Browsing by Author "Illing, Nicola"
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- ItemOpen AccessActivation of seed-specific genes in leaves and roots of the desiccation tolerant plant, Xerophyta humilis(2008) Walford, Sally-Ann; Illing, Nicola; Farrant, Jill M; Denby, Katherine JThe ability of tissues to survive almost complete loss of cellular water is a trait found throughout the plant kingdom. While this desiccation tolerance is common in seeds of most angiosperms it is rare in their vegetative tissues. Xerophyta humilis (Bak.) Dur and Schintz belongs to a small group of resurrection angiosperms and it possesses the ability to withstand extreme desiccation of greater than 90% in both its seeds and vegetative tissues and return to active metabolism upon rehydration. We have tested the hypothesis that vegetative desiccation tolerance in angiosperms has evolved as an adaptation of seed desiccation tolerance.
- ItemOpen AccessBat accelerated regions identify a bat forelimb specific enhancer in the HoxD locus(Public Library of Science, 2016) Booker, Betty M; Friedrich, Tara; Mason, Mandy K; VanderMeer, Julia E; Zhao, Jingjing; Eckalbar, Walter L; Logan, Malcolm; Illing, Nicola; Pollard, Katherine S; Ahituv, NadavAuthor Summary: The limb is a classic example of vertebrate homology and is represented by a large range of morphological structures such as fins, legs and wings. The evolution of these structures could be driven by alterations in gene regulatory elements that have critical roles during development. To identify elements that may contribute to bat wing development, we characterized sequences that are conserved between vertebrates, but changed significantly in the bat lineage. We then overlapped these sequences with predicted developing limb enhancers as determined by ChIP-seq, finding 166 bat accelerated sequences (BARs). Five BARs that were tested for enhancer activity in mice all drove expression in the limb. Testing the mouse orthologous sequence showed that three had differences in their limb enhancer activity as compared to the bat sequence. Of these, BAR116 was of particular interest as it is located near the HoxD locus, an essential gene complex required for proper spatiotemporal patterning of the developing limb. The bat BAR116 sequence drove robust forelimb expression but the mouse BAR116 sequence did not show enhancer activity. These experiments correspond to analyses of HoxD gene expressions in developing bat limbs, which had strong forelimb versus weak hindlimb expression for Hoxd10 - 11 . Combined, our studies highlight specific genomic regions that could be important in shaping the morphological differences that led to the development of the bat wing.
- ItemOpen AccessCharacterisation of the role of brain factor 1 in the olfactory neuroepithelium during neuronal development(2003) Linda, Pride; Illing, Nicola; Patterton, HughBrain Factor 1 (BF-1), a winged helix transcription factor, displays a restricted pattern of expression within the developing forebrain, playing a critical function in the regulation of neuronal progenitor cell proliferation and differentiation within the developing forebrain. The molecular mechanisms by which BF-1 carries out its function remain to be elucidated. Hence, this study aimed to investigate and characterise the molecular mechanisms by which BF-1 function is regulated during neuronal development.
- ItemOpen AccessCharacterization of the Xerophyta humilis desiccation induced-1 (Xhdsi-1voc) gene : a member of the Vicinal Oxygen Chelate (VOC) metalloenzyme superfamily upregulated in X. humilis (BAK) DUR and SCHINZ during desiccation(2008) Mulako, Inonge; Illing, Nicola; Farrant, Jill MThe resurrection plant, Xerophyta humilis is used as a model system to identify and characterise genes which play an important role in conferring desiccation tolerance in plants. In this study, the expression of a novel gene named desiccation induced-1 (dsi-1VOC) during desiccation in X. humilis and desiccationsensitive plants is characterised.
- ItemOpen AccessCharacterization of transcription factors and LncRNAs involved in the development of the bat wing(2016) Gill, Zoe; Illing, NicolaMammals have evolved a vast myriad of limb morphologies adapted for a wide range of activities. One of the most remarkable evolutionary adaptations of a mammalian limb is that of the forelimb wing of a bat used for powered flight. This capability evolved ~ 51 Mya from its arboreal ancestor without any fossil record of intermediate forms. To reconstruct how this transition occurred, an Evolutionary Developmental approach can be applied to investigate altered mechanisms present in bat limb development. Similar genes and signalling centres are present in both mice and bats, making mice a good model organism for comparison. This study used a pre-existing set of RNA-seq transcriptomes from three pivotal developmental stages (CS 15, CS 16 and CS 17) of bat development, to compare FL and HL gene expression. Of the list of differentially expressed genes, a subset was selected to characterise spatial expression patterns within the developing bat limb compared to mouse limbs by whole-mount in situ hybridisation. Five transcription factors: Lef1, Lhx8, HoxA10, Mllt3 and Tbx5, as well as two Long non-coding RNAs: Hottip and Tbx5-as1 were selected. Novel expression of Mllt3 was detected in FL autopods at CS15, in a region slated to expand with digit elongation. Lef1 in situ signal was more robust in HL autopods of CS 15 embryos compared to FLs and equivalently staged mice. Lhx8 displayed a strong signal in CS 16 and CS17 wrist tissue, as well as a faint signal in interdigital tissue in the FL autopods. The LncRNA Hottip displayed vastly different expression pattern between FL and HL, with staining being reduced in the digit and interdigital regions of the FL at CS 16L, whereas expression in the HL was robust in the digit, and even more so in the interdigital regions. The LncRNA Tbx5-as1, displayed a similar expression pattern to the known FL initiation transcription factor Tbx5 at late stages (CS 16L -CS 17L). Isoform characterization to validate the two LncRNAs, was performed on cDNA a CS 18L embryo. The cloned transcripts identified a new set of alternatively spliced isoforms for both LncRNAs. Unusual RNA-seq tracks in the HoxA10 locus were investigated using qPCR. It was discovered this region is variable amongst biological samples; however there is a large reduction in expression in this region from CS 15 to CS 16.
- ItemOpen AccessCharacterization of transcripts expressed from the Meis2 locus in the Natal Long-fingered bat (Miniopterus natalensis)(2014) Curry, Lyle; Illing, Nicola; Jacobs, David SThe Myeloid ecotropic insertion site 2 (Meis2) gene is an important transcriptional regulator involved in the pattering of the limb during vertebrate development. In the forelimbs (wings) of bats the expression Meis2 was shown to be differentially expressed when compared to mice forelimbs. Meis2 was found to be present in the interdigital webbing of the autopod (hand) of the developing bat forelimb. In both the mouse and the bat, two separate transcripts were discovered to be expressed from the 3’and 5’ region of the Meis2 locus. The 3’ transcripts corresponded to annotated Meis2 mRNA transcripts, the 5’ transcript corresponded to an mRNA transcript that did not code for any known protein. The 5’ transcript was thought to be a long non-coding RNA and was termed lncMeis2. In this study Random Amplification of cDNA Ends (RACE) analysis was used to identify the RNA transcripts expressed from the Meis2 locus and to verify the presence of lncMeis2. RACE was performed on the heads and forelimbs of the Natal Long-fingered bat (Miniopterus natalensis) and the mouse (Mus musculus) and the products sequenced and aligned to the mouse genome for verification.
- ItemOpen AccessCharacterization of two, desiccation linked, Group 1 LEA proteins from the resurrection plant Xerophyta humilis(2011) Ginbot, Zekarias Gebremedhin; Farrant, Jill M; Illing, NicolaStudies on resurrection plants and other anhydrobiotic organisms show expression of Late Embryogenesis Abundant (LEA) proteins associated with desiccation tolerance. However, the precise role of these proteins has not been described. This study was undertaken to investigate expression, structure and function of XhLEA1-4S1 and XhLEA1-1S2, Group 1 LEA proteins from Xerophyta humilis, in order to shed light on their role in desiccation tolerance. Complementary DNA (cDNA) of these XhLEAs were cloned into bacterial expression vectors and the recombinant proteins expressed in E. coli. Antibodies were generated and used in determination of expression conditions and immunolocalization studies.
- ItemOpen AccessCloning and characterisation of gonadotropin-releasing hormone (GnRH) receptors in the cichlid (Haplochromis burtoni) and the zebrafish (Danio rerio)(2003) Morley, Michelle Gaye; Illing, NicolaThe identification of multiple forms of gonadotropin-releasing hormone (GnRH) in a single species is becoming a common occurrence. The highly conserved chicken GnRH II is present along with one or two other GnRHs, composing a combination unique to particular species. This multifunctional peptide is widely distributed through the central nervous system and peripheral tissues. Also, endogenous GnRHs demonstrate distinct patterns of spatial expression within the brain, suggesting they may have separate functions. In addition to being the primary regulator of gonadotropin secretion in vertebrates, GnRH is also involved in the release of GH and prolactin and may fulfil a possible neuromodulatory role. GnRHs exert their actions through the stimulation of distinct GnRH receptors on pituitary gonadotrophs. The presence of multiple GnRH receptor subtypes has been demonstrated in several species and is likely to be a common characteristic of most vertebrates. This thesis describes the cloning and characterisation of GnRH receptors in two species of teleost fish, Haplochromis burtoni (cichlid) and Dania rerio (zebrafish). A type I GnRH receptor has previously been shown to exist in the cichlid. In the present study degenerate primers designed to extracellular loop three of the mammalian GnRH receptors were used to identify a second putative receptor subtype from cichlid (Haplochramis burtoni) genomic DNA. Furthermore, a near full-length cDNA, encompassing transmembrane domain 1 through to transmembrane domain 7 of the GnRH receptor, was cloned from cichlid RNA by reverse transcriptase PCR. This region of the receptor shares approximately 80% amino acid homology with corresponding regions of type III GnRH receptors previously identified in species of perciform fish. Partial sequences of a type IA and a type lB GnRH receptor have previously been identified in the zebrafish. Two sets of degenerate primers were used to elucidate the possible existence of a third receptor in the zebrafish using both genomic DNA and RNA. However, this strategy failed to result in the amplification of novel receptor subtypes in the zebrafish. Controversy surrounds the developmental origins of GnRH neurons and their temporal expression in relation to GnRH receptors. The zebrafish is a model organism, widely used for the study of reporter gene expression during development. Hence an attempt was made to isolate the zebrafish GnRH receptor genes using a genomic DNA library and identify the promoter regions for use as reporter genes in the study of GnRH and GnRH receptor expression during development. Southern blot analysis revealed six genomic clones with sequences homologous to zebrafish GnRH receptor cDNA. Comparison with genomic and cDNA sequences of other GnRH receptors revealed that those regions of the genomic clones that were sequenced only encoded exons 2 and 3. The presence of large introns in the GnRH receptor gene made it difficult to identify genomic clones containing the entire gene and the promoter region. The cloning of part of the zebrafish GnRH receptor genes will make their complete characterisation somewhat less problematic since an idea of their basic intron/exon structure has been obtained. Exons 2 and 3 of the zebrafish type IA and type IB GnRH receptor genes show a high degree of conservation when compared to the same regions of the goldfish type IA and type IB GnRH receptor cDNAs, demonstrating approximately 90% homology in both cases. In this study sequence information was obtained for the regions between transmembrane domains 4 and 7, and 3 and 7 of the zebrafish type IA and type IB GnRH receptor genes, respectively, and was subsequently used clone zebrafish GnRH receptor full-length cDNAs. This study describes the discovery of a type III GnRH receptor in the cichlid but suggests its presence may be restricted to only certain orders of teleost since a type III receptor was not identified in the zebrafish on this occasion. The information acquired from this study may help to reveal patterns, which relate the presence of particular GnRHs and GnRH receptors in single species to specific reproductive requirements.
- ItemOpen AccessCloning and characterisation of gonadotropin-releasing hormone receptors from species in non-mammalian vertebrate classes : amphibia and osteichthyes(1998) Troskie, Brigitte Elise; Illing, Nicola; Millar, Robert PTwo or more forms of gonadotropin-releasing hormone (GnRH) have been isolated from most vertebrate species. In most species, GnRH variants have been shown to occur in distinct areas of the peripheral and central nervous systems, the gonads and other peripheral organs. Although GnRH is a primary regulator of gonadotropin secretion, it has been shown to have additional roles such as the regulation of growth hormone secretion in goldfish and the inhibition of a potassium current (M-current) in amphibian sympathetic ganglia. This raises the possibility of the occurrence of multiple GnRH receptor subtypes. This thesis describes the cloning and characterisation of GnRH receptor subtypes from two nonmammalian vertebrates, the Amphibian, Xenopus laevis and the Osteichthyes, Carassius auratus (goldfish). Using degenerate primers designed to the mammalian GnRH receptors two putative receptor subtypes were identified from both X. laevis (X/a.1 and X/b.1) and goldfish (GfA and GfB) genomic DNA. The full-length cDNA for X/a.1, was cloned from pituitary cDNA. When transiently expressed in COS-1 cells, this clone showed a GnRH-dependent stimulation of inositol phosphates. No full-length clone for X/b.1 could be isolated using cDNA from several different tissues. A partially processed transcript was, however, amplified from sympathetic ganglia cDNA. These ganglia showed specific binding to a chicken GnRH II (cGnRH II) agonist and cGnRH II immunoreactivity was also detected in extracts from the ganglia. The expression, function and pharmacology of clone X/b.1, thus remains unknown, but the presence of cGnRH II-specific binding sites on membranes from the sympathetic ganglia with distinctly different pharmacology, implies the presence of a second GnRH receptor subtype in these neurons. Full-length cDNA clones of GfA and GfB were amplified from goldfish pituitary and brain cDNA respectively. These receptors had a 71% amino acid identity to each other and a 43% amino acid identity to the human GnRH receptor. The pharmacology of these two GnRH receptor subtypes was investigated by transient expression in COS-1 cells. The GfA and GfB receptors had different pharmacologies as demonstrated by their selectivities for GnRH analogues. In situ hybridisation revealed a distinct expression pattern of the goldfish GnRH receptor subtypes in the brain, gonads and liver (Dr R. Peter, University of Alberta). The full-length receptors cloned from the pituitaries and brain of X. /aevis and the goldfish have a low homology to the cloned mammalian GnRH receptors and have several different features, such as the presence of an intracellular carboxy-terminal tail. This thesis, describing the primary structure and characterisation of ligand selectivity of non-mammalian GnRH receptors, provides some useful foundations for future work towards understanding ligand recognition in the GnRH receptor. The description of multiple receptor subtypes in the goldfish and possibly in X. laevis also provides valuable information into alternative roles of GnRH and its receptor, which we are only beginning to understand.
- ItemOpen AccessCloning and characterisation of LEA1-EM genes in the resurrection plant, Xerophyta humilis(2008) Ngubane, Nqobile A C; Farrant, Jill M; Illing, NicolaThe presence and expressIon patterns of orthologues of LEA group 1 genes has been characterised in the resurrection plant, Xerophyta humilis. The group I LEAs (Em I and Em6) were first identified as proteins that were abundantly and specifically expressed during the desiccation and germination phase of angiosperm seed development. The group I LEA genes are characterised by the presence of one or more tandemly repeated 20-amino acid motifs that are particularly rich in Gly residues.
- ItemOpen AccessCloning and charaterisation of the Thyrotrophin-releasing hormone receptor and Gonadotrophin-relasing hormone receptor from chicken pituitary gland(1998) Sun, Yuh-Man; Millar, Robert P; Illing, NicolaThe hypothalamic hormones, thyrotrophin-releasing hormone (TRH) and gonadotrophin-releasing hormone (GnRH), play pivotal roles in the growth and sexual maturation of chickens. In chickens, TRH regulates the release and synthesis of thyrotrophin (TSH) and also acts as a growth hormone-releasing factor. GnRH stimulates the release and synthesis of gonadotrophins (LH and FSH). TRH and GnRH are released and stored in the median eminence, and both hormones are transported into the pituitary gland via the hypophysial portal circulation. TRH and GnRH exert their physiological functions by binding to their specific receptors (TRH receptor and GnRH receptor, respectively) on the surface of cells in the pituitary gland. The activated receptors couple to guanine nucleotide-binding regulatory proteins (G proteins), Gq and/or G11, which in turn triggers the secondary messenger [1,2- diacylglycerol (DAG) and inositoltrisphosphate (IP3)] signalling cascade. The signalling generates the physiological effects of the hormones. The TRH-R and GnRH-R are members of G-protein coupled receptor (GPCR) family. The objective of this thesis was to clone and characterise the chicken TRH and GnRH receptors as useful tools for investigating the regulatory roles of TRH and GnRH receptors in the growth and sexual maturation of chickens. In addition, sequence information of the receptors would potentially assist in elucidating the binding sites and the molecular nature of the processes involved in receptor activation.
- ItemOpen AccessCross-species microarray analysis of limb development in the bat, Miniopterus natalensis(2009) Mason, Mandy K; Jacobs, David S; Illing, NicolaThis study reports the first characterisation of the embryonic bat limb transcriptome, allowing the identification of novel candidate genes that were differentially expressed between the bat hand and foot plate. These genes may have played important roles in the evolution of the bat wing and hindlimb. The reproduction and development of an African bat species, Miniopterus natalensis, was characterised and three maternal features (mass, belly size and plasma progesterone levels) examined as potential predictors of embryonic stages of development. Belly palpitation was found to be a useful field method to distinguish between non-pregnancy, early development or late development in female bats. A microarray analysis between the hand and foot plates of CS 16 and CS 17 bat embryos, and the hand plates of E 13.5 mouse embryos, revealed high correlation between the transcriptomes of the bat autopods and the mouse hand plate (r > 0.88) and among all the bat autopods (r > 0.98). However, ten genes were found to be differentially expressed in both the CS 16 and the CS 17 bat hand plate as compared to the mouse hand plate while only three genes were identified as being significantly differentially expressed between bat foot plates and mouse hand plates. A comparison between the bat hand and foot plates identified fifteen genes that were differentially expressed at the stage CS 17 stage and six at the stage CS 16. Closer examination of gene families involved in limb development revealed novel expression of genes in the retinoic acid (RA) pathway, and the Hoxd family. This included the apparent co- regulation of the 5' Hoxd genes (Hoxd10, 11, 12 and 13). Of the genes characterised in bat limb development (Hoxd13, Bmp2, Fgf8 and Prrxl), higher mRNA transcript levels in the CS 17 bat hand plate relative to the mouse hand plate was found for Hoxd13 (FC = 2.6) and Prrxl (FC = 1.8). These differences were also found in a comparison to the CS 17 bat foot plate (Hoxd13: FC = 1.4; Prrxl: FC = 1.4). A potentially novel transcript of Meis2, a gene important in specifYing the proximal-distal (P-D) axis of the limb, was noted for its high fold changes in the bat hand plate as compared the foot plate (CSI7: FC = 7.0; CSI6: FC = 2.2) and the mouse hand plate (FC = 13.1).
- ItemOpen AccessDigit formation during embryonic development of bats and mice(2018) Parker, Ash; Illing, Nicola; Hockman, DoritThe evolution of a strikingly elongated and webbed FL in bats, which contrasts with a small, free-toed HL, has seen extensive research into bat wing development in an effort to determine the molecular mechanism driving limb development. A recent RNA-seq and ChIP-seq study carried out on M. natalensis showed differences in FL and HL activity for several genetic pathways known to be involved in bone formation during key bat development stages CS15-CS17. In this project the prediction made from the literature and the RNA-seq results was that the observed decreased Wnt/β-catenin signalling and increased BMP signalling in the bat FL may lead to elevated levels of Sox9 expression, and larger fields of mesenchymal condensations. This was tested by annotating Sox9 in the M. natalensis genome to further analyse the expression levels and associated ChIP-seq data. In addition the behaviour of condensing mesenchymal cells during bat and mouse limb development was observed by visualising the various stages of chondrogenesis, using H&E and PNA stains. In addition the RNA-seq study found 3000 genes to be differentially expressed. Thus, the project also set out to create an immortalised bat autopod cell line to facilitate future testing and predictions. The Sox9 gene was successfully annotated and revealed to not be differentially expressed between FL and HL as predicted. However downstream targets of Sox9 were further inspected as potential ideas for further investigation. The histological stains provided a set of data characterising mesenchymal condensation in both mouse and bat stages, revealing many interesting features such as the non-specific binding behaviours of PNA prior to digit formation. In addition, quantitative results demonstrated the bat FL digits are already longer than the HL digits at CS16. Cell line work established a working protocol for the storage, dissociation and plating of bat primary cells that retain their bat limb expression identity. Mouse cells were successfully immortalised and a cell line was established from a HL digit cell. This project has facilitated further studies in understanding extreme digit elongation in the bat FL autopod during development.
- ItemOpen AccessEvents that shape genomes(2018) Schlebusch, Stephen A.; Illing, Nicola; Wall, JeffThe invention and development of Next Generation Sequencing has opened up new possibilities for exploring the genomes of non-model organisms. For this thesis, a diverse range of non-model species from both plants and animals were used to identify and answer questions of evolutionary interest in four case studies. In doing so, a wide assortment of methodologies were used and developed, taking full advantage of the versatility that whole genome sequencing can provide. The genome of the Natal Long Fingered Bat, Miniopterus natalensis, was assembled to investigate the genetic mechanisms responsible for the evolution of the bat wing. The assembled genome was required to facilitate RNA-seq and ChIP-seq analysis. In addition to the genome assembly and annotation, dN/dS analysis and lncRNA prediction were also conducted. This resulted in a high quality genome assembly with just over 24000 genes being annotated and 227 putative lncRNAs being identified. None of the genetic pathways highlighted by the RNA-seq analysis showed any elevated dN/dS signal, suggesting this was not the loci of evolutionary change. The Amboseli National Park in Kenya has a local population of Yellow baboons (Papio cynocephalus) that has recently come into contact and hybridised with a population of Olive baboons (Papio anubis). A genome assembly of P. cynocephalus was created and used to align low coverage sequencing from 45 baboons, including admixed individuals along with unadmixed individuals from each species. By identifying SNPs that were predictive of the species, hybrid individuals were confirmed and evidence for previous admixture events discovered, such as P. anubis SNPs already at fixation in the P. cynocephalus population at Amboseli. The Ruschioideae are a clade of plants that encompasses the prolific tribe, the Ruschieae, which is comprised of approximately 1500 recently diverged species. An exploratory analysis sequenced two Ruschieae genomes (Polymita steenbokensis and Faucaria felina) along with a sister taxon (Cleretum herrei) from a neighbouring tribe (Dorotheantheae). The three plants were compared to each other in order to try and identify any genetic signatures that could be influencing the rapid speciation. The two Ruschieae species were found to have increased levels of non-tandem duplication within the genome as well as on going transposable element activity when compared to C. herrei. Xerohpyta humilis is a desiccation tolerant plant. In order to further facilitate research into how this is possible, the genome was sequenced and assembled. Irregular data led to the discovery that the plant had a genome duplication as well as a large amount of somatic mutations in its genome. Further analysis confirmed that this pattern of somatic mutations was only present in plants that had undergone multiple cycles of desiccation and rehydration. These apparently disparate topics explored the possibilities and limitations for whole genome sequencing in the study of non-model organisms. Mechanisms of genetic change were examined at the genomic scale, from adaptation and hybridisation to various forms of duplication and mutation. In this way, a large variety of events responsible for the evolutionary change of genomes in plants and animals were analysed in a diverse set of systems.
- ItemOpen AccessEvolution of the ZRS and the Regulation of SHH Expression in the Forelimbs of Bats.(2011) Brito, Denise Ribeiro Arthur; Illing, Nicola; Jacobs, David. SThe characterisation of sonic hedgehog (Shh) in the developing limbs of the two bat species Miniopterus natalensis and Carollia perspicillata showed that Shh expression pattern was different from that observed in mouse limbs. An interesting discovery was that bat embryos display a second round of expression at stage CS16/E12.5. The aim of this thesis is to investigate the cause of the Shh expression pattern in developing limbs of the bat. Sonic hedgehog expression in the limb is regulated by a long-distance cis-regulatory sequence called the ZPA-regulatory sequence (ZRS). Mutations in the ZRS are known to result in limb deformities in humans, mice and cats. I investigated whether conserved mutations occurring in the bat ZRS are the cause of the change in Shh expression in bat limbs during embryonic development. Comparison of the ZRS of eighteen bat species to the ZRS of twenty other vertebrates revealed five conserved mutations that are unique to the bat ZRS. To test if the bat ZRS has the ability to change the expression pattern of Shh, the ZRS from M. natalensis and Rhinolophus clivosus (which have different wing shapes) was linked to a β-globin-LacZ reporter construct and used in transgenic mice experiments. A mouse ZRS with bat mutations as well as a wild type mouse ZRS (the control) were also tested. Transgenic results suggest that the bat ZRS has the elements necessary to alter the spatial expression pattern of Shh, but that may not be able to induce a second round of expression. The two bat ZRS reporter construct species produce different β-galactosidase expression patterns - a result of having very different ZRS sequences - thus implying that there is interspecies variation of the Shh expression pattern. Phylogenetic analysis of bat ZRS grouped the bat species in three well supported clades, which may not only reflect differences in Shh expression but also differences in wing shape. To test this hypothesis, it was first necessary to identify which skeletal elements within the handwing vary the most across the bat species, and consequently account for the variation in wing shape. Analysis of wing morphology revealed that phalange I of digits 3 and 4, and phalange II of digits 3, 4 and 5 showed significant variation across 53 bat species, along with Aspect ratio, which is an indicator of wing shape. Comparisons of Aspect ratio between bat species in the ZRS clades imply a relationship between differences in the ZRS sequences of bats and wings. Analysis of the variation of lengths of five skeletal elements mentioned above between the ZRS clades suggest that the wing shape is determined by different combinations of the length of 3PII, 3PI, 4PI, 4PII, and 5PII. This suggests that the variation of the skeletal elements may be a result of post-natal development as juvenile bats learn to fly, or that there is another mechanism determining wing shape.
- ItemOpen AccessEvolutionary analysis and functional characterization of the forkhead transcription factor FoxG1(2006) Bredenkamp, Nicholas; Illing, NicolaForhead box G1 (FoxG1) is a winged-helix transcription factor that plays a crucial role in the development of the telecephalon, the most rostral region of the brain Here, FoxG1 acts as a transcriptional repressor and maintains the population of cortical progenitor cells by promoting their proliferation and inhibiting differrentiation. Vertebrate FoxG1 orthologs have highly conserved DNA-binding and corbosy-terminal domains that have functional roles. Conversely, no functional role has yet been assigned to the N-terminal domain which shows a high degree of variability across vertebrates, with a remarkable stretch of consecutive histidine, proline and glutamine (HPQ) residues in the mammalian orthologs. In this study it was tested whether differences in FoxG1 sequence amongst vertebrates might account for the increased cortex size of mammals compared to non-mammals. Furthermore, changes in the sub-cellular localization of FoxG1 in response to fibroblast growth factor 2 (FGF-2) were investigated in a neural precursor cell line.
- ItemOpen AccessA framework for the informed normalization of printed microarrays(Academy of Science of South Africa, 2007) van Heerden, Johan; Walford, Sally-Ann; Shen, Arthur; Illing, NicolaMicroarray technology has become an essential part of contemporary molecular biological research. An aspect central to any microarray experiment is that of normalization, a form of data processing directed at removing technical noise while preserving biological meaning, thereby allowing for more accurate interpretations of data. The statistics underlying many normalization methods can appear overwhelming to microarray newcomers, a situation which is further compounded by a lack of accessible, non-statistical descriptions of common approaches to normalization. Normalization strategies significantly affect the analytical outcome of a microarray experiment, and consequently it is important that the statistical assumptions underlying normalization algorithms are understood and met before researchers embark upon the processing of raw microarray data. Many of these assumptions pertain only to whole-genome arrays, and are not valid for custom or directed microarrays. A thorough diagnostic evaluation of the nature and extent to which technical noise affects individual arrays is paramount to the success of any chosen normalization strategy. Here we suggest an approach to normalization based on extensive stepwise exploration and diagnostic assessment of data prior to, and after, normalization. Common data visualization and diagnostic approaches are highlighted, followed by descriptions of popular normalization methods, and the underlying assumptions they are based on, within the context of removing general technical artefacts associated with microarray data.
- ItemOpen AccessIdentification of proteins that interact with brain factor-1 and characterization of these interactions(2000) Schwegmann, Anita; Illing, Nicolaabstract
- ItemOpen AccessImmortalisation, characterisation and differentiation of temperature sensitive cell lines from the Olfactory Neuroepithelium(1999) Boolay, Sihaam; Illing, NicolaEmbryonic olfactory neuroepithelium provides a useful experimental system for the study of olfactory neurogenesis. As a substrate for experimental neural cell biology, olfactory neuroepithelium is of unique interest since, unlike other neural cells, olfactory neurons are continually replaced - a feature that is dictated by their direct exposure to the damaging external environment. Basal cells in the olfactory placode are the source of this replacement. Each olfactory neuron expresses only one or a few of the many olfactory receptors that are encoded by the large array of olfactory genes. Despite this limited cellular display of receptors, vertebrates are able to distinguish many thousands of different odorants, implying a complicated need for perceptive neurological processing of signals coming from individual olfactory neurons. To study the events that take place during the differentiation of neuronal precursors - a process that sustains a diverse receptor repertoire - I felt that lines of conditionally immortalised cells that could be induced to differentiate would provide useful reagents. In this thesis I describe my successful attempts to immortalise olfactory cell lines from the neuroepithelium of E 10.5 mouse embryos. I used a conditionally immortalising retrovirus that included the coding sequence for the temperature-sensitive SY 40 large T antigen. Integration of this retrovirus into the genome of cells allowed continuous proliferation at the permissive temperature of 33°C. A shift to the nonpermissive temperature of 39°C inactivated the SV40 large T antigen, the cells ceased proliferation and differentiation commenced. Sixty cell lines were derived of which four were chosen for further characterisation. These four cell lines (OP6, OP27, OP47 and OP55) were clonally derived and were immortalised rather than transformed. They continued to express the SV40 large T antigen at 33°C but lost expression at 39°C concomitant with cessation of proliferation. When the OP cells were shifted to 39°C in the absence or presence of the morphogen, retinoic acid, morphological changes ensued that were consistent with the development of neuronal characteristics. The OP6, OP27 and the OP47 cells became phase-bright with neuritic extensions. The OP55 cells were the exception in that they did not develop extensions but instead differentiated to form compact epithelial islands when grown in DM-10 medium but not in RA medium. Differentiation of the OP cells at 39°C was further documented by the induced expression of a number of markers demonstrated by RT-PCR and/or immunocytochemistry. The OP cells differentiated at 39°C in DM-10 and in retinoic acid-containing medium to express olfactory receptor transcripts. Cloning and sequencing showed that each cell line expressed a single receptor type but that different receptors were expressed by different cell lines. Sequencing revealed that the receptors cloned from the OP27 cells were 98% homologous to the mouse-M65 olfactory receptor whereas OP55 had greatest homology to rat-Olf3 olfactory receptor. The transcripts induced in OP6 and the OP47 cells showed greatest homology with Gus58 - a taste receptor homologous to olfactory receptors. Sequences obtained from OP6, OP47 and OP55 cells were not 100% identical to published receptors and could thus represent members of different subfamilies. Interestingly, induced OP55 cells also expressed mRNA for clusterin - a molecule that has no homology with olfactory receptor transcripts but is involved in differentiation during embryogenesis.
- ItemOpen AccessInfection of two distinct Trichuris sp. genotypes within and among baboon (Papio ursinus) troops on the Cape Peninsula, South Africa(2013) Moxley, Courtney; Illing, Nicola; O'Riain, JustinThe chacma baboon (Papio ursinus) population on the Cape Peninsula, South Africa is divided into 16 geographically isolated troops, 14 of which are classified as being commensal with humans. Regular contact with humans in urban and agricultural settings may have increased the risk of transmission of the different parasite species identified within this population. The aim of the study was to identify whether two previously-identified genotypes of the whipworm, Trichuris sp., infect the same individual baboon simultaneously and whether both genotypes infect baboons of different troops on the Peninsula. Genomic DNA was extracted from adult Trichuris worms extracted from the gastrointestinal tract of six baboons from five different troops on the Peninsula. Two sets of primers were designed to amplify different sized products of the ITS1-5.8S-ITS2 region of the ribosomal DNA through PCR. Diagnostic PCR revealed the DG genotype among two Trichuris sp. specimens in a baboon from an unknown troop, while the CP-GOB genotype was observed among five specimens within a baboon from the Groot Olifantsbos troop. Sequence data confirmed the presence of a single genotype in each troop. This study suggests that the genotypes are specific to baboon troops but the potential for both genotypes to infect baboons within troops on the Peninsula cannot be ruled out. Knowledge of the specificity of the Trichuris genotypes to baboon hosts of different troops may inform our understanding of the evolution of diversity within this genus. Future research into the transmission of both genotypes within and between troops may also highlight the potential for two distinct species of Trichuris to exist among the baboons. Considering the close contact between baboons and humans on the Peninsula, clarification on host specificity of either genotype will also be important for managing zoonoses and preventing break-outs of infectious diseases between the species.