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Tom Strachan

Publications and source records attributed to Tom Strachan.

18 recordsLinked to original sources
PubMed2005

3D modelling, gene expression mapping and post-mapping image analysis in the developing human brain.

As human brain development proceeds, there are complex changes in size and shape, most notably in the developing forebrain. Molecular technologies enable us to characterise the gene expression patterns that underlie these changes. To interpret these patterns the location of expression must be identified and, often, gene expression patterns compared for several genes or across several developmental stages. To facilitate interpretation we have generated a set of three-dimensional models using a recently developed technique, optical projection tomography. The models act as a framework onto which gene expression patterns are mapped and anatomical domains identified using custom-designed software, MAPaint. Here, we demonstrate their use to compare forebrain development at two embryonic stages (Carnegie stages 18 and 21; 44 and 52 days post conception, respectively) and as a means of recording, storing and visualising gene expression data for three example genes EMX1, EMX2 and OTX2. Anatomical domains were also mapped to the models and the comparison of gene expression and anatomical data is demonstrated at Carnegie stage 21. The three-dimensional models and sophisticated software facilitate the analysis and visualisation of morphological changes and gene expression patterns during early brain development and can be applied to the development of other complex structures.

Brain↗
PubMed2005

Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages.

The homeobox transcription factor Nanog has been proposed to play a crucial role in the maintenance of the undifferentiated state of murine embryonic stem cells. A human counterpart, NANOG, has been identified, but its function and localization have not hitherto been described. We have used a combination of RNA interference and quantitative real-time polymerase chain reaction to study NANOG in human embryonic stem and embryonic carcinoma cells. Transfection of NANOG-specific small interfering RNAs reduced levels of NANOG transcript and protein and induced activation of the extraembryonic endoderm-associated genes GATA4, GATA6, LAMININ B1, and AFP as well as upregulation of trophectoderm-associated genes CDX2, GATA2, hCG-alpha, and hCG-beta. Immunostaining of preimplantation human embryos showed that NANOG was expressed in the inner cell mass of expanded blastocysts but not in earlier-stage embryos, consistent with a role in the maintenance of pluripotency. Taken together, our findings suggest that NANOG acts as a gatekeeper of pluripotency in human embryonic stem and carcinoma cells by preventing their differentiation to extraembryonic endoderm and trophectoderm lineages.

Blastocyst↗
PubMed2005

Cornelia de Lange Syndrome and the link between chromosomal function, DNA repair and developmental gene regulation.

Cornelia de Lange Syndrome (CdLS) is a rare multiple malformation disorder with characteristic facial features, growth and cognitive retardation, and many other abnormalities. CdLS individuals were recently shown to have heterozygous mutations in a previously uncharacterised gene, NIPBL, which encodes delangin, a homologue of fungal Scc2-type sister chromatid cohesion proteins and the Drosophila Nipped-B developmental regulator. Nipped-B and vertebrate delangins are also now known to regulate sister chromatid cohesion, probably as part of oligomeric complexes required to load cohesin subunits onto chromatin. CdLS is likely to be one of several developmental disorders resulting from defective expression of a multi-functional protein with roles in chromosome function, gene regulation and double-strand DNA repair - a combination of properties shared by certain bacterial proteins responsible for structural maintenance of chromatin.

Animals↗
PubMed2005

An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses. We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESC-derived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs.

Animals↗
PubMed2004

3 dimensional modelling of early human brain development using optical projection tomography.

BACKGROUND: As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. RESULTS: Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (approximately 300 microm by approximately 150 microm) to the rootlets of cranial nerve XII (approximately 20 microm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is approximately 30 microm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. CONCLUSION: In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of approximately 20 microm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

Brain↗
PubMed2004

A giant novel gene undergoing extensive alternative splicing is severed by a Cornelia de Lange-associated translocation breakpoint at 3q26.3.

Cornelia de Lange syndrome (CdLS) is a rare developmental malformation syndrome characterised by mental handicap, growth retardation, distinctive facial features and limb reduction defects. The vast majority of CdLS cases are sporadic. We carried out a high density bacterial artificial chromosome (BAC) microarray comparative genome hybridisation screen but no evidence was found for a consistent pattern of microdeletion/microduplication. As an alternative, we focused on identifying chromosomal regions spanning associated translocation breakpoints. We prioritised the distal 3q region because of the occurrence, in a classical CdLS patient, of a de novo balanced translocation with a breakpoint at 3q26.3 and of reports of phenotypic overlap between cases of mild CdLS and individuals trisomic for the 3q26-q27 region. We show that the 3q26.3 breakpoint severs a previously uncharacterised giant gene, NAALADL2, containing at least 32 exons spanning 1.37 Mb. Northern blot analysis identified up to six different transcripts in the 1-10 kb range with strongest expression in kidney and placenta; embryonic expression was largely confined to duodenal and stomach endoderm, mesonephros, metanephros and pancreas. Transcript analysis identified extensive alternative splicing leading to multiple 5' and 3' untranslated regions and variable coding sequences. Multiple protein isoforms were defined by different N-terminal regions (with at least four alternative initiating methionine codons), and by differential protein truncation/use of alternative C-terminal sequences attributable to alternative splicing/polyadenylation. Outside the N-terminal regions, the predicted proteins showed significant homology to N-acetylated alpha-linked acidic dipeptidase and transferrin receptors. Mutation screening of NAALADL2 in a panel of CdLS patient DNA samples failed to identify patient-specific mutations. We discuss the possibility that the 3q26.3 translocation could nevertheless contribute to pathogenesis.

Alternative Splicing↗
PubMed2004

NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a multiple malformation disorder characterized by dysmorphic facial features, mental retardation, growth delay and limb reduction defects. We indentified and characterized a new gene, NIPBL, that is mutated in individuals with CdLS and determined its structure and the structures of mouse, rat and zebrafish homologs. We named its protein product delangin. Vertebrate delangins have substantial homology to orthologs in flies, worms, plants and fungi, including Scc2-type sister chromatid cohesion proteins, and D. melanogaster Nipped-B. We propose that perturbed delangin function may inappropriately activate DLX genes, thereby contributing to the proximodistal limb patterning defects in CdLS. Genome analyses typically identify individual delangin or Nipped-B-like orthologs in diploid animal and plant genomes. The evolution of an ancestral sister chromatid cohesion protein to acquire an additional role in developmental gene regulation suggests that there are parallels between CdLS and Roberts syndrome.

Animals↗
PubMed2004

A perspective on inversin.

Over the past 5 years, there has been increasing evidence for the role of primary (9+0) cilia in renal physiology and in establishing the left-right axis. The cilia in the renal tract are immotile and thought to have a sensory function. Cilia at the murine embryonic node have a vortical movement that sets up a leftward flow. Inversin, the protein defective in the inv mouse and in patients with type-2 nephronophthisis, localizes to both renal and node primary cilia. However, we present evidence that it is also expressed before the node forms and that its subcellular localization in renal tubular cells is not confined to the cilia. Its role in both the pathway determining left-right axis and renal function remains to be elucidated.

Animals↗
PubMed2004

Derivation, growth and applications of human embryonic stem cells.

Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass cells of blastocysts with the potential to maintain an undifferentiated state indefinitely. Fully characterised hES cell lines express typical stem cell markers, possess high levels of telomerase activity, show normal karyotype and have the potential to differentiate into numerous cell types under in vitro and in vivo conditions. Therefore, hES cells are potentially valuable for the development of cell transplantation therapies for the treatment of various human diseases. However, there are a number of factors which may limit the medical application of hES cells: (a) continuous culture of hES cells in an undifferentiated state requires the presence of feeder layers and animal-based ingredients which incurs a risk of cross-transfer of pathogens; (b) hES cells demonstrate high genomic instability and non-predictable differentiation after long-term growth; and (c) differentiated hES cells express molecules which could cause immune rejection. In this review we summarise recent progress in the derivation and growth of undifferentiated hES cells and their differentiated progeny, and the problems associated with these techniques. We also examine the potential use of the therapeutic cloning technique to derive isogenic hES cells.

Blastocyst↗
PubMed2004

Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture.

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.

Adult↗
PubMed2003

Mutations in INVS encoding inversin cause nephronophthisis type 2, linking renal cystic disease to the function of primary cilia and left-right axis determination.

Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, leads to chronic renal failure in children. The genes mutated in NPHP1 and NPHP4 have been identified, and a gene locus associated with infantile nephronophthisis (NPHP2) was mapped. The kidney phenotype of NPHP2 combines clinical features of NPHP and polycystic kidney disease (PKD). Here, we identify inversin (INVS) as the gene mutated in NPHP2 with and without situs inversus. We show molecular interaction of inversin with nephrocystin, the product of the gene mutated in NPHP1 and interaction of nephrocystin with beta-tubulin, a main component of primary cilia. We show that nephrocystin, inversin and beta-tubulin colocalize to primary cilia of renal tubular cells. Furthermore, we produce a PKD-like renal cystic phenotype and randomization of heart looping by knockdown of invs expression in zebrafish. The interaction and colocalization in cilia of inversin, nephrocystin and beta-tubulin connect pathogenetic aspects of NPHP to PKD, to primary cilia function and to left-right axis determination.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Expression analyses and interaction with the anaphase promoting complex protein Apc2 suggest a role for inversin in primary cilia and involvement in the cell cycle.

Homozygous inv mice lack a functional inversin protein and exhibit situs inversus plus severe cystic changes in the kidney and pancreas. Although the inversin sequence has provided few clues to its function, we and others have previously identified calmodulin as a binding partner. We now provide evidence that inversin interacts with the anaphase promoting complex protein Apc2. As expected of an Apc2 target, inversin possesses D-boxes and site-directed mutagenesis of the well-conserved D-box residues abrogates inversin-Apc2 interaction. An inversin-specific antibody reveals a dynamic expression pattern throughout the cell cycle and strong expression in the primary cilia of renal epithelium. Our data support a role for inversin in primary cilia and involvement in the cell cycle. Mutations of the proteins polaris, cystin and polycystin-2 which are expressed in renal epithelium primary cilia, lead to renal cystic changes. Aberrant cell proliferation is also involved in cyst development. The data reported here suggest that inversin may provide a link between these two mechanisms.

Amino Acid Motifs↗
PubMed2002

Craniofacial expression of human and murine TBX22 correlates with the cleft palate and ankyloglossia phenotype observed in CPX patients.

Cleft palate with ankyloglossia (CPX; MIM 303400) is inherited as a Mendelian, semidominant X-linked disorder and has been described in several large families from different ethnic origins. It is a useful genetic model for non-syndromic cleft palate, a common congenital disorder. Recently, the underlying genetic defect in CPX was identified, where unique mutations were found in the T-box-containing transcription factor TBX22. Here we report two new familial cases with novel missense and insertion mutations, each occurring within the T-box domain and highlighting the functional significance of this DNA-binding motif. We describe TBX22 expression in early human development, where expression is found in the palatal shelves and is highest prior to elevation to a horizontal position above the tongue. mRNA is also detected in the base of the tongue in the region of the frenulum that corresponds to the ankyloglossia seen in CPX patients. Other sites of expression include the inferior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop, and the tooth buds themselves. We have also identified the orthologous mouse Tbx22 gene and performed expression analysis in E12.5-E17.5 mouse embryos. The location of mRNA expression closely correlates between mouse and human, while at later stages of development, we also detected expression in mouse lung and whisker follicles. We conclude that expression of TBX22 is entirely consistent with the CPX phenotype and that the mouse should provide a useful model for elucidating its role in craniofacial development.

Amino Acid Sequence↗
PubMed2002

Identification and characterisation of novel mammalian homologues of Drosophila polyhomeoticpermits new insights into relationships between members of the polyhomeotic family.

We have identified and characterised novel members of the mammalian polyhomeotic gene family, comprising a third human homologue, PHC3, and its mouse counterpart, Phc3. The two new genes have essentially the same genomic organisation, with 15 exons specifying proteins of 983 (PHC3) or 981 (Phc3) amino acids, with an overall sequence identity of 93%. Northern blot hybridisations have identified a single large transcript in a variety of adult mouse tissues and in situ hybridisation analyses indicate apparently ubiquitous expression during early human and mouse development. Exhaustive database screening suggests a maximum of three polyhomeotic homologues in humans and in mice and, for completeness, we have also derived the full-length coding sequence of the PHC2 gene (formerly called EDR2). The availability of full-length coding sequences for PHC2, PHC3 and Phc3 completes the profile of human and mouse polyhomeotic homologues and has enabled comprehensive comparative analyses of the human, mouse and Drosophilapolyhomeotic proteins. The mammalian paralogues are located on three different chromosomes and pairwise comparisons of their protein products typically show about 34% amino acid sequence identity, except for some highly conserved domains for which we present consensus mammalian sequences. The data indicate that the novel PHC3 and Phc3 proteins are significantly more closely related to PHC2/Phc2 than either is to PHC1/Phc1 and reveal two hitherto unknown but highly conserved N-terminal domains that are shared by the PHC2/Phc2 and PHC3/Phc3 proteins but that are poorly conserved or absent in other polyhomeotic family members.

Amino Acid Sequence↗
PubMed2002

The left-right determinant inversin has highly conserved ankyrin repeat and IQ domains and interacts with calmodulin.

All vertebrates have a left-right body axis with invariant asymmetries of the heart and the positions of the abdominal viscera. Major advances have recently been made in defining molecular components of the pathway specifying the vertebrate left-right axis, but our knowledge of the early determinants is extremely limited. In the invmouse the left-right axis is consistently reversed, unlike other vertebrate mutants where randomisation of situs is apparent. The gene disrupted in this mouse encodes a 1062-amino-acid protein, inversin. We previously reported 16 tandem ankyrin repeats, spanning amino acids 13-557, and two putative nuclear localisation sequences, but otherwise the sequence offered few clues to the possible function. In order to identify regions likely to be functionally important, we have identified and characterised orthologous sequences in several species, including chick, Xenopus and zebrafish. Sequence comparisons show strong conservation of the ankyrin repeat region and also a lysine-rich domain spanning amino acids 558-604. Further analysis identified a highly conserved IQ calmodulin-binding domain in the latter region and another such domain in an otherwise poorly conserved C-terminal region. A yeast two-hybrid screen identified calmodulin in one third of the positive clones, and we confirmed this interaction by immunoprecipitation.

Amino Acid Sequence↗
PubMed2002

Breakpoint position on 17q identifies the most aggressive neuroblastoma tumors.

Gain of chromosome arm 17q is a powerful prognostic factor in neuroblastoma, and the distribution of 17q breakpoints suggests that the dosage of one or more genes in 17q22-23 to 17qter is critical for tumor progression. To identify the smallest region of 17q gain, we used eight probes to map translocation breakpoints in 48 primary neuroblastoma tumors. We identified at least five different breakpoints, all localized within the proximal part of 17q (from D17Z1 to MPO). The shortest region of gain identified by these probes extends from MPO (17q23.1) to 17qter. Surprisingly, we found that breakpoints localized proximal to ERBB2 (17q12) were associated with significantly better patient survival than breakpoints localized distal to ERBB2. Breakpoints localized distal to ERBB2 identified patients with a particularly poor prognosis, higher mitotic karyorrhectic index, and stage 4 disease. This implies that breakpoint position on 17q is a discriminative factor within this prognostically poor group of patients. This result also suggests that the biological effect of 17q gain during neuroblastoma progression has a complex basis. We propose that this involves dosage alterations of genes localized on both sides of the 17q breakpoints, with a gene or genes mapping between 17cen and 17q12 acting to suppress progression, and a gene or genes mapping between 17q23.1 and 17qter acting to promote tumor progression.

Biomarkers, Tumor↗
PubMed2002

Hailey-Hailey disease: molecular and clinical characterization of novel mutations in the ATP2C1 gene.

Hailey-Hailey disease is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Mutations in ATP2C1, the gene encoding a novel, P-type Ca2+-transport ATPase, were recently found to cause Hailey-Hailey disease. In this study, we used conformation-sensitive gel electrophoresis to screen all 28 translated exons of ATP2C1 in 24 Hailey-Hailey disease families and three sporadic cases with the disorder. We identified 22 different mutations, 18 of which have not previously been reported, in 25 probands. The novel mutations comprise three nonsense, six insertion/deletion, three splice-site, and six missense mutations and are distributed throughout the ATP2C1 gene. Six mutations were found in multiple families investigated here or in our previous study. Haplotype analysis revealed that two of these are recurrent mutations that have not been inherited from a common ancestor. Comparison between genotype and phenotype in 23 families failed to yield any clear correlation between the nature of the mutation and clinical features of Hailey-Hailey disease. The extensive interfamilial and intrafamilial phenotypic variability observed suggests that modifying genes and/or environmental factors may greatly influence the clinical features of this disease.

Calcium-Transporting ATPases↗