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Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.ORCID iD: 0000-0002-2671-3645
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The central nervous system (CNS) consists of an enormous number of cells, and large cellular variance, integrated into an elaborate network. The CNS is the most complex animal organ, and therefore its establishment must be controlled by many different genetic programs. Considering the high level of complexity in the human CNS, addressing issues related to human neurodevelopment represents a major challenge. Since comparative studies have revealed that neurodevelopmental programs are well conserved through evolution, on both the genetic and functional levels, studies on invertebrate neurodevelopmental programs are often translatable to vertebrates. Indeed, the basis of our current knowledge about vertebrate CNS development has been greatly aided by studies on invertebrates, and in particular on the Drosophila melanogaster (fruit fly) model system.

This thesis attempted to identify novel genes regulating neural cell specification and proliferation in the CNS, using the Drosophila model system. Moreover, I aimed to address how those genes govern neural progenitor cells (neuroblasts; NBs) to obtain/maintain their stemness identity and proliferation capacity, and how they drive NBs through temporal windows and series of programmed asymmetric division, which gradually reduces their stemness identity in favor of neural differentiation, resulting in appropriate lineage progression. In the first project, we conducted a forward genetic screen in Drosophila embryos, aimed at isolating genes involved in regulation of neural proliferation and specification, at the single cell resolution. By taking advantage of the restricted expression of the neuropeptide FMRFa in the last-born cell of the NB lineage 5-6T, the Ap4 neuron, we could monitor the entire lineage progression. This screen succeeded in identifying 43 novel genes controlling different aspects of CNS development. One of the genes isolated, Ctr9, displayed extra Ap4/FMRFa neurons. Ctr9 encodes a component of the RNA polymerase II complex Paf1, which is involved in a number of transcriptional processes. The Paf1C, including Ctr9, is highly conserved from yeast to human, and in the past couple of years, its importance for transcription has become increasingly appreciated. However, studies in the Drosophila system have been limited. In the screen, we isolated the first mutant of Drosophila Ctr9 and conducted the first detailed phenotypic study on its function in the Drosophila embryonic CNS. Loss of function of Ctr9 leads to extra NB numbers, higher proliferation ratio and lower expression of neuropeptides. Gene expression analysis identified several other genes regulated by Ctr9, which may explain the Ctr9 mutant phenotypes. In summary, we identified Ctr9 as an essential gene for proper CNS development in Drosophila, and this provides a platform for future study on the Drosophila Paf1C. Another interesting gene isolated in the screen was worniou (wor), a member of the Snail family of transcription factors. In contrast to Ctr9, whichdisplayed additional Ap4/FMRFa neurons, wor mutants displayed a loss of these neurons. Previous studies in our group have identified many genes acting to stop NB lineage progression, but how NBs are pushed to proliferate and generate their lineages was not well known. Since wor may constitute a “driver” of proliferation, we decided to study it further. Also, we identified five other transcription factors acting together with Wor as pro-proliferative in both NBs and their daughter cells. These “drivers” are gradually replaced by the previously identified late-acting “stoppers.” Early and late factors regulate each other and the cell cycle, and thereby orchestrate proper neural lineage progression.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2017. , p. 68
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1558
National Category
Developmental Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cell Biology Genetics Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-134459DOI: 10.3384/diss.diva-134459ISBN: 9789176856055 (print)OAI: oai:DiVA.org:liu-134459DiVA, id: diva2:1074014
Public defence
2017-03-17, Hasselquist, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2017-02-14 Created: 2017-02-14 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
Open this publication in new window or tab >>Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
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2015 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 200, no 4, p. 1229-1244Article in journal (Refereed) Published
Abstract [en]

The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system.

Place, publisher, year, edition, pages
Genetics Society of America, 2015
Keywords
Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-121318 (URN)10.1534/genetics.115.178483 (DOI)000359917000020 ()26092715 (PubMedID)
Available from: 2015-09-16 Created: 2015-09-14 Last updated: 2017-12-04Bibliographically approved
2. Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System
Open this publication in new window or tab >>Ctr9, a Key Component of the Paf1 Complex, Affects Proliferation and Terminal Differentiation in the Developing Drosophila Nervous System
2016 (English)In: G3: Genes, Genomes, Genetics, ISSN 2160-1836, E-ISSN 2160-1836, Vol. 6, no 10, p. 3229-3239Article in journal (Refereed) Published
Abstract [en]

The Paf1 protein complex (Paf1C) is increasingly recognized as a highly conserved and broadly utilized regulator of a variety of transcriptional processes. These include the promotion of H3K4 and H3K36 trimethylation, H2BK123 ubiquitination, RNA Pol II transcriptional termination, and also RNA-mediated gene silencing. Paf1C contains five canonical protein components, including Paf1 and Ctr9, which are critical for overall complex integrity, as well as Rtf1, Leo1, and Cdc73/Parafibromin(Hrpt2)/Hyrax. In spite of a growing appreciation for the importance of Paf1C from yeast and mammalian studies, there has only been limited work in Drosophila. Here, we provide the first detailed phenotypic study of Ctr9 function in Drosophila. We found that Ctr9 mutants die at late embryogenesis or early larval life, but can be partly rescued by nervous system reexpression of Ctr9. We observed a number of phenotypes in Ctr9 mutants, including increased neuroblast numbers, increased nervous system proliferation, as well as downregulation of many neuropeptide genes. Analysis of cell cycle and regulatory gene expression revealed upregulation of the E2f1 cell cycle factor, as well as changes in Antennapedia and Grainy head expression. We also found reduction of H3K4me3 modification in the embryonic nervous system. Genome-wide transcriptome analysis points to additional downstream genes that may underlie these Ctr9 phenotypes, revealing gene expression changes in Notch pathway target genes, cell cycle genes, and neuropeptide genes. In addition, we find significant effects on the gene expression of metabolic genes. These findings reveal that Ctr9 is an essential gene that is necessary at multiple stages of nervous system development, and provides a starting point for future studies of the Paf1C in Drosophila.

Place, publisher, year, edition, pages
Genetics Society of America, 2016
Keywords
neuroblast, lineage tree, cell cycle, epigenetics, terminal differentiation, FlyBook
National Category
Genetics
Identifiers
urn:nbn:se:liu:diva-132856 (URN)10.1534/g3.116.034231 (DOI)000386581200018 ()27520958 (PubMedID)
Note

Funding Agencies|Swedish Research Council [621-2013-5258]; Knut and Alice Wallenberg Foundation [KAW2011.0165]; Swedish Cancer Foundation [120531]; Swedish Royal Academy of Sciences

Available from: 2016-12-06 Created: 2016-11-30 Last updated: 2017-11-29
3. Neural Lineage Progression Controlled by a Temporal Proliferation Program.
Open this publication in new window or tab >>Neural Lineage Progression Controlled by a Temporal Proliferation Program.
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2017 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 43, no 3, p. 332-348Article in journal (Refereed) Published
Abstract [en]

Great progress has been made in identifying transcriptional programs that establish stem cell identity. In contrast, we have limited insight into how these programs are down-graded in a timely manner to halt proliferation and allow for cellular differentiation. Drosophila embryonic neuroblasts undergo such a temporal progression, initially dividing to bud off daughters that divide once (type I), then switching to generating non-dividing daughters (type 0), and finally exiting the cell cycle. We identify six early transcription factors that drive neuroblast and type I daughter proliferation. Early factors are gradually replaced by three late factors, acting to trigger the type I→0 daughter proliferation switch and eventually to stop neuroblasts. Early and late factors regulate each other and four key cell-cycle genes, providing a logical genetic pathway for these transitions. The identification of this extensive driver-stopper temporal program controlling neuroblast lineage progression may have implications for studies in many other systems.less thanbr /greater than (Copyright © 2017 Elsevier Inc. All rights reserved.)

Place, publisher, year, edition, pages
Cell Press, 2017
National Category
Developmental Biology
Identifiers
urn:nbn:se:liu:diva-143117 (URN)10.1016/j.devcel.2017.10.004 (DOI)000414584300011 ()29112852 (PubMedID)
Note

Funding agencies: Swedish Research Council [621-2013-5258]; Knut and Alice Wallenberg Foundation [KAW2011.0165, KAW2012.0101]; Swedish Cancer Foundation [140780, 150633]

Available from: 2017-11-20 Created: 2017-11-20 Last updated: 2017-11-20Bibliographically approved

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