/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX7/genética , TranscriptomaRESUMO
Biphenotypic sinonasal sarcoma (BSNS) is a recently described spindle cell sarcoma with neural and myogenic differentiation which arises exclusively in the sinonasal region. A man presented with swelling of left eyelid, and history of resection of a low-grade spindle cell mesenchymal tumor of left sinonasal cavity performed 15 years before. The original diagnosis was synovial sarcoma. Current MRI showed left supraorbital mass with intracranial extension. IR biopsy confirmed recurrence, and a left orbital craniotomy was done. NGS identified PAX3-MAML3 fusion in both, current and original tumor. The sarcoma was reclassified as BSNS, recurrent, with higher grade transformation. While the morphology, phenotype and molecular signature were in keeping with BSNS, the tumor showed biological progression towards a high-grade sarcoma. High-grade transformation of low-grade BSNS has not been described so far. High-grade transformation was not appreciated at the time of initial diagnosis, and it occurred in the local recurrence 15 years after.
Assuntos
Neoplasias dos Seios Paranasais , Sarcoma Sinovial , Sarcoma , Fusão Gênica , Humanos , Neoplasias dos Seios Paranasais/diagnóstico por imagem , Neoplasias dos Seios Paranasais/genética , Neoplasias dos Seios Paranasais/patologia , Fenótipo , Sarcoma/diagnóstico , Sarcoma/genética , Sarcoma/patologiaRESUMO
Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Piridinas/farmacologia , Rabdomiossarcoma Alveolar/patologia , Animais , Linhagem Celular Tumoral , Linhagem da Célula , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The mammalian heart is a four-chambered organ with systemic and pulmonary circulations to deliver oxygenated blood to the body, and a tightly regulated genetic network exists to shape normal development of the heart and its associated major arteries. A key process during cardiovascular morphogenesis is the septation of the outflow tract which initially forms as a single vessel before separating into the aorta and pulmonary trunk. The outflow tract connects to the aortic arch arteries which are derived from the pharyngeal arch arteries. Congenital heart defects are a major cause of death and morbidity and are frequently associated with a failure to deliver oxygenated blood to the body. The Pax transcription factor family is characterised through their highly conserved paired box and DNA binding domains and are crucial in organogenesis, regulating the development of a wide range of cells, organs and tissues including the cardiovascular system. Studies altering the expression of these genes in murine models, notably Pax3 and Pax9, have found a range of cardiovascular patterning abnormalities such as interruption of the aortic arch and common arterial trunk. This suggests that these Pax genes play a crucial role in the regulatory networks governing cardiovascular development.
Assuntos
Cardiopatias Congênitas , Crista Neural , Animais , Aorta Torácica , Região Branquial , Redes Reguladoras de Genes , Cardiopatias Congênitas/metabolismo , Mamíferos , Camundongos , Crista Neural/metabolismoRESUMO
Mouse models provide opportunities to investigate genetic interactions that cause or modify the frequency of neural tube defects (NTDs). Mutation of the PAX3 transcription factor prevents neural tube closure, leading to cranial and spinal NTDs whose frequency is responsive to folate status. Canonical Wnt signalling is implicated both in regulation of Pax3 expression and as a target of PAX3. This study investigated potential interactions of Pax3 mutation and canonical Wnt signalling using conditional gain- and loss-of-function models of ß-catenin. We found an additive effect of ß-catenin gain of function and Pax3 loss of function on NTDs and neural crest defects. ß-catenin gain of function in the Pax3 expression domain led to significantly increased frequency of cranial but not spinal NTDs in embryos that are heterozygous for Pax3 mutation, while both cranial and spinal neural tube closure were exacerbated in Pax3 homozygotes. Similarly, deficits of migrating neural crest cells were exacerbated by ß-catenin gain of function, with almost complete ablation of spinal neural crest cells and derivatives in Pax3 homozygous mutants. Pax3 expression was not affected by ß-catenin gain of function, while we confirmed that loss of function led to reduced Pax3 transcription. In contrast to gain of function, ß-catenin knockout in the Pax3 expression domain lowered the frequency of cranial NTDs in Pax3 null embryos. However, loss of function of ß-catenin and Pax3 resulted in spinal NTDs, suggesting differential regulation of cranial and spinal neural tube closure. In summary, ß-catenin function modulates the frequency of PAX3-related NTDs in the mouse.
Assuntos
Crista Neural/metabolismo , Defeitos do Tubo Neural/genética , Fator de Transcrição PAX3/genética , Via de Sinalização Wnt , Animais , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Crista Neural/anormalidades , Crista Neural/embriologia , Fator de Transcrição PAX3/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Pax3 and Pax7 are closely related transcription factors that are widely expressed in the developing nervous system and somites. During the normal development in the central nervous system (CNS), Pax3 and Pax7 are mainly expressed in the dorsal part of the neural tube. Further analysis revealed that Pax3 and Pax7 shared redundant functions in the spinal cord development. However, it is still unknown whether Pax3 and Pax7 play a role in neuronal differentiation. In this study, Pax3 and Pax7 genes were overexpressed in Neuro-2a, the mouse neuroblastoma cell line. CCK-8 and EdU assay results showed that overexpression of Pax3 inhibited cell viability and proliferation of Neuro-2a cells, whereas the overexpression of Pax7 had no significant difference on their cell viability and proliferation. Overexpression of Pax3 not only increased the percentage of cells in the S phase and G0/G1 phase, but also decreased that in the G2 phase. Moreover, the total neurite lengths of Neuro-2a cells were significantly shorter in Pax3 overexpressed group than those in negative control group and showed no significant difference between Pax7 overexpressed group and negative control group. These results suggested that Pax3 not only inhibited the cell viability and proliferation but also affected the cell cycle and the neurite outgrowth of Neuro-2a cells. RNA sequencing analysis showed up-regulated genes in Pax3 overexpressed group were involved in cell cycle machinery, which may reveal the potential mechanism of Neuro-2a cells proliferation.
Assuntos
Crescimento Neuronal , Fator de Transcrição PAX3/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Regulação da Expressão Gênica , Ontologia Genética , Camundongos , Fator de Transcrição PAX7/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: The objective of this analysis was to evaluate the clinical factors influencing survival outcomes in patients with localized (clinical group I-III), FOXO1 fusion-positive rhabdomyosarcoma (RMS). METHODS: Patients with confirmed FOXO1 fusion-positive RMS who were enrolled on 3 completed clinical trials for localized RMS were included in the analytic cohort. Outcomes were analyzed using the Kaplan-Meier method to estimate event-free survival (EFS) and overall survival (OS), and the curves were compared using the log-rank test. A Cox proportional hazards regression model was used to perform multivariate analysis of prognostic factors that were significant in the univariate analysis. RESULTS: The estimated 4-year EFS and OS of 269 patients with localized, FOXO1 fusion-positive RMS was 53% (95% CI, 47%-59%) and 69% (95% CI, 63%-74%), respectively. Univariate analysis revealed that several known favorable clinical characteristics, including age at diagnosis between 1 and 9 years, complete surgical resection, tumor size ≤5 cm, favorable tumor site, absence of lymph node involvement, confinement to the anatomic site of origin, and PAX7-FOXO1 fusion, were associated with improved outcomes. Multivariate analysis identified older age (≥10 years) and large tumor size (>5 cm) as independent, adverse prognostic factors for EFS within this population, and patients who had both adverse features experienced substantially inferior outcomes. CONCLUSIONS: Patients with localized, FOXO1 fusion-positive RMS can be further risk stratified based on clinical features at diagnosis, and older patients with large primary tumors have the poorest prognosis.