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Genetic mechanisms controlling cell specification and cell numbers in the Drosophila CNS
Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 77 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1299
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-76157ISBN: 978-91-7519-942-9 (print)OAI: oai:DiVA.org:liu-76157DiVA: diva2:512780
Public defence
2012-04-19, Eken, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2012-03-29 Created: 2012-03-29 Last updated: 2016-11-30Bibliographically approved
List of papers
1. A genetic screen for genes controlling Ap neuron specification
Open this publication in new window or tab >>A genetic screen for genes controlling Ap neuron specification
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A central theme in developmental biology pertains to how the diversity of different cell types is generated. In addition, it is important to understand how the numbers of each cell type are regulated. In the developing Drosophila ventral nerve cord, only six neurons, the Ap4 neurons, express the neuropeptide gene FMRFamide (FMRFa). This is the result of proper lineage development and a cascade of regulatory information leading to final cell specification. In addition to these cascades, FMRFa expression is critically dependent upon a retrogarade TGFβ/BMP signal from the axonal target. Its restricted expression pattern and the wealth of information regarding its gene regulation, makes FMRFa a useful marker for understanding cell specification, as well as axon path finding and retrograde signaling. To identify novel genes acting at any level of neuronal development, including pattern formation, stem cell competence, cell cycle control, cell specification, axon transport and retrograde signaling, we have conducted a single cell resolution, forward genetic screen utilizing an FMRFa-EGFP reporter as our read-out. A total of 9,781 EMS-mutated chromosomes were screened for perturbations in FMRFa-EGFP expression, and 611 mutants were identified. Complementation tests showed that many of the previously known regulators had been isolated, which confirmed the validity of the screen. However, in addition to these known genes, the majority of mutants represent novel genes with previously undefined functions in neural development.

Keyword
Drosophila, CNS development, neural cell fate specification, forward genetic screening, FMRFamide
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-76155 (URN)
Available from: 2012-03-29 Created: 2012-03-29 Last updated: 2016-11-30Bibliographically approved
2. Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development
Open this publication in new window or tab >>Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development
Show others...
2011 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 138, no 24, 5311-5320 p.Article in journal (Refereed) Published
Abstract [en]

Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb -greater than Kr -greater than Pdm -greater than Cas -greater than Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a switching factor. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, we have isolated a novel allele of svp. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage.

Place, publisher, year, edition, pages
Company of Biologists, 2011
Keyword
Temporal genes; COUP-TFI/II; Cell specification; Lineage progression; Drosophila
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-73311 (URN)10.1242/dev.070946 (DOI)297329900004 ()
Available from: 2012-01-03 Created: 2012-01-02 Last updated: 2017-12-08Bibliographically approved
3. 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
Show others...
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.

Place, publisher, year, edition, pages
Company of Biologists, 2012
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: 2017-12-07

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