Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

BACKGROUND: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. METHODS: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. RESULTS: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. CONCLUSION: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

Independent regulation of Sox3 and Lmx1b by FGF and BMP signaling influences the neurogenic and non-neurogenic domains in the chick otic placode.

The development of neural tissue starts with the activation of early neural genes such as the SoxB1 transcription factors, which are expressed in response to signaling molecules. Neural progenitors in the inner ear are only generated in the anterior placodal domain, but the mechanisms that determine when and how otic neural fate is acquired are still unknown. Here, we show that Sox3 expression becomes restricted to the anterior territory of the chick otic field and that misexpression of Sox3 induces Sox2 and Delta1 in the non-neurogenic otic territory. This suggests that Sox3 plays a central role in the establishment of an otic neural fate. Furthermore, Sox3 down-regulates the expression of Lmx1b, a marker of the posterior non-neurogenic otic epithelium. The expression of Sox3 is maintained by the positive action of FGF8 derived from the otic ectoderm. On the contrary, BMP signaling does not have a major influence on neural commitment but instead regulates Lmx1b expression in the otic placode. Together, the data support the notion that Sox3 is critical for the development of the neural elements of the inner ear, and they highlight the importance of localized signaling from the ectoderm in establishing the neurogenic vs. non-neurogenic anteroposterior asymmetry that characterizes the early otic placode.

De-regulation of the sonic hedgehog pathway in the InsGas mouse model of gastric carcinogenesis.

This study investigated sonic hedgehog (Shh) signalling in gastric metaplasia in the insulin-gastrin (InsGas) hypergastrinaemic mouse +/- Helicobacter felis (H. felis) infection. Sonic hedgehog gene and protein expression was reduced in pre-metaplastic lesions from non-infected mice (90% gene reduction, P<0.01) compared to normal mucosa. Sonic hedgehog was reactivated in gastric metaplasia of H. felis-infected mice (3.5-fold increase, P<0.01) compared to pre-metaplastic lesions. Additionally, the Shh target gene, glioma-associated oncogene (Gli)-1, was significantly reduced in the gastric glands of InsGas mice (75% reduction, P<0.05) and reactivated with H. felis infection (P<0.05, base of glands, P<0.01 stroma of metaplastic glands). The ability of H. felis to activate the Shh pathway was investigated by measuring the effect of target cytokine, interleukin-8 (IL-8), on Shh expression in AGS and MGLVA1 cells, which was shown to induce Shh expression at physiological concentrations. H. felis induced the expression of NF-kappaB in inflammatory infiltrates in vivo, and the expression of the IL-8 mouse homologue, protein KC, in inflammatory infiltrates and metaplastic lesions. Sonic hedgehog pathway reactivation was paralleled with an increase in proliferation of metaplastic lesions (15.75 vs 4.39% in infected vs non-infected mice, respectively, P<0.001). Furthermore, Shh overexpression increased the growth rate of the gastric cancer cell line, AGS. The antiapoptotic protein, bcl-2, was expressed in the stroma of infected mice, along with a second Shh target gene, patched-1 (P=0.0001, stroma of metaplastic gland). This study provides evidence suggesting reactivation of Shh signalling from pre-metaplastic to advanced metaplastic lesions of the stomach and outlines the importance of the Shh pathway as a potential chemoprophylactic target for gastric carcinogenesis.

Adult mesenchymal stem cells: differentiation potential and therapeutic applications.

Adult mesenchymal stem cells (MSCs) are a population of multipotent cells found primarily in the bone marrow. They have long been known to be capable of osteogenic, adipogenic and chondrogenic differentiation and are currently the subject of a number of trials to assess their potential use in the clinic. Recently, the plasticity of these cells has come under close scrutiny as it has been suggested that they may have a differentiation potential beyond the mesenchymal lineage. Myogenic and in particular cardiomyogenic potential has been shown in vitro. MSCs have also been shown to have the ability to form neural cells both in vitro and in vivo, although the molecular mechanisms underlying these apparent transdifferentiation events are yet to be elucidated. We describe here the cellular characteristics and differentiation potential of MSCs, which represent a promising stem cell population for future applications in regenerative medicine.

Are cranial germ cell tumours really tumours of germ cells?

Germ cell tumours of the brain and those that occur in the gonads are believed to share a common origin from germ cell progenitors. This 'germ cell theory' rests upon similar histopathology between these tumours in different locations and the belief that endogenous somatic cells of the brain could not give rise to the range of cell types seen in germ cell tumours. An alternative 'embryonic cell theory' has been proposed for some classes of cranial germ cell tumours, but this still relies on the misplacement of cells in the brain (in this case the earliest embryonic stem cells) during early embryonic development. Recent evidence has demonstrated that neural stem cells of the brain can also give rise to many of the cell types seen in germ cell tumours. These data suggest that endogenous progenitor cells of the brain are a plausible alternative origin for these tumours. This idea is of central importance for studies aiming to elucidate the mechanisms of tumour development. The application of modern molecular analyses to reveal how tumour cells have altered with respect to their cell of origin relies on the certain identification of the cell from which the particular tumour arose. If the identity of this cell is mistaken, then studies to elucidate the mechanisms by which the progenitor cell has been subverted from its normal behaviour will not yield useful information. In addition, it will prove impossible to generate an appropriate animal model in which to study the underlying causes of those tumours. This article makes the case that current assumptions of the origins of cranial germ cell tumours are unreliable. It reviews the evidence in favour of the 'germ cell theory' and argues in favour of a 'brain cell theory' in which endogenous neural progenitor cells of the brain are the likely origin for these tumours. Thus, the case is made that cranial germ cell tumours, like other brain tumours, arise by the transformation of progenitor cells normally resident in the brain.

Expression of Sox8, Sox9 and Sox10 in the developing valves and autonomic nerves of the embryonic heart.

We describe the expression pattern of Sox8, Sox9 and Sox10 during the development of the chick embryo heart. These Sox genes constitute the group E of the large Sox family of transcription factors. We show that the expression of Sox8, Sox9 and Sox10 in the developing heart correlates with heart septation and with the differentiation of the connective tissue of the valve leaflets. Sox10 appears also as a specific marker of developing heart nerves. These findings fit with the occurrence of morphological and functional anomalies of the heart reported in humans deficient for Sox9 and Sox10.

The transcription factor cSox2 and Neuropeptide Y define a novel subgroup of amacrine cells in the retina.

The retina has been extensively used as a model to study the mechanisms responsible for the production of different neural cell phenotypes. The importance of both extrinsic and intrinsic cues in these processes is now appreciated and numerous transcription factors have been identified which are required for both neuronal determination and cell differentiation. In this study we have analysed the expression of the transcription factor Sox2 during development of the chick retina. Expression was found in the proliferating cells of the retina during development and was down regulated by nearly all cell types as they started to differentiate and migrate to the different layers of the retina. In one cell type, however, Sox2 expression was retained after the cells have ceased division and migrated to their adult location. These cells formed two rows located on either side of the inner plexiform layer and were also positive for Neuropeptide Y, characteristics which indicate that they were a subpopulation of amacrine cells. The expression of Sox2 by only this population of post-mitotic neurones makes it possible to follow these cells as they migrate to their adult location and shows that they initially form a single row of cells which subsequently divides to form the double row seen in the adult tissue. We suggest that retained expression of Sox2 is involved in directing the differentiation of these cells and is an early marker of this cell type.

cSox3 expression and neurogenesis in the epibranchial placodes.

Epibranchial placodes are local thickenings of the surface ectoderm, which give rise to sensory neurons of the distal cranial ganglia. The development of these placodes has remained unclear due to the lack of any definitive marker for these structures. We show here that the chick transcription factor, cSox3, is expressed in four lateral patches at the rostral edge of the epibranchial arches and that these mark the epibranchial placodes. These patches of cSox3 expression arise by gradual thinning from broader areas of cSox3 expression with concomitant loss of cSox3 in nonplacodal regions. Cells leaving the epithelial placodes as they initiate neurogenesis, lose cSox3 expression and sequentially express Ngn1, NeuroD, NeuroM, and Phox2a, but do not express Ngn2. This is in contrast to studies in the mouse where it is Ngn2, rather than Ngn1, that is predominantly expressed in epibranchial-derived neuroblasts. Overexpression of cSox3 interferes with normal neuroblast migration and results in changes in ectodermal morphology. Thus, cSox3 provides a useful tool for the study of placode formation, and loss of cSox3 expression appears to be a necessary event in normal neurogenesis from the epibranchial placodes.

Sox8 gene expression identifies immature glial cells in developing cerebellum and cerebellar tumours.

Sox8 is a member of the E subgroup of Sox genes, the other members of which are Sox9 and Sox10, both of which are implicated in specific human disorders. Recently, Sox8 homologues have been cloned in chick, mouse and human and have been shown to be strongly expressed in the embryonic and adult brain. Nevertheless, the cell types that express Sox8 have not been determined. We show here that Sox8 is expressed in immature glia in the developing cerebellum. Sox8 is also expressed in scattered cells in the cerebellar tumour, medulloblastoma. This gene therefore provides an early glial marker that may provide more detailed insight into the cellular makeup and consequent behaviour of medulloblastomas.

Sox3 expression defines a common primordium for the epibranchial placodes in chick.

The epibranchial placodes are ectodermal thickenings that generate sensory neurons of the distal ganglia of the branchial nerves. Although significant advances in our understanding of neurogenesis from the placodes have recently been made, the events prior to the onset of neurogenesis remain unclear. We found that chick Sox3 (cSox3) shows a highly dynamic pattern of expression before becoming confined to the final placodes: one pre-otic (geniculate) and three post-otic (one petrosal and two nodose) placodes. A fate-mapping study using lipophilic dyes revealed that all post-otic placodes arise within a single broad cSox3-positive domain, where cSox3 expression and epithelial thickness will be retained only in much smaller final neurogenic placodes. The data presented here suggest that post-otic placodes are remnants of a common primordium defined as a discrete domain of cSox3 expression.

Novel ERBB4 juxtamembrane splice variants are frequently expressed in childhood medulloblastoma.

We recently reported a significant relationship between tumor cell expression of the ERBB4 receptor, the most recently described member of the epidermal growth factor receptor family, and aggressive tumor phenotype in childhood medulloblastoma. Two alternative juxtamembrane (JM) isoforms of the ERBB4 receptor have been described. Termed JMa and JMb, these variants possess different receptor processing and ligand-binding characteristics. In the current study, we employed an RT-PCR and sequencing strategy to determine the pattern of ERBB4 JM isoform expression in a large (n = 78) series of pediatric medulloblastomas. JMa and JMb transcript expression was detected in 53% and 28% of tumor samples, respectively. In addition, two novel ERBB4 JM isoforms, which we have termed JMc and JMd, were isolated from 10% and 36% of tumors, respectively. Sequence analysis revealed the JMc transcript to contain a deletion of the entire JM region. In contrast, JMd includes an extended coding region, retaining both the JMa and JMb sequences. Neither of these novel isoforms was detected in normal human adult cerebellum, but expression of JMd was observed in developing fetal cerebellum, suggesting that this later isoform may represent an ERBB4 transcript restricted to primitive neuroectoderm-derived tissue. To confirm that the four ERBB4 JM isoforms arise by alternative RNA splicing, we sequenced the intron-exon junctions of the human ERBB4 gene within the JM region. This demonstrated the four ERBB4 JM variants to be encoded by two short exons containing the JMb and JMa sequences positioned in the order 5' to 3' and separated by a 121 bp intron.

Roles of Sox4 in central nervous system development.

The transcription factor-encoding gene, Sox4, is expressed in a wide range of tissues and has been shown to be functionally involved in heart, B-cell and reproductive system development. Sox4 shows a high degree of sequence homology with another group C Sox gene, Sox11, which is predominantly expressed in the CNS. Since the expression of Sox4 in the CNS has not been described we have carried out such a study. Sox4 and Sox11 expression increased simultaneously in the same early differentiating cells of the developing CNS except in the external granule layer of the cerebellum where Sox11 expression preceded that of Sox4. As development proceeded, their expression always appeared to relate to the maturational stage of the cell population, with Sox11 expression more transient than Sox4, except in the spinal cord where the reverse was true. Sox4 knock-out mice have been shown to die of a heart defect half way through gestation with no observable CNS phenotype. Our more detailed analysis showed no abnormality in the spatial restriction of expression of Sox2, Sox11, Mash1, neurogenin1 or neurogenin2, although the level of expression of Sox11 and Mash1 appeared a little different from the wild-type, implying that Sox4 might indeed have a functional role in CNS development. However, since Sox4 and Sox11 expression is so similar, we propose that Sox11 might compensate for the loss of Sox4 function in the CNS such that the phenotype is extremely mild in the Sox4 null mutant.

Chick sox10, a transcription factor expressed in both early neural crest cells and central nervous system.

Human SOX10 and mouse Sox10 have been cloned and shown to be expressed in the neural crest derivatives that contribute to formation of the peripheral nervous system during embryogenesis. Mutations in Sox10 have been identified as a cause of the Dominant megacolon mouse and Waardenburg-Shah syndrome in human, both of which include defects in the enteric nervous system and pigmentation (and in the latter, sometimes hearing). We have cloned a chick Sox10 ortholog (cSox10) in order to study its role in neural crest cell development. This cDNA reveals a 1383 bp open reading frame encoding 461 amino acids which is highly conserved with human SOX10 and mouse Sox10. In situ hybridization showed cSox10 is expressed in migrating neural crest cells just after the zinc finger transcription factor Slug, but is lost as cells undergo neuronal differentiation in ganglia of the peripheral nervous system. In addition, cSox10 is expressed in the developing otic vesicle, the developing central nervous system and pineal gland.

Paediatric brain tumours: an embryological perspective.

Primitive neuroectodermal tumours are amongst the most common paediatric tumours of the central nervous system. These tumours are composed of undifferentiated cells and a variable component of more differentiated cell types. Most analysis of these tumours has focused on molecules normally found in the differentiated cells or those found in all primitive neuronal precursors. In this article we describe recent advances in understanding of the molecular processes involved in normal neurogenesis. We discuss the relevance of these data to the biology of neuronal tumours and describe strategies we and others have adopted to investigate the usefulness of molecules found in undifferentiated neuronal tissues in understanding the events which underlie oncogenesis in this tumour type.

Role for cGATA-5 in transcriptional regulation of the embryonic chicken pepsinogen gene by epithelial-mesenchymal interactions in the developing chicken stomach.

A gene encoding embryonic chicken pepsinogen (ECPg), a zymogen of the digestive enzyme pepsin, is expressed specifically in epithelial cells of glands of embryonic stage proventriculus (glandular stomach) under the influence of mesenchyme. We found four GATA and one Sox binding motifs in 1.1 kb of the 5' flanking region of the ECPg gene which are essential to the organ-specific expression of the gene. The expression of cGATA-5 and cSox2 in the proventriculus from day 6 to day 12 of incubation was therefore analyzed. cGATA-5 was more strongly expressed in glandular epithelial cells than in luminal epithelial cells, while cSox2 gene expression was weaker in glandular epithelial cells. Using heterologous recombination explants we also discovered that the expression of cGATA-5 and cSox2 in epithelial cells was affected by mesenchyme when the latter induced ECPg gene expression in epithelial cells. Introduction of expression constructs into epithelial cells by electroporation demonstrated that cGATA-5 upregulated transcription of a reporter luciferase gene via a cis element in the 5' flanking region of the ECPg gene. The gel mobility shift assay revealed that the cGATA-5 protein specifically binds to the GATA binding sites. cSox2 downregulated the activity of luciferase but it was not through the Sox binding motif. These results suggest that cGATA-5 positively regulates transcription of the ECPg gene and is involved in spatial regulation of the pepsinogen gene during development.

Rat embryonic myoblasts are restricted to forming primary fibres while later myogenic populations are pluripotent.

Three populations of myoblasts, embryonic, foetal and adult, appear sequentially during myogenesis. The present study uses retroviruses to mark myoblasts clones in vivo from these populations. Myoblasts labelled at E15 (embryonic) contributed to primary fibres only. The majority of marked primary fibres were slow but a small number of clones contained marked primaries which were no longer slow at E19. Myoblasts labelled at E17 (foetal) fused with both primary and secondary fibres and most clones contained both fast and slow fibres. Similarly, adult myoblasts marked at P0 fused with all fibre types. These results indicate that embryonic myoblasts are restricted to producing only primary fibres which are initially slow but which can convert to being fast. Clones of foetal and adult myoblasts fuse with both primary and secondary fibres which may be either fast or slow.

Region-specific expression of chicken Sox2 in the developing gut and lung epithelium: regulation by epithelial-mesenchymal interactions.

In situ analysis of the chicken cSox2 gene, a member of the transcription factor family containing an Sry-like high-mobility group (HMG) box, demonstrated localized expression in the embryonic endoderm. Transcripts of cSox2 appeared before commencement of morphogenesis and cytodifferentiation in the rostral gut epithelium from the pharynx to the stomach. The caudal limit of cSox2 expression coincided with that of the region competent for proventricular differentiation and to the rostral limit of the domain of CdxA, a homologue of Drosophila caudal. During morphogenesis, the level of transcripts of cSox2 decreased in epithelia invaginating into surrounding mesenchyme to form glandular or tubular structures, such as the primordia of the thyroid and lung, glandular epithelium of the proventriculus, and secondary bronchus of the lung. Tissue recombination experiments demonstrated that cSox2 expression is regulated by the underlying mesenchyme as well as morphogenesis and cytodifferentiation. The results suggest that cSox2 plays pivotal roles in generating morphologically and physiologically distinct types of epithelial cells in the gut.

Granule cell development in the cerebellum is punctuated by changes in Sox gene expression.

Development of the vertebrate cerebellum is unusual compared to most other regions of the brain since it involves two germinal regions. Most cell types arise from the luminal, ventricular zone as in other brain regions, but granule cells arise from the second germinal layer, the external granular layer (EGL). Our analysis of the temporal and positional expression of three members of the Sox gene family of transcription factors in the cerebellum shows that granule cell development is unusual compared to most other neurons of the central nervous system (CNS). We show that granule cell precursors lose expression of cSox2 and cSox3 as they migrate to form the EGL. The EGL is the first example of a germinal layer in the CNS which does not exhibit expression of these genes. Throughout most of the CNS cSox11 expression is very low in the ventricular zone but increases dramatically as cells cease proliferation and migrate to form the subventricular zone. We also find that cSox11 expression increases when cells of the cerebellum migrate to form the EGL, but levels of expression as high as that in the subventricular zone are only seen when cells cease proliferation and migrate inwards to form the deep EGL. These observations demonstrate that cells of the proliferative superficial EGL differ qualitatively from cells of the ventricular zone in their expression of Sox genes whereas the post-proliferative cells of the deep EGL appear analogous, in their expression of Sox genes, to cells of the subventricular zone.

Non-isotopic in situ hybridization to detect chick Sox gene mRNA in plastic-embedded tissue.

In situ hybridization techniques have rapidly become widely used by the molecular biologist for the localization of specific nucleic acid sequences in individual cells or tissues. We describe the demonstration of Sox gene mRNA in chick tissue that has been embedded in the plastic methyl methacrylate to permit the preparation of sections for high-resolution light microscopy. Polymerization of the plastic was induced by using either N,N-dimethylaniline or N,N-3,5-tetramethylaniline. The in situ hybridization technique used was non-isotopic and used a digoxigenin-labelled probe detected with an antibody bound to alkaline phosphatase, which was then localized using X-phosphate-Nitro BT as a substrate-chromogen mix. Various pretreatments of the tissue sections were investigated, including the use of proteinase K, and heat-mediated techniques using a microwave oven and a pressure cooker. The best results were produced using pressure cooking on tissue in which the plastic had been chemically polymerized with N,N-3,5-tetramethylaniline. For the demonstration of Sox 11, this combination had a critical influence on the staining results, but for Sox 21 all protocols used produced good staining.