RESUMEN
Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.
Asunto(s)
Disfunción Cognitiva , Endofenotipos , Animales , Ratones , Humanos , Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Lactatos/metabolismo , Concentración de Iones de HidrógenoRESUMEN
BACKGROUND: During mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta's intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17 is expressed in IAHCs, and that, among them, CD45lowc-Kithigh cells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity. METHODS: RNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study, and firstly, the whole-mount immunostaining was done. We conducted luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells. RESULTS: The increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity. CONCLUSIONS: Rasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.
RESUMEN
Cancer stem cells are a promising target for cancer eradication due to their responsibility for therapy-resistance and cancer recurrence. Previously, we have demonstrated that glioma stem cells (GSCs) recruit and induce the differentiation of bone marrow (BM) monocytes into tumor-infiltrating macrophages, which phagocytose hemorrhaged erythrocytes and store GSC-beneficial iron in mouse xenografts, suggesting a self-expanding strategy of GSCs that exploits host hematopoiesis of myeloid cells. However, it remains unclear whether a self-advantageous effect of GSCs also occurs on erythroid cells during glioma development. Here, we found that, in the primary cultures of mouse fetal liver proerythroblasts (proEs), conditioned media prepared from glioma cells including patient-derived glioblastoma (GBM) cells significantly facilitated the differentiation of proEs into erythroblasts. Importantly, in-vivo erythroid analysis in intracranially GSC-transplanted mice showed an enhanced erythropoiesis in the BM. In addition, the sphere forming ability of patient-derived GBM cells was significantly suppressed by hypoxia treatment and iron chelation, suggesting higher demands of GSCs for oxygen and iron, which may be supplied by GSCs- and their progeny-induced erythrocyte production. Our findings provide a new insight into survival and expanding strategies of GSCs that systemically exploit host erythropoiesis.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Animales , Eritropoyesis , Humanos , Ratones , Células Madre NeoplásicasRESUMEN
Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.
Asunto(s)
Antígenos CD/genética , Cadherinas/genética , Moléculas de Adhesión Celular/genética , Diferenciación Celular/genética , Proteínas HMGB/genética , Hematopoyesis/genética , Factores de Transcripción SOXF/genética , Animales , Aorta/crecimiento & desarrollo , Aorta/metabolismo , Cadherinas/antagonistas & inhibidores , Moléculas de Adhesión Celular/antagonistas & inhibidores , Embrión de Mamíferos , Células Endoteliales/citología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas HMGB/antagonistas & inhibidores , Células Madre Hematopoyéticas/citología , Humanos , Ratones , Embarazo , ARN Interferente Pequeño/farmacología , Factores de Transcripción SOXF/antagonistas & inhibidoresRESUMEN
Spontaneous necrosis is a defining feature of glioblastomas (GBMs), the most malignant glioma. Despite its strong correlations with poor prognosis, it remains unclear whether necrosis could be a possible cause or mere consequence of glioma progression. Here we isolated a particular fraction of necrotic products spontaneously arising from glioma cells, morphologically and biochemically defined as autoschizis-like products (ALPs). When administered to granulocyte macrophage colony-stimulating factor (GM-CSF)-primed bone marrow-derived macrophage/dendritic cells (Mφ/DCs), ALPs were found to be specifically engulfed by Mφs expressing a tumor-associated macrophage (TAM) marker CD204. ALPs from glioma stem cells (GSCs) had higher activity for the TAM development than those from non-GSCs. Of note, expression of the Il12b gene encoding a common subunit of IL-12/23 was upregulated in ALPs-educated Mφs. Furthermore, IL-12 protein evidently enhanced the sphere-forming activity of GBM patient-derived cells, although interestingly IL-12 is generally recognized as an antitumoral M1-Mφ marker. Finally, in silico analysis of The Cancer Genome Atlas (TCGA) transcriptome data of primary and recurrent GBMs revealed that higher expression of these IL-12 family genes was well correlated with more infiltration of M1-type TAMs and closely associated with poorer prognosis in recurrent GBMs. Our results highlight a role of necrosis in GSC-driven self-beneficial niche construction and glioma progression, providing important clues for developing new therapeutic strategies against gliomas.
Asunto(s)
Glioma/genética , Proteína Goosecoide/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Macrófagos Asociados a Tumores/metabolismo , Animales , Femenino , Humanos , Ratones , Transducción de SeñalRESUMEN
A transcription factor Sry-related high mobility group box (Sox) 17 is involved in developmental processes including spermatogenesis, cardiovascular system, endoderm formation, and so on. In this article, we firstly review the studies on the relation between the Sox17 expression and tumor malignancy. Although Sox17 positively promotes various tissue development, most of the cancers associated with Sox17 show decreased expression levels of Sox17, and an inverse correlation between Sox17 expression and malignancy is revealed. We briefly discuss the mechanism of such Sox17 down-regulation by focusing on DNA methylation of CpG sites located in the Sox17 gene promoter. Next, we overview the function of Sox17 in the fetal hematopoiesis, particularly in the dorsal aorta in midgestation mouse embryos. The Sox17 expression in hematopoietic stem cell (HSC)-containing intra-aortic hematopoietic cell cluster (IAHCs) is important for the cluster formation with the hematopoietic ability. The sustained expression of Sox17 in adult bone marrow HSCs and the cells in IAHCs of the dorsal aorta indicate abnormalities that are low lymphocyte chimerism and the aberrant proliferation of common myeloid progenitors in transplantation experiments. We then summarize the perspectives of Sox17 research in cancer control.
RESUMEN
The aorta-gonad-mesonephros region, from which definitive hematopoiesis first arises in midgestation mouse embryos, has intra-aortic hematopoietic clusters (IAHCs) containing hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We previously reported expression of the transcription factor Sox17 in IAHCs, and overexpression of Sox17 in CD45lowc-KIThigh cells comprising IAHCs maintains the formation of cell clusters and their multipotency in vitro over multiple passages. Here, we demonstrate the importance of NOTCH1 in IAHC formation and maintenance of the HSC/HPC phenotype. We further show that Notch1 expression is positively regulated by SOX17 via direct binding to its gene promoter. SOX17 and NOTCH1 were both found to be expressed in vivo in cells of IAHCs by whole mount immunostaining. We found that cells transduced with the active form of NOTCH1 or its downstream target, Hes1, maintained their multipotent colony-forming capacity in semisolid medium. Moreover, cells stimulated by NOTCH1 ligand, Jagged1, or Delta-like protein 1, had the capacity to form multilineage colonies. Conversely, knockdown of Notch1 and Hes1 led to a reduction of their multipotent colony-forming capacity. These results suggest that the Sox17-Notch1-Hes1 pathway is critical for maintaining the undifferentiated state of IAHCs.
Asunto(s)
Aorta/metabolismo , Proteínas HMGB/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Receptor Notch1/metabolismo , Factores de Transcripción SOXF/metabolismo , Factor de Transcripción HES-1/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Feto/metabolismo , Gónadas/metabolismo , Mesonefro/metabolismo , Ratones , Ratones Endogámicos ICR , Regiones Promotoras Genéticas/fisiologíaRESUMEN
In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.
Asunto(s)
Aorta/citología , Gónadas/citología , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Mesonefro/citología , Trombopoyetina/fisiología , Animales , Aorta/embriología , Aorta/metabolismo , Células Cultivadas , Gónadas/embriología , Gónadas/metabolismo , Interleucina-3/fisiología , Mesonefro/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Receptores de Trombopoyetina/metabolismo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Transducción de Señal , Factor de Células Madre/fisiología , Transducción GenéticaRESUMEN
Cancer stem cells (CSCs) are maintained under special microenvironment called niche, and elucidation and targeting of the CSC niche will be a feasible strategy for cancer eradication. Tumor-associated macrophages (TAMs) are known to be involved in cancer progression and thus can be a component of CSC niche. Although TAMs are known to play multiple roles in tumor progression, involvement of CSCs in TAM development fully remains to be elucidated. Using rat C6 glioma side population (SP) cells as a model of glioma CSCs, we here show that CSCs induce the TAM development by promoting survival and differentiation of bone marrow-derived monocytes. CSC-induced macrophages can be separated into two distinct subsets of cells, CD11c(low) and CD11c(high) cells. Interestingly, only the CD11c(high) subset of cells have protumoral activity, as shown by intracranial transplantation into immune-deficient mice together with CSCs. These CD11c(high) macrophages were observed in the tumor formed by co-transplantation with CSCs. Furthermore, CSCs produced GM-CSF and anti-GM-CSF antibody inhibited CSC-induced TAM development. In conclusion, CSCs have the ability to self-create their own niche involving TAMs through CSC-derived GM-CSF, which can thus be a therapeutic target in view of CSC niche disruption.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Antígenos CD11/metabolismo , Glioma/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Macrófagos/citología , Células Madre Neoplásicas/trasplante , Animales , Neoplasias Encefálicas/patología , Antígenos CD11/genética , Diferenciación Celular , Línea Celular Tumoral , Femenino , Glioma/patología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Monocitos/citología , Monocitos/metabolismo , Células Madre Neoplásicas/metabolismo , Ratas , Nicho de Células MadreRESUMEN
Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. Stem Cells 2016;34:1151-1162.
Asunto(s)
Neoplasias Encefálicas/patología , Glioma/patología , Polímeros/farmacología , Nicho de Células Madre , Andamios del Tejido/química , Animales , Neoplasias Encefálicas/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/genética , Humanos , Hierro/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Poliuretanos/farmacología , Ratas , Receptores Depuradores de Clase A/genética , Receptores Depuradores de Clase A/metabolismo , Células de Población Lateral/citología , Células de Población Lateral/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Nicho de Células Madre/genética , Transferrina/metabolismo , Resultado del TratamientoRESUMEN
Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45(low) c-Kit(high) cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45(low) c-Kit(high) cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and ß-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.
Asunto(s)
Proteínas HMGB/metabolismo , Células Mieloides/citología , Factores de Transcripción SOXF/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Técnicas de Cocultivo , Proteínas HMGB/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Progenitoras de Megacariocitos y Eritrocitos/citología , Células Progenitoras de Megacariocitos y Eritrocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXF/genéticaRESUMEN
During mouse development, definitive hematopoiesis is first detected around embryonic day 10.5 (E10.5) in the aorta-gonad-mesonephros (AGM) region, which exhibits intra-aortic cell clusters. These clusters are known to contain hematopoietic stem cells (HSCs). On the other hand, it is not clear how the cells in such clusters maintain their HSC phenotype and how they are triggered to differentiate. Here we found that an endodermal transcription factor marker, Sox17, and other F-group (SoxF) proteins, Sox7 and Sox18, were expressed in E10.5 intra-aortic cell clusters. Forced expression of any of these SoxF proteins, particularly Sox17, in E10.5 AGM CD45(low) c-Kit(high) cells, which are the major component of intra-aortic clusters, led to consistent formation of cell clusters in vitro during several passages of cocultures with stromal cells. Cluster-forming cells with constitutive Sox17 expression retained long-term bone marrow reconstitution activity in vivo. Notably, shutdown of exogenously introduced Sox17 gene expression resulted in immediate hematopoietic differentiation. These results indicate that SoxF proteins, especially Sox17, contribute to the maintenance of cell clusters containing HSCs in the midgestation AGM region. Furthermore, SoxF proteins play a pivotal role in controlling the HSC fate decision between indefinite self-renewal and differentiation during fetal hematopoiesis.
Asunto(s)
Trasplante de Médula Ósea , Proteínas HMGB/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Factores de Transcripción SOXF/genética , Animales , Aorta/embriología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Linaje de la Célula/genética , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Gónadas/embriología , Proteínas Fluorescentes Verdes/genética , Proteínas HMGB/metabolismo , Mesonefro/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Interferencia de ARN , ARN Interferente Pequeño , Factores de Transcripción SOXF/metabolismoRESUMEN
Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.
Asunto(s)
Atresia Biliar/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/metabolismo , Haploinsuficiencia , Factores de Transcripción SOXF/metabolismo , Animales , Animales Recién Nacidos , Conductos Biliares/metabolismo , Conductos Biliares/patología , Atresia Biliar/patología , Proliferación Celular , Colestasis/genética , Colestasis/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Estrés del Retículo Endoplásmico , Epitelio/metabolismo , Epitelio/patología , Femenino , Vesícula Biliar/metabolismo , Vesícula Biliar/ultraestructura , Proteínas HMGB/genética , Hepatitis Animal/genética , Hepatitis Animal/metabolismo , Hepatitis Animal/patología , Hepatocitos/metabolismo , Heterocigoto , Inmunohistoquímica , Hígado/metabolismo , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Embarazo , Factores de Transcripción SOXF/genética , Factores de TiempoRESUMEN
Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45(low)c-Kit(+) cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined six cell populations (CD45(-)c-Kit(-), CD45(-)c-Kit(low), CD45(-)c-Kit(high), CD45(low)c-Kit(high), CD45(high)c-Kit(high), and CD45(high)c-Kit(very low)). Among these six populations, CD45(low)c-Kit(high) cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45(low)c-Kit(high) cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45(low)c-Kit(high) cells. Here, we determined that CD45(low)c-Kit(high) cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45(low)c-Kit(high) cells are the major hematopoietic cells of mouse AGM.
Asunto(s)
Aorta/citología , Gónadas/citología , Células Madre Hematopoyéticas/citología , Antígenos Comunes de Leucocito/metabolismo , Mesonefro/citología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Aorta/embriología , Aorta/metabolismo , Diferenciación Celular , Gónadas/embriología , Gónadas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mesonefro/embriología , RatonesRESUMEN
During murine embryonic development, primitive hematopoiesis occurs in the yolk sac (YS). Recent studies have shown that the YS also harbors definitive hematopoietic activity. However, the population of YS cells contributing to definitive hematopoiesis has not been identified. In this study, we characterized the hematopoietic cell populations in the YS of mouse embryos from E9.5 to E14.5 in view of the expression profiles of CD45 and c-Kit. The YS cells from E9.5 to E11.5 could be divided into six populations: CD45(-) c-Kit(-) , CD45(-) c-Kit(low) , CD45(-) c-Kit(high) , CD45(low) c-Kit(high) , CD45(high) c-Kit(high) and CD45(high) c-Kit(very low) . Among these populations, CD45(low) c-Kit(high) cells showed the highest multilineage hematopoietic colony-forming activity. Later in development, the YS cells from E12.5 to E14.5 lost the second and fourth populations (i.e., they retained CD45(-) c-Kit(-) , CD45(-) c-Kit(high) , CD45(high) c-Kit(high) and CD45(high) c-Kit(very low) cells), and concurrently with the disappearance of the CD45(low) c-Kit(high) population, no significant hematopoietic activity was found in any of the populations on and after E12.5. CD45(low) c-Kit(high) YS cells, which had a round morphology with a large nucleus, possessed the ability to differentiate into myeloid and B lymphoid cells when cultured with stromal cells. These findings suggest that CD45(low) c-Kit(high) YS cells include more undifferentiated cells than the other YS cell populations and possess in vitro potency to differentiate into multilineage hematopoietic cells. Furthermore, this cell population disappears from the YS at around E12.5, when the site of hematopoiesis has already shifted to the fetal liver and the placenta.
Asunto(s)
Embrión de Mamíferos/citología , Hematopoyesis , Antígenos Comunes de Leucocito/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Saco Vitelino/citología , Animales , Linfocitos B/citología , Biomarcadores , Técnicas de Cultivo de Célula , Diferenciación Celular , Separación Celular , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Citometría de Flujo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Monocitos/citología , Células del Estroma/citología , Células del Estroma/metabolismo , Saco Vitelino/metabolismoRESUMEN
The discovery of a major hematopoietic stem cell pool in midgestation mouse embryo has defined the placenta as an important hematopoietic anatomical site. In this study, we examined the flow cytometric pattern of mouse placenta cells on embryonic days (E) 10.5 to E15.5, in view of CD45 and c-Kit expression. We also determined which population of these cells shows differentiation potential toward multiple hematopoietic lineages by performing coculture with OP9 stromal cells and colony-forming assay in methylcellulose. Only CD45(+)c-Kit(+) population showed the ability to form hematopoietic colonies including multiple lineages. To distinguish which fraction of placenta cells have the hematopoietic activity, we used GFP transgenic mice in which the fetal part of the placenta is GFP positive and the maternal part is GFP negative. E11.5 and E13.5 CD45(+)c-Kit(+) placental cells that have ability to form hematopoietic colonies are the fetal GFP positive placental cells. E11.5 and E13.5 CD45(+)c-Kit(+) placental cells that have an ability to form hematopoietic colonies mainly reside in Hoechst dye-effluxing side population area (SP). Taken together, in the placenta of mouse embryo, we conclude that SP cells in the CD45(+)c-Kit(+) fetal placental cells have the ability to form hematopoietic colonies.
Asunto(s)
Células Madre Hematopoyéticas/citología , Antígenos Comunes de Leucocito/metabolismo , Placenta/citología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Antígenos CD34/metabolismo , Línea Celular , Separación Celular , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Placenta/metabolismo , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Embarazo , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de TiempoRESUMEN
Members of the homeodomain-interacting protein kinase (HIPK) family are involved in various intracellular regulatory mechanisms. The present study focused on clarifying the functions of HIPK family members in ocular organization during late embryogenesis. HIPK1 and HIPK2 were expressed in the inner retina during late embryogenesis. Hipk1(+/-)Hipk2(-/-) mice had a greater frequency of small eyes with a lens deficiency and abnormally laminated and thickened retinas than did wild-type littermates. These data indicate that Hipk1 and Hipk2 are involved in regulation of eye size, lens formation and retinal lamination during late embryogenesis.
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Proteínas de Homeodominio/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Ojo/metabolismo , Proteínas de Homeodominio/genética , Cristalino/metabolismo , Ratones , Ratones Noqueados , Morfogénesis/genética , Retina/metabolismo , Retinaldehído/genética , Retinaldehído/metabolismoRESUMEN
A cDNA encoding a novel phospholipase A(2) (PLA(2)) inhibitor (PLI) was isolated from a Protobothrops flavoviridis snake (Tokunoshima island, Japan) liver cDNA library. This cDNA encoded a signal peptide of 19 amino acids followed by a mature protein of 181 amino acids. Its N-terminal amino acid sequence was completely in accord with that of a PLI, named PLI-II, previously found in P. flavoviridis serum. PLI-II showed a high similarity in sequence to the B subtype of gammaPLI, denoted gammaPLI-B, isolated from Agkistrodon blomhoffii siniticus serum. Thus, PLI-II is P. flavoviridis serum gammaPLI-B. Since PLI-I, previously isolated from P. flavoviridis serum, can be assigned as gammaPLI-A, P. flavoviridis serum contains both A and B subtypes of gammaPLI. Phylogenetic analysis of gammaPLIs from the sera of various kinds of snakes, Elapinae, Colubrinae, Laticaudinae, Acanthophiinae, Crotalinae, and Pythonidae, based on the amino acid sequences revealed that A and B subtypes of gammaPLIs are clearly separated from each other. It was also found that phylogenetic topologies of gammaPLIs are in good agreement with speciation processes of snakes. The BLAST search followed by analyses with particular Internet search engines of proteins with Cys/loop frameworks similar to those of PLI-II and PLI-I revealed that gammaPLI-Bs, including PLI-II and PLI-II-like proteins from mammalian sources, form a novel PLI-II family which possesses the common Cys/loop frameworks in the anterior and posterior three-finger motifs in the molecules. Several lines of evidence suggest that PLI-II is evolutionarily ancestral to PLI-I.
Asunto(s)
Bothrops/sangre , Inhibidores Enzimáticos/farmacología , Evolución Molecular , Inhibidores de Fosfolipasa A2 , Animales , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Inhibidores Enzimáticos/química , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45(low) c-Kit(+) cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45(low) c-Kit(-) cells that showed a granulocyte morphology; CD45(high) c-Kit(low/-) that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45(low) c-Kit(+) cells from the AGM culture had the abilities to reproduce CD45(low) c-Kit(+) cells and differentiate into CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) cells, whereas CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) did not produce CD45(low) c-Kit(+) cells. Furthermore, CD45(low) c-Kit(+) cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45(low) c-Kit(+) cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.
Asunto(s)
Aorta/citología , Gónadas/citología , Células Madre Hematopoyéticas/fisiología , Mesonefro/citología , Técnicas de Cultivo de Tejidos/métodos , Animales , Aorta/embriología , Diferenciación Celular , Citocinas/farmacología , Gónadas/embriología , Células Madre Hematopoyéticas/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Mesonefro/embriología , Ratones , Ratones Endogámicos ICR , Proteínas Proto-Oncogénicas c-kit/metabolismo , Trasplante de Células Madre , Células del Estroma/fisiologíaRESUMEN
Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45(+) hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45(low)c-Kit(+) cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.