| 1. |
- Berg, Ina, 1982-
(författare)
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Modeling Amyloid Disease in Drosophila melanogaster
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Amyloid diseases are caused by protein misfolding and aggregation. To date there are 27 known proteins causing amyloid disorders involving brain and peripheral protein deposition. The proteins involved in this mechanism do not share sequence homology, but the amyloid fibrils share biophysical properties and possibly a common pathogenic mechanism. Amyloid deposits are known to be involved in a broad range of neurodegenerative diseases, such as Alzheimer’s disease and Creutzfeldt-Jakob disease, as well as in non-neuropathic diseases, such as senile systemic amyloidosis and type II diabetes.During the last decade the fruit fly, Drosophila melanogaster (Drosophila), have increasingly been used as a model for neurodegenerative disease, such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and familial amyloidotic polyneuropathy. The advantages of using the Drosophila model are the well-defined genetic characteristics, the quantity, short life span, simplicity in genetic manipulation and the powerful binary UAS-Gal4 transgenic system. The UAS-Gal4 system allows for rapid generation of individual strains in which expression of a specific gene of interest can be directed to different tissues or cell types. The system allows the target gene to be activated in different cell- and tissue-types by altering the activator-expressing lines.This thesis has been focused on modeling amyloid diseases in Drosophila. This has been performed by:Creating new model systems of senile systemic amyloidosis and familial amyloidotic polyneuropathy in DrosophilaDeveloping a new staining protocol for detection of amyloid in DrosophilaInitiate a compound screen of Alzheimer’s disease modeled in Drosophila
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| 2. |
- Ulvklo, Carina
(författare)
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Genetic mechanisms controlling cell specification and cell numbers in the Drosophila CNS
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A central theme in developmental neurobiology pertains to how the diversity of different cell types is generated. In addition, it is equally important to understand how the specific numbers of each cell type is regulated. The developing Drosophila central nervous system (CNS) is a widely used system in which to study the genetic mechanisms underlying these events. Earlier studies have shown that a small number of progenitors produce the daunting number of cells that builds the mature CNS. This is accomplished by a series of events that in an increasingly restricted manner results in different combinatorial transcription factor codes that act to specify the different cell types in the CNS. However the factors controlling the progressive restriction in developmental potential and the ultimate fate of cells have not been completely elucidated.My PhD project has been focused on a specific stem cell in the embryonic Drosophila CNS, the neuroblast 5-6 (NB 5-6), and the lineage of neural cells that is produced by that stem cell. Earlier work have provided both a lot of knowledge and a multitude of genetic tools regarding this specific stem cell, which allowed us to address these issues at single cell resolution in an identifiable lineage. In particular, a late-born group of neurons expressing the apterous gene, the Apterous neurons, had been extensively studied in the past. One particular Apterous neuron, Ap4, expresses the neuropeptide gene FMRFamide (FMRFa), and the selective expression of this gene makes it a powerful marker for addressing many aspects of NB 5-6 development.To identify novel genes acting to control neuronal development, a large scale forward genetic screen was performed utilizing an FMRFa-GFP transgenic reporter construct, thereby using a marker that reports perturbations of NB 5-6-lineage development. Flies were treated with EMS, a chemical that induces random point mutations and the progeny where screened for aberrant FMRFa-GFP expression. From a total of ~ 10,000 mutated chromosomes ~600 mutants where isolated and further characterized. One group of mutants displayed additional Apterous neurons when compared to wild type, and a number of them represented new alleles of three previously known genes: neuralized (neur), kuzbanian (kuz), and seven up (svp). Neur and Kuz are parts of the Notch signaling pathway and Svp is the Drosophila COUP-TF1/2 ortholog; an orphan member of the steroid/thyroid receptor superfamily. These findings initiated two separate studies regarding the roles of these genes in the NB 5-6 lineage.Mutants in the Notch pathway i.e., neur and kuz displayed an excess number of Apterous neurons, born from NB 5-6. We initiated detailed studies regarding the origin of these ectopic neurons and could show that Notch signaling is critical for controlling a switch in proliferation mode in the latter part of the NB 5-6 lineage. With this new mechanism we could independently and simultaneously manipulate cell proliferation and temporal progression, and thereby predictable control cell fate and cell numbers born from the NB 5-6.The screen further identified additional mechanisms acting to specify the Ap cluster neurons. During NB 5-6 lineage development several temporal transitions acts to specify neurons born in different time windows. The temporal gene castor is expressed in a fairly large temporal window and the Ap neurons are sub-specified during that window by several combinatorial feed forward loops of transcription factors. In the screen, we identified a novel allele of the svp gene. We found that svp acts as a sub-temporal factor, fine-tuning the castor window into three different temporal parts. Previous studies have shown a role for svp earlier in the temporal cascade and we could confirm this in the NB 5-6 lineage. Together these data for the first time identify dual temporal roles of the same gene in a single NB lineage.In summary, my thesis has helped identify novel genetic mechanisms controlling neuron subtype specification and numbers.
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| 3. |
- Eriksson, Therese, 1980-
(författare)
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Exploiting Drosophila as a model system for studying anaplastic lymphoma kinase in vivo
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and an oncogene associated with several human diseases, but its normal function in humans and other vertebrates is unclear. Drosophila melanogaster has an ALK homolog, demonstrating that the RTK has been conserved throughout evolution. This makes Drosophila a suitable model organism for studying not only Drosophila ALK function, but also to study mammalian forms of ALK. In Drosophila the ligand Jeb activates ALK, initiating signaling crucial for visceral mesoderm development. The activating ligand for mammalian ALK is unclear, and for this reason Drosophila was employed in a cross-species approach to investigate whether Drosophila Jeb can activate mouse ALK. Jeb is unable to activate mouse ALK, and therefore mouse ALK is unable to substitute for and rescue the Drosophila ALK mutant phenotype. This suggests that there has been significant evolution in the ALK-ligand relationship between the mouse and Drosophila. In humans ALK has recently been shown to be involved in the development of neuroblastoma, a cancer tumor in children. I have developed a Drosophila model for examining human gain of function ALK mutants found in neuroblastoma patients. The various ALK variants have acquired point mutations in the kinase domain that have been predicted to activate the RTK in a constitutive and ligand independent manner. When expressed in the fly eye, active human ALK mutants result in a rough eye phenotype, while inactive wild type ALK does not, due to the lack of an activating ligand in the fly. In this way several of the ALK mutations identified in neuroblastoma patients could be confirmed to be activated in a ligand independent manner. Moreover, a novel ALK mutant; ALKF1174S, was discovered in a neuroblastoma patient and was in the Drosophila model shown to be a gain of function mutation, and a previously predicted gain of function mutation; ALKI1250T, was shown to be a kinase dead mutation. This fly model can also be used for testing ALK selective inhibitors, for identifying activating ligands for human ALK and for identifying conserved components of the ALK signaling pathway. Gut musculature development in Drosophila is dependent on ALK signaling, while somatic muscle development is not. Proteins of the Wasp-Scar signaling network regulate Arp2/3-complex mediated actin polymerization, and I have investigated their function in visceral and somatic muscle fusion. I found that Verprolin and other members of this protein family are essential for somatic but not visceral muscle development. Despite fusion defects in both tissues in Verprolin and other examined mutants, gut development proceeds, suggesting that fusion is not crucial for visceral mesoderm development. Hence the actin polymerization machinery functions in both somatic and visceral muscle fusion, but this process only appears to be essential in somatic muscle development.
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| 4. |
- Karlsson, Daniel
(författare)
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Specification of unique neuronal sub-types by integration of positional and temporal cues
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The nervous system contains vast numbers of neuronal sub-types, generated at specific time points, in the proper location, and in proper numbers. One of the fundamental issues in neurobiology is to understand the molecular genetic mechanisms that underlie the generation of this daunting neuronal diversity.To help shed light upon these fundamental questions, my PhD project has addressed the generation and specification of a certain group of neurons, the Ap cluster. This group of four neurons is found only in thoracic segments within the Drosophila melanogaster central nervous system, and consists of three different cell types. Mapping of the neuroblast (stem cell) that generates the Ap cluster neurons, neuroblast 5-6, and the highly restricted appearance of this cluster allowed me to address the following questions: How does NB 5-6 change its temporal competence over time to generate the Ap cluster neurons late in the lineage, and how is temporal competence altered to ensure diversity among the Ap neurons? What are the mechanisms that allow these Ap cluster neurons to emerge only in the thoracic segments?My studies have helped identify a number of mechanisms acting to specify the Ap cluster neurons. One type of mechanism involves several of different feed-forward loops that play out during NB 5-6 lineage development. These are triggered within the stem cell, where the temporal gene castor activates a number of genes. These castor targets are subsequently involved in several regulatory feed-forward loops, that ultimately result in the unique combinatorial expression of cell fate determinants in the different Ap neurons, which in turn ultimately lead to the activation of unique terminal differentiation genes. In addition, I have identified three different mechanisms by which the NB 5-6 lineage is modulated along the neuroaxis. In the abdomen I find that an early cell cycle exit is initiated by the Bx-C gene members and Pbx/Meis cofactors, which result in the truncation of the NB 5-6 lineage, preventing the Ap cluster neurons from being generated. In thoracic segments Hox, Pbx/Meisand temporal genes act in concert to specify Ap cluster neurons, by integrating with the castor temporal gene. In anterior segments, improper Hox and temporal coding results in a failure to specify bona fide Ap cluster neurons, even though equivalents of Ap cluster neurons are generated.In summary, my thesis work has helped identify a number of mechanisms acting to specify this unique neuronal sub-type, including: feed-forward combinatorial coding, opposing feed-forward loops and integrated temporal/Hox mediated specification throughout different axial levels. I suggest that these mechanisms may be widely used within the animal kingdom, hence contributing to the great cellular diversity observed within the central nervous system of most animal species.
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