N6-Methyladenosine detected in RNA of testicular germ cell tumors is controlled by METTL3, ALKBH5, YTHDC1/F1/F2, and HNRNPC as writers, erasers, and readers.

BACKGROUND: Type II testicular germ cell tumors (GCTs) arise from a common precursor lesion (germ cell neoplasia in situ) and are stratified into seminomas and non-seminomas, which differ considerably in morphology, gene expression, and epigenetic landscape. The N6-methyladenosine (6mA) epigenetic modification is the most abundant modification in mRNA and is also detectable in eukaryotic DNA. The functional role of 6mA is not fully understood, but 6mA residues may influence transcription by affecting splicing, miRNA processing, and mRNA stability. Additionally, the methyl group of 6mA destabilizes Watson-Crick base-pairing affecting RNA structure and protein binding. OBJECTIVES: Here, we analyzed the presence of the 6mA epigenetic modification in germ cells and GCT tissues and cell lines. MATERIALS AND METHODS: We screened for the presence of 6mA in DNA and RNA by immunohistochemistry, mass spectrometry or ELISA-based quantification assays. Additionally, expression of 6mA writer-, eraser- and reader-factors was analyzed by microarrays, qRT-PCR, western blotting and screening of public databases. RESULTS: We demonstrate that 6mA is detectable in RNA, but not DNA, of GCT cell lines and tissues, fibroblasts, and Sertoli cells as well as germ cells of different developmental stages. Based on expression analyses, our results suggest METTL3, ALKBH5, YTHDC1, YTHDF1, YTHDF2 and HNRNPC as main writers, erasers, and readers of the 6mA modification in GCTs. DISCUSSION: Owing to the lack of 6mA in DNA of GCTs, a functional role in regulating DNA transcription can be excluded. Interestingly, expression levels of 6mA regulators are comparable between tumor and normal tissues/cells, suggesting a similar mechanism of 6mA regulation in RNA. Finally, we demonstrate that 6mA levels in RNA increase upon differentiation of GCT cell lines, suggesting a role of 6mA in cell fate decisions. CONCLUSION: In summary, our data provide the starting point for further experiments deciphering the role of 6mA in the RNA of GCTs.

AP2γ controls adult hippocampal neurogenesis and modulates cognitive, but not anxiety or depressive-like behavior.

Hippocampal neurogenesis has been proposed to participate in a myriad of behavioral responses, both in basal states and in the context of neuropsychiatric disorders. Here, we identify activating protein 2γ (AP2γ, also known as Tcfap2c), originally described to regulate the generation of neurons in the developing cortex, as a modulator of adult hippocampal glutamatergic neurogenesis in mice. Specifically, AP2γ is present in a sub-population of hippocampal transient amplifying progenitors. There, it is found to act as a positive regulator of the cell fate determinants Tbr2 and NeuroD, promoting proliferation and differentiation of new glutamatergic granular neurons. Conditional ablation of AP2γ in the adult brain significantly reduced hippocampal neurogenesis and disrupted neural coherence between the ventral hippocampus and the medial prefrontal cortex. Furthermore, it resulted in the precipitation of multimodal cognitive deficits. This indicates that the sub-population of AP2γ-positive hippocampal progenitors may constitute an important cellular substrate for hippocampal-dependent cognitive functions. Concurrently, AP2γ deletion produced significant impairments in contextual memory and reversal learning. More so, in a water maze reference memory task a delay in the transition to cognitive strategies relying on hippocampal function integrity was observed. Interestingly, anxiety- and depressive-like behaviors were not significantly affected. Altogether, findings open new perspectives in understanding the role of specific sub-populations of newborn neurons in the (patho)physiology of neuropsychiatric disorders affecting hippocampal neuroplasticity and cognitive function in the adult brain.

Kit transduced signals counteract erythroid maturation by MAPK-dependent modulation of erythropoietin signaling and apoptosis induction in mouse fetal liver.

Signaling by the stem cell factor receptor Kit in hematopoietic stem and progenitor cells is functionally associated with the regulation of cellular proliferation, differentiation and survival. Expression of the receptor is downregulated upon terminal differentiation in most lineages, including red blood cell terminal maturation, suggesting that omission of Kit transduced signals is a prerequisite for the differentiation process to occur. However, the molecular mechanisms by which Kit signaling preserves the undifferentiated state of progenitor cells are not yet characterized in detail. In this study, we generated a mouse model for inducible expression of a Kit receptor carrying an activating mutation and studied its effects on fetal liver hematopoiesis. We found that sustained Kit signaling leads to expansion of erythroid precursors and interferes with terminal maturation beyond the erythroblast stage. Primary KIT(D816V) erythroblasts stimulated to differentiate fail to exit cell cycle and show elevated rates of apoptosis because of insufficient induction of survival factors. They further retain expression of progenitor cell associated factors c-Myc, c-Myb and GATA-2 and inefficiently upregulate erythroid transcription factors GATA-1, Klf1 and Tal1. In KIT(D816V) erythroblasts we found constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, elevated expression of the src kinase family member Lyn and impaired Akt activation in response to erythropoietin. We demonstrate that the block in differentiation is partially rescued by MAPK inhibition, and completely rescued by the multikinase inhibitor Dasatinib. These results show that a crosstalk between Kit and erythropoietin receptor signaling cascades exists and that continuous Kit signaling, partly mediated by the MAPK pathway, interferes with this crosstalk.

Are testicular mast cells involved in the regulation of germ cells in man?

Protease activated receptor-2 (PAR-2) is the receptor for the prototype mast cell product tryptase. PAR-2 expression by cells of the human germinal epithelium was reported, but the exact cellular sites of testicular expression remained unknown. That became of interest, because mast cells, expressing tryptase, were found in the walls of seminiferous tubules of patients suffering from sub- and infertility. This location suggested that mast cells via tryptase might be able to influence PAR-2-expressing cells in the germinal epithelium. To explore these points, we used testicular paraffin-embedded sections for immunohistochemistry. PAR-2-positive cells were mostly basally located cells of the seminiferous epithelium, namely spermatogonia. Some stained for the receptor for GDNF (GFRalpha-1), and possibly represent spermatogonial stem cells (SSCs). As true human SSCs could not be examined, we turned to TCam-2 seminoma cells, expressing PAR-2 and stem cell markers, including GFRalpha-1. TCam-2 cells robustly responded to stimulation with a specific PAR-2 agonist (SLIGKV) by increased intracellular Ca(2+) levels. Recombinant tryptase and trypsin, but not a control peptide (VKGILS) evoked this response, implying functional PAR-2. Video imaging and caspase 3/7 assays showed that SLIGKV and tryptase prevented spontaneous apoptosis and increased proliferation of TCam-2 cells. The expression of the marker of pluripotency OCT3/4 was unchanged upon activation of PAR-2, suggesting that the stem cell-like character is not changed. Furthermore, human germ cell cancers were examined. A subset of seminoma and carcinoma in situ samples expressed PAR-2, indicating that yet unknown subgroups exist. Collectively, the descriptive data obtained in human testicular sections, in germ cell cancers and the functional results in TCam-2 cells imply a trophic role of mast cell-derived tryptase for human germ cells. This may be relevant for subtypes of human germ cell cancers, and possibly SSCs. It also raises the possibility that PAR-2 agonists might be useful for the in vitro propagation of human SSCs.

MYCN and ALKF1174L are sufficient to drive neuroblastoma development from neural crest progenitor cells.

Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALK(F1174L). For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycER(T)). Expression of MYCN or ALK(F1174L), one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycER(T) activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.

TGF-ß1, EGF and FGF4 synergistically induce differentiation of the seminoma cell line TCam-2 into a cell type resembling mixed non-seminoma.

Malignant germ-cell tumours arise from a neoplastic precursor, the carcinoma in situ, and develop into seminomas and/or non-seminomas (embryonal carcinomas, teratomas, yolk-sac tumours and choriocarcinomas). Based on histological and clinical findings, it has been postulated that seminomas can eventually transform into non-seminomas. Here, we used the cell line TCam-2 as model for seminomas and interrogated their differentiation potential. We demonstrate that TCam-2 cells are able to differentiate into mixed non-seminomatous lineages after supplementing the media with TGF-ß1, EGF and FGF4. On a molecular level, the differentiation is initiated by repression of BMP/SMAD signalling. As a consequence, BLIMP1, a molecule known to inhibit the differentiation of murine primordial germ cells, is down-regulated and differentiation-inhibiting histone modifications are lost. The appearance of multinucleated giant cells and the expression of marker genes indicate that cells differentiate predominantly into extra-embryonic choriocarcinoma-like cells. This is most likely due to the presence of components of the Hippo pathway, TEAD4 and YAP1. These molecules have been described to trigger extra-embryonic fate determination in the murine system. This study supports the model that seminomas indeed have an intrinsic ability to transform into a non-seminoma. In addition, the data suggest that the transformation does not require an additional mutation, but can be triggered by changes in the tumour microenvironment.

The role of BLIMP1 and its putative downstream target TFAP2C in germ cell development and germ cell tumours.

During the past years, much information has been gathered regarding the genetic and epigenetic programmes leading to the specification and maintenance of primordial germ cells. Expression of the transcriptional regulator BLIMP1 (PRDM1) is regarded as the main event in germ cell specification. BLIMP1 induces a set of target genes, one of them being transcription factor TFAP2C (AP-2γ, Tcfap2c). In murine loss of function models Blimp1 and Tcfap2c share an identical phenotype, strengthening the assumption that they act in the same pathway. One major role of this pathway is the inhibition of somatic differentiation in germ cells. BLIMP1 and TFAP2C are also expressed in carcinoma in situ (CIS, IGCNU, TIN) and in seminoma. As pointed out herein, the presence of both proteins helps to explain the undifferentiated nature of these germ cell tumours. In addition, we performed a meta-analysis of high-throughput datasets searching for TFAP2C/Tcfap2c target genes. This analysis leads us to suggest Nanos3, Dmrt1 and Dnmt3b as potential TFAP2C/Tcfap2c target genes with relevance to germ cell development and germ cell tumours.

TCam-2 but not JKT-1 cells resemble seminoma in cell culture.

Of all malignancies diagnosed in men between 17 and 45 years of age, 60% are germ cell tumors (GCT). GCT arise from carcinoma in situ cells, which are thought to originate from a transformed fetal germ cell, the gonocyte. Seminoma together with embryonal carcinoma represent the most frequent subtypes of GCT. However, the nature of the molecular pathways involved in seminoma formation remains elusive. Therefore, analysis of appropriate cell culture systems is an important prerequisite for further understanding of the etiology of this tumor entity. Although several cell lines for embryonal carcinoma have been established and analyzed, so far only two cell lines from seminoma patients have been reported. In the present study, we have analyzed these seminoma cell lines (TCam-2 and JKT-1) and compared the gene-expression profiles with those of normal tissue and of seminoma and embryonal carcinoma by using DNA Array technology. We have found that TCam-2 clusters with the group of classical seminoma, whereas JKT-1 clusters with the group of embryonal carcinoma. Using reverse transcription/polymerase chain reaction, Western blot, and immunohistochemistry, we have confirmed the seminoma-like nature of TCam-2, whereas JKT-1 lacks expression for most of the genes detectable in GCTs, thus making doubtful the germ cell nature of this cell line. The data represent the first genome-wide expression analysis of the two cell lines and comparison/clustering with subgroups of germ cell tumors. Only TCam-2 seems to represent a suitable in vitro model for seminoma.

c-KIT is frequently mutated in bilateral germ cell tumours and down-regulated during progression from intratubular germ cell neoplasia to seminoma.

Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men; 5% of these patients develop a second TGCT in the contralateral testis. The pathogenesis of TGCT is closely linked to primordial germ cells (PGCs) or gonocytes. The receptor tyrosine kinase (c-KIT) is necessary for migration and survival of PGCs and is expressed in intratubular neoplastic germ cells (IGCNUs) and seminomas. We studied the frequency of c-KIT exon 11 and 17 mutations in 155 unilateral (108 seminomas and 47 non-seminomas) and 22 bilateral (18 seminomas, two embryonal carcinomas, two IGCNU) cases. While no mutations were detected in exon 11, the mutation frequency in exon 17 was significantly higher in bilateral (14/22, 63.6%) compared to unilateral TGCT (10/155, 6.4%) (p < 0.001). Different activating mutations (Y823D, D816V, D816H and N822K) were detected in bilateral TGCT. Y823D mutation was identical in both testes in three cases and quantitative pyrosequencing showed that up to 76% of the cells analysed in tumour samples carried this mutation. One bilateral synchronous seminoma revealed a S821F mutation in one testis and a Y823D mutation contralaterally. To study the role of c-KIT in TGCT progression, we compared its expression in 41 seminomas and adjacent IGCNUs. Immunohistochemical analysis revealed that c-KIT expression was significantly reduced in seminomas compared to IGCNUs (p < 0.006) and that there were no significant changes in c-KIT mRNA copy numbers in progressed compared to low-stage seminomas. In summary, our study shows that patients with c-KIT mutations are more prone to develop a bilateral TGCT and suggests that in a portion of bilateral TGCTs, c-KIT mutations occur early during embryonal development, prior to the arrival of PGCs at the genital ridge. Furthermore, our findings show that c-KIT down-regulation occurs during the progression of IGCNU to seminoma.

[Embryonal germ cells and germ cell tumors]. / Embryonale Keimzellen und Keimzelltumoren.

Testicular germ cell tumors comprise of group of pluripotent tumors including seminomas and nonseminomas, arise from intratubular germ cell neoplasia and originate from the primordial germ cells/ gonocytes. Many well characterized markers of embryonic stem cells including CD9, PODXL and centromere-specific histone-H3-like protein CENPA are consistently expressed in TGCTs. In embryonic stem cells, pluripotency and self renewal capacities are provided by a network of OCT3/4, NANOG and SOX2. In testicular germ cell tumors, pluripotency genes OCT3/4 und NANOG are upregulated both, in seminomas and non-seminomas, while SOX2 is differentially upregulated in embryonal carcinomas only. Similar to embryonic stem cells, most histological elements of type II GCTs are sensitive to chemotherapy and irradiation. Furthermore, all invasive TGCTs show a consistent gain of the short arm of chromosome 12, as found in ES cells upon extensive in vitro culturing. Moreover, the genetic constitution of testicular germ cell tumors can also be linked to characteristics of embryonic stem cells, likely related to their specific inability to repair DNA damage and their high sensitivity to apoptotic cell death. In conclusion, testicular germ cell tumors represent embryonic cancers found in adults. Both the seminomas and nonseminomas have their specific population of stem cells representative of the primordial germ cells/gonocytes and for embryonic stem cells, respectively.

[On the role of transcription factor family AP-2 for development and disease--lessons from mouse models]. / Uber die Rolle der AP-2 Transkriptionsfaktoren in Entwicklung und bei Krankheitsgeschehen--was wir von Mausmodellen lernen können.

To analyze the consequence of ,gain of function' and ,loss of function' of transcription factor AP-2gamma in mice Using pronuclear injection, mice harbouring a mammary gland specific transgene were established and analyzed. Constitutive and conditional knockout mice complement the analysis, showing the consequences of loss of function for specific tissues. Gain of AP-2 expression on it's own in mammary gland results in enhanced proliferation which is compensated by accelerated apoptosis. Only after mating with a second transgenic mouseline, the MMTV-ErbB2 oncomouse model, an effect on tumor progression was observed. Hence, AP-2 might influence progression, but not initiation of mammary tumors. Knockout mouse models reveal that AP-2gamma is essential for the extraembryonic cells indicating a role for trophoblast cell lineage These and other studies indicate that AP-2gamma might be a molecule which enables proliferation of undifferentiated precursors or transient amplifying cells. Reexpression in tumors correlates with dedifferentiation and progression.

Diagnostic value of markers M2A, OCT3/4, AP-2gamma, PLAP and c-KIT in the detection of extragonadal seminomas.

AIMS: To compare the suitability of new seminoma markers including transcription factors AP-2gamma, OCT3/4 and M2A for detection of metastatic and extragonadal seminomas with the two well-known markers c-KIT and PLAP. MATERIALS AND METHODS: The immunohistochemical distribution of PLAP, c-KIT, M2A, AP-2gamma and OCT3/4 was examined in two pineal germinomas, 28 metastatic seminomas and 10 of their testicular primaries. Evaluation of specificity was achieved by additional tissue array studies on 75 malignancies other than germ cell tumours (GCT). Clinical data including serum PLAP were available in 18 patients. RESULTS: Compared with other markers, significantly better staining results were observed with antibodies to M2A and AP-2gamma in all seminomatous GCT. In contrast, the staining pattern with antibodies to c-KIT, PLAP and OCT3/4 was variable or absent. The lowest specificity was obtained with c-KIT, which was expressed in a variety of non-GCT. The only M2A+ mesothelioma expressed no other seminoma markers. No correlation between serum PLAP level and tissue PLAP expression was found. CONCLUSIONS: M2A and AP-2gamma are the most sensitive markers for seminoma metastases or primary extragonadal seminomas. Combination of these markers provides highly specific and clear results for detection of a seminomatous GCT.

Normal embryonic development and cardiac morphogenesis in mice with Wnt1-Cre-mediated deletion of connexin43.

Mice harboring a null mutation in the gap junction protein connexin43 (Cx43) die shortly after birth due to an obstruction of the right ventricular outflow tract of the heart. These hearts exhibit prominent pouches at the base of the pulmonary outlet, i.e., morphological abnormalities that were ascribed to Cx43-deficiency in neural crest cells. In order to examine the Cx43 expression pattern in neural crest cells and derived tissues and to test whether neural crest-specific deletion of Cx43 leads to the conotruncal defects seen in Cx43null mice, we ablated Cx43 using a Wnt1-Cre transgene. Deletion of Cx43 was complete and occurred in neural crest cells as well as in neural crest-derived tissues. Nevertheless, hearts of mice lacking Cx43 specifically in neural crest cells were indistinguishable from controls. Thus, the morphological heart abnormalities of Cx43 null mice are most likely not caused by lack of Cx43 in neural crest cells.

Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects.

Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is a congenital anomaly disorder associated with hemizygous 22q11 deletions. We previously showed that bacterial artificial chromosome (BAC) transgenic mice overexpressing four transgenes, PNUTL1, (CDCrel-1), GP1B beta, TBX1 and WDR14, had reduced viability, cardiovascular malformations and thymus gland hypoplasia. Since these are hallmark features of VCFS/DGS, we analyzed the mice for additional anomalies. We found that the mice have important defects in the middle and inner ear that are directly relevant to the disorder. The most striking defect was the presence of chronic otitis media, a common finding in VCFS/DGS patients. In addition, the mice had a hyperactive circling behavior and sensorineural hearing loss. This was associated with middle and inner ear malformations, analogous to Mondini dysplasia in humans reported to occur in VCFS/DGS patients. We propose that overexpression of one or more of the transgenes is responsible for the etiology of the ear defects in the mice. Based upon its pattern of expression in the ear and functional studies of the gene, TbX1 likely plays a central role. Haploinsufficiency of TBX1 may be responsible for ear disorders in VCFS/DGS patients.

TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome.

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.

Expression of Cdcrel-1 (Pnutl1), a gene frequently deleted in velo-cardio-facial syndrome/DiGeorge syndrome.

The murine Cdcrel-1 (Pnutl1) gene belongs to the family of septins, which are thought to be involved in cytokinesis in yeast, Drosophila and vertebrates. Recent studies implicate Cdcrel-1 in the regulation of vesicle transport in neurons of the adult brain. The human homologue, hCDCREL-1 maps to chromosome 22q11.2, a region commonly deleted in patients displaying velo-cardio-facial syndrome (VCFS) or DiGeorge syndrome (DGS). During development, Cdcrel-1 transcripts are expressed from E10.5 on in the nervous system such as the dorsal root ganglia and the cranial ganglia as well as the lateral layer of the neural tube, the area where terminally differentiated neurons are located. Low level expression is found in the mesenchyme of the frontonasal mass and the limb bud mesenchyme of E11.5 and E13.5 murine embryos. At E15.5, expression is detected in the nervous tissue and in the neural layer of the eye. Based on the expression pattern as well as clinical data, Cdcrel-1 may be involved in the etiology of VCFS/DGS.

Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control.

AP-2 transcription factors represent a family of three closely related and evolutionarily conserved sequence-specific DNA-binding proteins, AP-2alpha, -beta and -gamma. Subsequent studies have identified spatially and temporally regulated embryonic expression patterns in a number of different tissues including neural crest derivatives, neural, epidermal and urogenital tissues. Here, we review the current understanding of developmental defects in AP-2-deficient mice and consider regulatory functions of AP-2 in control of apoptosis, cell cycle, and gene expression. Recently, the first inherited human disorder, Char syndrome, was identified to be caused by AP-2beta missense mutations. In light of the manifold and essential functions of AP-2 proteins in cell growth, differentiation and programmed death, mutations or changes in precisely programmed expression patterns are likely to contribute to other congenital malformations or neoplastic diseases.

Spatiotemporal expression pattern of keratins in skin of AP-2alpha-deficient mice.

Transcription factor AP-2alpha has been implicated as being a cell-type-specific regulator of gene expression during vertebrate embryogenesis based on its expression pattern in neural crest cells, ectoderm, and the nervous system in mouse and frog embryos. In mice, AP-2alpha is expressed in surface ectoderm beginning at the single cell layer state around E8.75. AP-2alpha-deficient mice, derived by targeted mutagenesis, display a severe ventral closure defect resulting in cranio-abdominoschisis and a hypoplasia of the cranial ganglia. This study analyzed the effect of a targeted disruption of the AP-2alpha gene on the architecture and the expression of intermediate filaments in skin. We analyzed skin samples from newborn mice and found no difference in either the morphology of the skin or the amount of intermediate filaments expressed. This suggests that despite the results from other analyses, loss of transcription factor AP-2alpha does not affect the expression of intermediate filaments in the skin of newborn animals. We found an altered spatial distribution of intermediate filament expression in the single layered cranial ectoderm during days 9-12 of gestation leading to an evenly distributed expression of keratin 5 and 15 in the mutants. Furthermore, the mutants lack a ring of ectodermal cells highly positive for keratin 15 in the area where lens induction occurs, indicating a defect in the inductive interactions underlying eye formation.