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Mechanisms of floor plate formation in the developing chick embryoPatten, Iain January 2002 (has links)
No description available.
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Chromatin dynamics at the Sonic Hedgehog locus : a study using limb derived Sonic Hedgehog inducible cell lines to investigate chromatin architectureDouglas, Adam Thomas January 2017 (has links)
Enhancers are cis-regulatory sequences which promote the expression of target genes in a spatial and temporal fashion. They can be located within genes or between them and can act at distances of over 1 Mb. There are several different mechanisms by which enhancers regulate gene expression. Some, such as those regulating the Hox genes, are located close to each other in the genome in a structure referred to as a regulatory archipelago. These come together and act in combination to regulate gene expression, with different enhancer combinations resulting in different patterns of expression. On the other hand, enhancers can act individually, with designated enhancers responsible for regulating the expression of the same gene in different tissues or at different stages of development. Indeed, this is the case for the Sonic Hedgehog gene (Shh) where several different enhancers located within a gene sparse region referred to as a gene desert, act separately leading to Shh expression in areas such as the brain, the lungs, the notochord and neural tube and the limbs. Within the developing mouse embryo, Shh is expressed over roughly a two day period from E10 to E12 in a posterior distal region referred to as the Zone of Polarising Activity (ZPA). Ectopic expression in anterior regions has been observed in some common congenital diseases which affect the limbs, sometimes resulting in the formation of extra digits. The reason for this mis-expression is largely due to defects in the Shh limb enhancer commonly referred to as the Zone of Polarising Activity Regulatory Sequence (ZRS). Mutations within this highly conserved sequence create additional protein binding sites thus activating the enhancer in the wrong locations. The associated diseases are known collectively as the ZRS associated syndromes and can range from the less severe phenotype of preaxial polydactyly type II (characterised by an extra digit near the thumb) to the more severe Werner Mesomelic Syndrome (WMS), where patients present with a clear displacement of their tibia. The mechanism by which the ZRS functions is yet to be fully elucidated, with current studies producing conflicting data. What is known, is that the region encapsulating the Shh gene is highly compact, with both the gene and its enhancers located in a highly conserved Toplogical Associated Domain (TAD) as proven by Hi-C experiments. The boundaries of this domain are likely created by the binding of the protein CTCF to specified binding sites located at the either end of the locus. This restricts the ability of the enhancers to regulate the expression of genes outside the TAD. To study the exact mechanism by which the ZRS is activated and regulates Shh expression, the Hill laboratory has used cultured cell lines derived from the posterior regions of an E11.5 limb bud. Gene expression in these cells is highly reflective of the posterior limb bud, with the key exception being Shh, which is not expressed. However, using different drug treatments or biological manipulations Shh can be activated thereby making this the perfect system to analyse the mechanisms leading to Shh activation. In this investigation the cell lines were used to determine how the position of the ZRS changes upon activation. Using techniques such as Fluorescent in situ hybridisation (FISH) with either fosmid probes or directly labelled probes called MYtags, it was confirmed that the Shh locus is indeed highly compact in both Shh expressing and non-expressing cells. However, no differences were observed in terms of the distance between the ZRS and Shh between these two conditions in our cell lines. Next, both carbon copy chromosome conformation capture (5C) and circular chromosome conformation capture (4C) were used to look at changes to the Shh locus in different conditions. This confirmed Hi-C experiments and other recent publications suggesting that Shh is located within a TAD, the position of which is highly conserved between different conditions and cell lines. Furthermore, treatments activating the Shh gene resulted in significant deviations to the chromatin interactions within the locus suggesting a repositioning of structures when the gene is active. It is believed that the use of Shh inducible limb derived cell lines will prove extremely useful in future scientific endeavours to study the mechanisms of mammalian limb development. These provide a quick and easy means of accessing large numbers of Shh expressing cells, a feature which is increasingly important in an era where large cell numbers are needed for conducting chromosome conformation capture experiments such as Hi-C, 5C and 4C.
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Identification of Sox8 and Ndp as Novel Targets of the Hedgehog Signaling Pathway in the RetinaMcNeill, Brian 19 March 2012 (has links)
During embryonic development, the Hedgehog (Hh) signaling pathway plays an
important role in the growth and patterning of numerous tissues and organs. In the
developing retina, Hh signaling regulates the proliferation and differentiation of retinal
progenitor cells (RPC) through mechanisms that are not completely understood. The
principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3),
which regulate the expression of target genes responsible for the effects of the Hh pathway
on RPC. The network of genes targeted by this pathway in neural progenitor cells however,
remains unknown. The objective of this thesis was to identify and characterize novel targets
of Hh/Gli during retinal development. Using a computation approach, 390 genes were
identified as having at least one conserved Gli binding motif within the vicinity of the coding
sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected
targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5
retinal explants and that the induction of 25 of these were significantly different between the
two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp,
two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to
determine a role for Sox8 during retinal development which could reflect compensation by
the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient
and required for Hh mediated induction in progenitor cell proliferation and cell fate
determination. Therefore, in this thesis Hh target genes have been identified which could
provide some insight into the mechanisms that are responsible for the cellular outcome of a
response to the pathway.
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Identification of Sox8 and Ndp as Novel Targets of the Hedgehog Signaling Pathway in the RetinaMcNeill, Brian 19 March 2012 (has links)
During embryonic development, the Hedgehog (Hh) signaling pathway plays an
important role in the growth and patterning of numerous tissues and organs. In the
developing retina, Hh signaling regulates the proliferation and differentiation of retinal
progenitor cells (RPC) through mechanisms that are not completely understood. The
principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3),
which regulate the expression of target genes responsible for the effects of the Hh pathway
on RPC. The network of genes targeted by this pathway in neural progenitor cells however,
remains unknown. The objective of this thesis was to identify and characterize novel targets
of Hh/Gli during retinal development. Using a computation approach, 390 genes were
identified as having at least one conserved Gli binding motif within the vicinity of the coding
sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected
targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5
retinal explants and that the induction of 25 of these were significantly different between the
two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp,
two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to
determine a role for Sox8 during retinal development which could reflect compensation by
the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient
and required for Hh mediated induction in progenitor cell proliferation and cell fate
determination. Therefore, in this thesis Hh target genes have been identified which could
provide some insight into the mechanisms that are responsible for the cellular outcome of a
response to the pathway.
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Investigate the role bromodomain- and plant homeodomain-linked zinc finger-containing protein 1 (BRPF1) plays in medulloblastomaDrozdowicz, Kelly 12 July 2017 (has links)
BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for 15-20% of all pediatric brain tumors. In patients with MB, prognosis depends heavily on the molecular makeup of the tumor. New genomic approaches over the last decade have enabled researchers to sub-classify MB based on differences in the transcriptome: WNT, Sonic hedgehog (SHH), Group 3 (MYC-amplified), and Group 4 (heterogeneous). SHH tumors represent a third of all MB cases, and small-molecule inhibitors have already been developed that target SHH signaling. Most notably, vismodegib has shown great promise in the treatment of MB and other SHH-driven cancers by targeting Smoothened (SMO), an upstream regulator of GLI activity. However, most patients who had initially responded to the drug quickly acquired point mutations in SMO that led to treatment resistance. In addition, patients who harbored mutations downstream of SMO had no response to treatment and were found to be intrinsically resistant. Although most patients with SHH-MB can be cured, current treatments often require broad base therapies, such as radiation and chemotherapy, which can have harmful and long-lasting side effects. These observations underscore the need for less toxic, more targeted therapies that act at the level of the GLI family of transcription factors themselves. However, as transcription factors are generally considered undruggable, Dr. Robbins’ group at the University of Miami Miller School of Medicine sought to address this need by using focused screens of siRNAs or small molecules that target epigenetic GLI regulators. They identified several candidates that act as readers, writers, and/or erasers of protein acetylation and methylation and showed that a subset of these candidates act downstream of SMO to attenuate GLI signaling (data not yet published). Bromodomain- and Plant Homeodomain-linked Zinc Finger-containing Protein 1 (BRPF1) was one of these candidates and further analysis revealed that its knockdown reduced Gli1 expression by more than 50%. Recent studies link BRPF1 to cerebellar development and tumor formation in SHH-MB and may be suggestive of its role as a negative regulator.
OBJECTIVES: We sought to compare basal levels of Brpf1 expression in normal versus MB in mice; to characterize Brpf1 knockdown versus overexpression in SHH cell lines; and to determine if BRPF1 merits further investigation as a candidate for future drug targeting therapies in MB and other SHH-driven cancers.
METHODS: We used RT-qPCR and immunoblotting analysis to look at Brpf1 expression in Ptch+/- and adult wild-type mice. cDNA and protein samples were donated by colleagues in the lab. We also grew and maintained SHH Light2 cells in culture and then used these cells to carry out siRNA and plasmid DNA transfections. RNA extraction, RT-PCR, and RT-qPCR were used to examine transfection efficiency and its effect on Gli1 expression.
RESULTS: Brpf1 levels were higher in SHH-MB compared to normal cerebellum. However, BRPF1 proteins were not detected in either normal or tumor samples. Brpf1 knockdown in Light2 cells correlated with an overall decrease in Gli1 expression while overexpression had no obvious affect on Gli1 expression.
CONCLUSIONS: Our findings suggest that BRPF1 may function as a positive regulator of GLI activity. Recent studies verify this claim at least partially stating that BRPF1 acts as both a positive and negative regulator of gene expression depending on the context. Thus, before we can draw any final conclusions, more research is needed to look at BRPF1 in the specific context of the SHH pathway and developing cerebellum.
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Identification of Sox8 and Ndp as Novel Targets of the Hedgehog Signaling Pathway in the RetinaMcNeill, Brian January 2012 (has links)
During embryonic development, the Hedgehog (Hh) signaling pathway plays an
important role in the growth and patterning of numerous tissues and organs. In the
developing retina, Hh signaling regulates the proliferation and differentiation of retinal
progenitor cells (RPC) through mechanisms that are not completely understood. The
principal downstream mediators of the Hh pathway are the Gli transcription factors (Gli1-3),
which regulate the expression of target genes responsible for the effects of the Hh pathway
on RPC. The network of genes targeted by this pathway in neural progenitor cells however,
remains unknown. The objective of this thesis was to identify and characterize novel targets
of Hh/Gli during retinal development. Using a computation approach, 390 genes were
identified as having at least one conserved Gli binding motif within the vicinity of the coding
sequence between humans and mice. During validation, I demonstrate that 30 of 46 selected
targets were modulated in response to Hh pathway activation in either E14.5 and/or P0.5
retinal explants and that the induction of 25 of these were significantly different between the
two developmental stages. Included in this list of Hh-modulated genes were Sox8 and Ndp,
two highly inducible genes that are direct targets of Gli2. Functionally, I was unable to
determine a role for Sox8 during retinal development which could reflect compensation by
the closely related Sox9 and Sox10 genes. Ndp on the other hand was found to be sufficient
and required for Hh mediated induction in progenitor cell proliferation and cell fate
determination. Therefore, in this thesis Hh target genes have been identified which could
provide some insight into the mechanisms that are responsible for the cellular outcome of a
response to the pathway.
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Primary Cilia in the Oligodendrocyte LineageHao, Yung-Chia 05 1900 (has links)
oligodendrocytes migrate from the corpus callosum into the overlying cortex. The incidence of cilia did not change markedly across age groups, and did not vary consistently with the number of processes per cell, which was used as an indication of the maturation stage of OPCs and young OLs. The mean percent of Olig1 immunopositive (Olig1+) cells having cilia across ages was 33.1% + 16.5%, with all ages combined. In O4+ cells of these mice, 56.7 + 3.6% had primary cilia. If it is the case that adult OLs do not have cilia, the point in the lineage when primary cilia are lost is still unknown. Adult mice that had been injected with cyclopamine to block cilia-dependent Shh signaling were examined to determine whether the rate of generating new OPCs was influenced. In the CC of control mice, the numerical density of Olig1+/BrdU+ cells was 1.29 + 0.07/mm2 was reduced to 0.68 + 0.38/mm2 in the cyclopamine-injected group, and the numerical density of all BrdU+ cells (including both Olig1+ and Olig1- cells) of 4.55 + 1.50/mm2 in the control group was reduced to 3.14 + 1.27/mm2 in the cyclopamine-injected group. However, there were only 2 mice in each group and the differences were not statistically significant.
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MACROPHAGE AEBP1 CONTRIBUTES TO MAMMARY EPITHELIAL CELL HYPERPLASIA AS A NOVEL REGULATOR OF SONIC HEDGEHOG SIGNALLINGHolloway, Ryan 27 November 2012 (has links)
Chronic inflammation stimulates mammary tumourigenesis by disrupting signalling interactions between the epithelial ducts and the surrounding stromal microenvironment. Adipocyte enhancer-binding protein 1 (AEBP1) promotes mammary epithelial cell hyperplasia as a stromal factor that enhances activity of the proinflammatory transcription factor Nuclear Factor-?B (NF-?B) in macrophages. Aberrant NF-?B activity in macrophages elevates production of proinflammatory signals and the ligand sonic hedgehog (Shh), a significant contributor to tumourigenesis. In this study, Shh expression was elevated in macrophages isolated from transgenic mice (AEBP1TG) that overexpress AEBP1. Transient overexpression of AEBP1 in a macrophage cell line resulted in increased Shh expression. Furthermore, hedgehog target genes Gli1 and Bmi1 were up-regulated in mammary epithelium of AEBP1TG mice and HC11 mammary epithelial cells co-cultured with AEBP1TG macrophages. Growth of HC11 cells and mammary tumours was enhanced in response to AEBP1TG macrophages. These findings suggest that macrophage AEBP1 overexpression contributes to mammary hyperplasia through enhanced hedgehog signalling.
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Investigating Tom1 as a Candidate Regulator of Ptch1Crawford, Michelle Audrey 03 December 2012 (has links)
Sonic hedgehog (Shh) is a signaling molecule that is involved in patterning the embryo and regulates adult stem cell homeostasis. Patched1 (Ptch1) is the receptor for Shh and upon binding to Shh is endocytosed, allowing downstream signaling to occur. Ptch1 is critical to the cellular response to Shh because it is both a negative regulator of the Shh signaling pathway and a transcriptional target of the pathway. Therefore, the regulation of Ptch1 levels will directly affect the ability of cells to respond to Shh. Understanding this process requires the characterization of novel Ptch1-interacting proteins that regulate Ptch1 levels in the cell. This thesis investigated a role for the adapter protein Tom1 as a putative Ptch1-interacting protein involved in regulating Ptch1 levels through endocytic cycling. It was found that Tom1 overexpression did not regulate the patterning of vertebrate nervous system, but did play a role the sub-cellular localization of Ptch1.
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SPATIAL-TEMPORAL EXPRESSION OF SONIC HEDGEHOG REGULATES GROWTH, PATTERNING AND BRANCHING MORPHOGENESIS OF THE EMBRYONIC MOUSE LUNGMILLER, LEIGH-ANNE DEBORAH January 2003 (has links)
No description available.
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