Biomedical subjects

Paul J Scotting

Publications and source records attributed to Paul J Scotting.

12 recordsLinked to original sources

PubMed2007

A change in response to Bmp signalling precedes ectodermal fate choice.

Bone morphogenetic protein (Bmp) signalling plays a central role in the decision of ectoderm to adopt either neural or non-neural fates. The effects of this signalling are seen at mid-gastrulation in the activation of genes such as the Gata factors and the repression of genes such as the SoxB1 transcription factors in the non-neural regions. Using zebrafish embryos, we show that this Bmp signalling does not repress the expression of these same neural markers just 2-3 hours earlier. Since expression of the Bmp signalling effector, Smad1, only begins during early gastrulation, we tested the role of Smad1 and Smad5 (which is maternally expressed) in controlling gene expression both before and during gastrulation. This showed that the absence of Smad1 does not explain the lack of response of neural genes to Bmp signalling at early stages. However, these experiments showed that expression of the non-neural marker, gata2, is mediated by Smad5 in the absence of Smad1 at early stages, but is dependent upon Smad1 at later stages. Hence, we have shown a dynamic change in the molecular machinery underlying the Bmp response in the ectoderm during gastrulation stages of development.

Animals↗

PubMed2006

Epibranchial and otic placodes are induced by a common Fgf signal, but their subsequent development is independent.

The epibranchial placodes are cranial, ectodermal thickenings that give rise to sensory neurons of the peripheral nervous system. Despite their importance in the developing animal, the signals responsible for their induction remain unknown. Using the placodal marker, sox3, we have shown that the same Fgf signaling required for otic vesicle development is required for the development of the epibranchial placodes. Loss of both Fgf3 and Fgf8 is sufficient to block placode development. We further show that epibranchial sox3 expression is unaffected in mutants in which no otic placode forms, where dlx3b and dlx4b are knocked down, or deleted along with sox9a. However, the forkhead factor, Foxi1, is required for both otic and epibranchial placode development. Thus, both the otic and epibranchial placodes form in a common region of ectoderm under the influence of Fgfs, but these two structures subsequently develop independently. Although previous studies have investigated the signals that trigger neurogenesis from the epibranchial placodes, this represents the first demonstration of the signaling events that underlie the formation of the placodes themselves, and therefore, the process that determines which ectodermal cells will adopt a neural fate.

Animals↗

PubMed2006

Stem cell marker expression in the Bergmann glia population of the adult mouse brain.

Recent evidence suggests that the postnatal cerebellum contains cells with characteristics of neural stem cells, which had so far only been identified in the subventricular zone of the lateral ventricles and the subdentate gyrus of the hippocampus. In order to investigate the identity of these cells in the adult cerebellum, we have analyzed the expression of Sox1, a transcription factor from the SoxB1 subgroup and widely used marker of neural stem cells. In situ hybridization and the use of a transgenic mouse model show that, in the adult cerebellum, Sox 1 is only expressed in the Bergmann glia, a population of radial glia present in the Purkinje cell layer. Furthermore, another neural stem cell marker, Sox2 (also member of the SoxB1 subgroup), is also expressed in the Bergmann glia. We have previously shown that these same cells express Sox9, a member of the SoxE subgroup known for its role in glial development. Here we show that Sox9 is in fact also expressed in other regions harboring adult neural stem cells, suggesting that Sox9 represents a novel stem cell marker. Finally, using a Sox1-null mouse, we show that the formation of this Sox2/Sox9 positive Bergmann glia population does not require the presence of a functional Sox1. Our results identify these radial glia as a previously unreported Sox1/Sox2/Sox9 positive adult cell population, suggesting that these cells may represent the recently reported stem cells in the adult cerebellum.

Animals↗

PubMed2005

Sox group E gene expression distinguishes different types and maturational stages of glial cells in developing chick and mouse.

The SoxE genes, Sox8, Sox9 and Sox10, have been shown to modulate glial specification and differentiation in the peripheral nervous system and spinal cord. However, little is known of their expression during brain development. We have therefore analyzed their expression in developing brain, focused on the cerebellum. In situ hybridization showed a striking degree of temporo-spatial variation in their expression in different glial lineages in both chick and mouse. Sox8 appeared both in oligodendrocytes and astrocytes, Sox9 was more abundant in astrocytes and Sox10 was restricted to later stages of oligodendrocyte development.

Animals↗

PubMed2005

CIC, a gene involved in cerebellar development and ErbB signaling, is significantly expressed in medulloblastomas.

In children, the majority of brain tumors arise in the cerebellum. Medulloblastomas, the most common of these, are believed to originate from the granule cell lineage. We have recently identified a mammalian gene, capicua (Cic), the ortholog of a Drosophila gene implicated in c-erbB (Egfr) signaling, which is predominantly expressed during mouse granule cell development. Its expression in medulloblastoma is therefore of particular interest. In the present study the expression of human CIC in medulloblastoma was analyzed. In silico SAGE analysis demonstrated that medulloblastomas exhibited the highest level of CIC expression and expression was most common in tumors of the CNS in general. RT-PCR and in situ hybridization verified the expression of CIC in tumor cells, although the level of expression varied between different medulloblastoma subtypes. The expression of CIC did not correlate with other markers, such as neurofilament, GFAP and Mib-1. In postnatally developing cerebellum, in silico analysis and in situ hybridization both indicated a strong correlation between Cic expression and the maturation profile of cerebellar granule cell precursors. Expression of CIC is therefore a feature shared between immature granule cells and the tumors derived from them. Cic has been implicated as a mediator of ErbB signaling and this pathway has been associated with a poor prognosis for medulloblastomas. Therefore, further analysis of the role of Cic is likely to provide valuable insight into the biology of these tumors. Additionally, study of genes such as CIC should provide objective criteria by which, in combination with other markers and clinical data, to categorize these tumors into subgroups that might allow better allocation into specific treatment regimes.

Biomarkers↗

PubMed2005

Childhood solid tumours: a developmental disorder.

Several lines of evidence demonstrate that the biology, genetics and environment of childhood solid tumours (CSTs) sets them apart from adult solid tumours. The nature of the progenitor cells from which these tumours arise, and their immature tissue environment, allows CSTs to develop with fewer defects in cell regulatory processes than adult cancers. These differences could explain why CSTs are more susceptible to therapeutic intervention than adult tumours. How does the aetiology of these cancers differ from those occurring in adults and how might this affect the development of more effective therapies?

Cell Proliferation↗

PubMed2005

Strategies to investigate gene expression and function in granule cells.

Studying gene expression in granule cells is a major route to understanding the factors required for many key cellular processes such as specification, proliferation, migration, differentiation, apoptosis, tumour formation and neurodegeneration. A greater understanding of these processes will not only provide insight into cerebellum development, but also diseases of the cerebellum. Granule cells can be readily grown in culture and both viral and non-viral strategies have been optimised to allow gene transfer and expression in cultured cells. However, granule cell migration and maturation are inherent parts of cerebellum development and these rely on interactions with other cells. Hence, a true picture of gene function in these cells can only be obtained when tissue context is maintained. Studies of gene function in this context can be achieved by creation of mouse models. Conditional mouse models, where loss of gene expression is restricted as far as possible to granule cells, are by far the most informative resource in this respect. Despite their obvious benefits, the production of mouse models is both costly and time-consuming and this may be further compounded by a potential lack of phenotype due to redundancy of gene function. Organotypic slice cultures, on the other hand, are a comparatively cheap and accessible model for studies of gene function where tissue context is maintained. Recent technologies have provided the means to manipulate gene expression in such systems and are beginning to yield valuable insights into the molecular regulation of cerebellum development.

Animals↗

PubMed2004

Novel strategy to study gene expression and function in developing cerebellar granule cells.

The advent of techniques for global analyses of cell biology, such as genomics and proteomics, opens the way to rapid progress in understanding the molecular control of developing tissues. However, such studies in the CNS are hindered by the complexity of this tissue. In particular, few approaches allow cells to be isolated that are enriched for specific stages of their maturation. We describe a new strategy to study gene expression and function in cerebellar granule cells. In these experiments, we have used square pulse electroporation to introduce fluorescent dye or DNA constructs into immature granule cell precursors in situ. This method only labels granule cell precursors in the superficial part of the external granule layer. Combining this labelling with fluorescent activated cell sorting (FACS) allows the transfected cells to be isolated at any time during their subsequent development, thus providing a means of analysing granule cells as they undergo maturation. This transfection method can be used to study events in the normal maturation of granule cells or the effects of introduced transgenes. Such studies can be carried out on cells purified from primary cultures or cells in situ using cerebellar slice cultures. Our strategy provides a new route to detailed analysis of the role of genes in controlling many aspects of granule cell biology. These approaches will allow recent global analyses to be more readily applied to subpopulations of cells in complex tissues.

Animals↗

PubMed2004

Gene transfer into intact fetal skeletal muscle grown in vitro.

The development of an organ culture system for growing prenatal intercostal muscle in vitro and its use to study gene function is described. Fetal skeletal muscle is relatively inaccessible during the key stages of its development, and this method enables DNA transfections and other manipulations to be carried out. The system allows cell proliferation and differentiation to continue and also maintains the morphology and fiber types of developing muscle. Gene transfer into cultured embryonic intercostal muscle was achieved by square-pulse electroporation of intact pieces of tissue. Expression of a marker gene (GFP) was found within 5 h and maintained for 2 days in muscle fibers and cells. The technique should enable the function of genes implicated in muscle development and disease to be studied at stages when access is difficult and in a controlled environment.

Animals↗

PubMed2002

CIC, a member of a novel subfamily of the HMG-box superfamily, is transiently expressed in developing granule neurons.

We describe here the identification and characterization of a new gene, Cic, in both human and mouse genomes. These are orthologs of the Drosophila gene capicua, and represent a new subfamily of the HMG-box superfamily. Expression of the Cic gene is predominantly restricted to immature granule cells in the cerebellum, hippocampus and olfactory bulb in the CNS. This gene is therefore implicated in CNS development, in particular in granule cell development.

Amino Acid Sequence↗

PubMed2002

Pre-gut endoderm of chick embryos is regionalized by 1.5 days of development.

In this study, we set out to test the ability of endoderm from 1.5-day-old chick embryos (just before digestive tube formation) to develop region-specific characteristics when cultured heterotopically. Various parts of the 1.5-day endoderm were cultured in combination with the flank somatic mesoderm of 3- to 3.5-day chick embryos, and these cultures were analyzed for the expression of several transcription factors and the differentiation of the endoderm. By 1.5 days of normal development, the transcription factors, which are expressed in specific digestive organs, cSox2, CdxA, and cHoxb9/a13 were already expressed in the endodermal cells of the presumptive areas of their later expression domains. When 1.5-day pre-gut endoderm was cultured for 14-15 days, it showed specific differentiation into appropriate organ structures. In general, the more anterior part of the pre-gut endoderm formed the more rostral digestive organ structures while the posterior part became the caudal gut. The differentiation of these regions of endoderm matches their normal fate as recently elucidated (Matsushita [1996a] Rouxs Arch. Dev. Biol. 205:225-231; Matsushita [1999] Dev. Growth Differ. 41:313-319). Expression of cSox2, CdxA, and cHoxb9/a13 in endoderm cultured for 4-5 days is also consistent with their normal fate. Thus, each part of the pre-gut endoderm appears to be already regionally committed to some extent, in accordance with its fate by 1.5 days of development.

Animals↗

PubMed2002

Differential expression of SOX4 and SOX11 in medulloblastoma.

Primitive neuroectodermal tumors (PNETs) are composed of immature neuronal precursor cells and sometimes more mature neuronal cell types. Medulloblastomas, occuring in the cerebellum, represent the most common PNET and are broadly classified into two subgroups: classical and desmoplastic. Desmoplastic medulloblastomas exhibit a slightly better prognosis than classical medulloblastomas. However, there are currently no good molecular markers available to distinguish clinical outcome and similar treatment is used for most patients with associated complications. It has been shown that neoplastic cells in these tumors recapitulate stages in maturation of normal human neuroblasts; therefore, embryological studies of the earliest events in the development of the cerebellum may provide useful information about the molecular behavior of the tumor. Transcription factors such as Sox proteins involved in neural development may also play a role in the etiology of brain tumors. Sox4 in particular has been implicated in the biology of several other types of cancer. We have studied the expression of Sox4, and the closely related Sox11 gene, in medulloblastomas. Sox4 and Sox11 were strongly expressed in most classical medulloblastomas but only weakly in desmoplastic medulloblastomas. The expression profile of these two genes in developing cerebellum was also analyzed. Our results suggest that strong Sox4 and Sox11 expression in classical medulloblastomas reflects their maturation-dependent expression during normal cerebellum development, and that they may therefore provide markers to divide tumors into clinically relevant subgroups.

Biomarkers, Tumor↗