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Genetic pathways controlling CNS development: The role of Notch signaling in regulating daughter cell proliferation in Drosophila
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human central nervous system (CNS) displays the greatest cellular diversity of anyorgan system, consisting of billions of neurons, of numerous cell sub-types, interconnectedin a vast network. Given this enormous complexity, decoding the genetic programscontrolling the multistep process of CNS development remains a major challenge. Whilegreat progress has been made with respect to understanding sub-type specification,considerably less is known regarding how the generation of the precise number of eachsub-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: urn:nbn:se:liu:diva-132743DOI: 10.3384/diss.diva-132743ISBN: 9789176856659 (Print)OAI: oai:DiVA.org:liu-132743DiVA: diva2:1048888
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: 2016-11-30Bibliographically 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, 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
Keyword
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: 2016-11-30Bibliographically approved
2. Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression
Open this publication in new window or tab >>Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression
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2012 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 139, no 4, 678-689 p.Article in journal (Refereed) Published
Abstract [en]

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-74790 (URN)10.1242/dev.074500 (DOI)000300259800005 ()
Note

funding agencies|Swedish Research Council||Knut and Alice Wallenberg foundation||Swedish Cancer Foundation||

Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2016-11-30
3. Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
Open this publication in new window or tab >>Control of Neural Daughter Cell Proliferation by Multi-level Notch/Su(H)/E(spl)-HLH Signaling
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2016 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 4, e1005984Article in journal (Refereed) Published
Abstract [en]

The Notch pathway controls proliferation during development and in adulthood, and is frequently affected in many disorders. However, the genetic sensitivity and multi-layered transcriptional properties of the Notch pathway has made its molecular decoding challenging. Here, we address the complexity of Notch signaling with respect to proliferation, using the developing Drosophila CNS as model. We find that a Notch/Su(H)/E(spl)-HLH cascade specifically controls daughter, but not progenitor proliferation. Additionally, we find that different E(spl)-HLH genes are required in different neuroblast lineages. The Notch/Su(H)/E(spl)-HLH cascade alters daughter proliferation by regulating four key cell cycle factors: Cyclin E, String/Cdc25, E2f and Dacapo (mammalian p21(CIP1)/p27(KIP1)/p57(Kip2)). ChIP and DamID analysis of Su(H) and E(spl)-HLH indicates direct transcriptional regulation of the cell cycle genes, and of the Notch pathway itself. These results point to a multi-level signaling model and may help shed light on the dichotomous proliferative role of Notch signaling in many other systems.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2016
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-128759 (URN)10.1371/journal.pgen.1005984 (DOI)000375231900032 ()27070787 (PubMedID)
Note

Funding Agencies|Knut and Alice Wallenberg Foundation [KAW2012.0101]; Swedish Research Council [621-2010-5214]; Swedish Cancer Foundation [120531]

Available from: 2016-05-31 Created: 2016-05-30 Last updated: 2016-11-30
4. sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
Open this publication in new window or tab >>sequoia controls the type I>0 daughter proliferation switch in the developing Drosophila nervous system
2016 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 143, no 20, 3774-3784 p.Article in journal (Refereed) Published
Abstract [en]

Neural progenitors typically divide asymmetrically to renew themselves, while producing daughters with more limited potential. In the Drosophila embryonic ventral nerve cord, neuroblasts initially produce daughters that divide once to generate two neurons/glia (type I proliferation mode). Subsequently, many neuroblasts switch to generating daughters that differentiate directly (type 0). This programmed type I>0 switch is controlled by Notch signaling, triggered at a distinct point of lineage progression in each neuroblast. However, how Notch signaling onset is gated was unclear. We recently identified Sequoia (Seq), a C2H2 zinc-finger transcription factor with homology to Drosophila Tramtrack (Ttk) and the positive regulatory domain (PRDM) family, as important for lineage progression. Here, we find that seq mutants fail to execute the type I>0 daughter proliferation switch and also display increased neuroblast proliferation. Genetic interaction studies reveal that seq interacts with the Notch pathway, and seq furthermore affects expression of a Notch pathway reporter. These findings suggest that seq may act as a context-dependent regulator of Notch signaling, and underscore the growing connection between Seq, Ttk, the PRDM family and Notch signaling.

Place, publisher, year, edition, pages
The Company of Biologists Ltd, 2016
Keyword
Lineage tree, Cell cycle, Asymmetric division, Combinatorial control, Notch
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology Cell Biology Medical Biotechnology
Identifiers
urn:nbn:se:liu:diva-132739 (URN)10.1242/dev.139998 (DOI)27578794 (PubMedID)
Available from: 2016-11-22 Created: 2016-11-22 Last updated: 2016-11-28Bibliographically approved

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