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Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
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
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences.
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2015 (English)In: Genetics, ISSN 0016-6731, E-ISSN 1943-2631, Vol. 200, no 4, 1229-1244 p.Article 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. Vol. 200, no 4, 1229-1244 p.
Keyword [en]
Drosophila; CNS development; neural cell fate specification; forward genetic screening; FMRFamide
National Category
Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-121318DOI: 10.1534/genetics.115.178483ISI: 000359917000020PubMedID: 26092715OAI: oai:DiVA.org:liu-121318DiVA: diva2:854130
Available from: 2015-09-16 Created: 2015-09-14 Last updated: 2017-02-14Bibliographically approved
In thesis
1. Genetic pathways controlling CNS development: The role of Notch signaling in regulating daughter cell proliferation in Drosophila
Open this publication in new window or tab >>Genetic pathways controlling CNS development: The role of Notch signaling in regulating daughter cell proliferation in Drosophila
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human central nervous system (CNS) displays the greatest cellular diversity of any organ system, consisting of billions of neurons, of numerous cell sub-types, interconnected in a vast network. Given this enormous complexity, decoding the genetic programs controlling the multistep process of CNS development remains a major challenge. While great progress has been made with respect to understanding sub-type specification, considerably less is known regarding how the generation of the precise number of each sub-type is controlled.

The aim of this thesis was to gain deeper knowledge into the regulatory programs controlling cell specification and proliferation. To address these questions I have studied the Drosophila embryonic CNS as a model system, to thereby be able to investigate the genetic mechanisms at high resolution. Despite the different size and morphology between the Drosophila and the mammalian CNS, the lineages of their progenitors share similarity. Importantly for this thesis, both species progenitors show elaborate variations in their proliferation modes, either giving rise to daughters that can directly differentiate into neurons or glia (type 0), divide once (type I), or multiple times (type II).

The studies launched off with a comprehensive chemical forward genetic screen, for the very last born cell in the well-studied lineage of progenitor NB5-6T: the Ap4 neuron, which expresses the neuropeptide FMRFa. NB5-6T is a powerful model to use, because it undergoes a programmed type I>0 daughter cell proliferation switch. An FMRF-eGFP transgenic reporter was utilized as readout for successful terminal differentiation of Ap4/FMRFa and thereby proper lineage progression of the ∼20 cells generated. The strongest mutants were mapped to genes with both known and novel essential functions e.g., spatial and temporal patterning, cell cycle control, cell specification and chromatin modification. Subsequently, we focused on some of the genes that showed a loss of function phenotype with an excess of lineage cells. We found that Notch is critical for the type I>0 daughter cell proliferation switch in the NB5-6T lineage and globally as well. When addressing the broader relevance of these findings, and to further decipher the Notch pathway, we discovered that selective groups of E(spl) genes is controlling the switch in a close interplay with four key cell cycle factors: Cyclin E, String, E2F and Dacapo, in most if not all embryonic progenitors. The Notch mediation of the switch is likely to be by direct transcriptional regulation. Furthermore, another gene identified in the screen, sequoia, was investigated. The analysis revealed that sequoia is also controlling the daughter cell switch in the CNS, and this partly through context dependent interactions with the Notch pathway.

Taken together, the findings presented in this thesis demonstrate that daughter cell proliferation switches in Drosophila neural lineages are genetically programmed, and that Notch contributes to the triggering of these events. Given that early embryonic processes is frequently shown to be evolutionary conserved, you can speculate that changeable daughter proliferation programs could be applied to mammals, and contribute to a broader understanding of proliferation processes in humans as well.

 

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. 80 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1542
National Category
Cell and Molecular Biology Cell Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Immunology
Identifiers
urn:nbn:se:liu:diva-132743 (URN)10.3384/diss.diva-132743 (DOI)9789176856659 (ISBN)
Public defence
2016-12-15, Berzeliussalen, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2017-01-13Bibliographically approved
2. Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS
Open this publication in new window or tab >>Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS
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. 68 p.
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:nbn:se:liu:diva-134459 (URN)10.3384/diss.diva-134459 (DOI)9789176856055 (ISBN)
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: 2017-02-21Bibliographically approved

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