Role of growth-differentiation factor (GDF) 15 in the regulation of embryonic neural precursors [Elektronische Ressource] / presented by Carmen Carrillo García

Dissertation submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences.
















presented by
Diplom Carmen Carrillo García
Born in Santander, España.
th
Oral-examination: 12 November 2008.





I





Role of Growth/Differentiation Factor (GDF) 15 in the regulation
of embryonic neural precursors.

















Referees:
Prof. Dr. Klaus Unsicker
Prof. Dr. Hilmar Bading





II







A mis padres y hermano.

To Francesca, with all my gratitude.

















III Index
Index:

Summary...............................................................................................1
Zusammenfassung. ..............................................................................2
Articles from this PhD thesis:.............................................................3
Chapter 1: Introduction......................................................................4
1.1- Definition of neural stem cells. .................................................................4
1.2- Neural precursor cells during development............................................6
1.3- FGF-2/EGF responsiveness during embryonic development................9
1.4- Transforming growth factor-beta (TFG- ) superfamily.....................13
1.4.1- Bone morphogenetic proteins (BMPs)......................................................... 15
1.4.2- Growth/differentiation factor 15 (GDF15)................................................... 16
1.5- Aims of the study. ....................................................................................19
Chapter 2: Materials and Methods................................................. 21
2.1- Materials...................................................................................................21
2.1.1- General reagents, buffers and solutions. ...................................................... 21
2.1.2- Cell Culture Reagents and Media. ............................................................... 21
2.1.3- Reagents for immunostaining. ..................................................................... 22
2.1.4- RNA isolation............................................................................................... 23
2.1.5- cDNA synthesis reagents and Reverse Transcriptase-PCR. ........................ 23
2.1.6- qPCR reagents.............................................................................................. 23
2.1.7- Primary Antibodies. ..................................................................................... 24
2.1.8- Secondary Antibodies. ................................................................................. 24
2.1.9- Software. ...................................................................................................... 24
2.1.10- Genotyping primers.................................................................................... 25
2.2- Methods. ...................................................................................................26
2.2.1- Dissection of the tissue................................................................................. 26
2.2.2- Primary neural precursor cell cultures. ........................................................ 27
2.2.3- Fluorescence activated cell sorting (FACS)................................................. 28
2.2.4- Differentiation of neurosphere-derived precursors. ..................................... 28
2.2.5- BrdU incorporation on differentiating neurosphere-derived precursors...... 29
2.2.6- Immunocytochemistry.................................................................................. 29
IV
bbbb Index
2.2.7- Immunohistochemistry................................................................................. 30
2.2.8- Quantitative analysis of immunolabelled cells in vivo. ............................... 31
2.2.9- Fluorescence microscopy. ............................................................................ 31
2.2.10- RNA isolation............................................................................................. 31
2.2.11- cDNA synthesis by RT PCR (Reverse transcription polymerase chain
reaction).................................................................................................................. 32
2.2.12- Quantitative PCR. ...................................................................................... 32
2.2.13- BrdU cumulative labelling. ........................................................................ 33
Chapter 3: Results. ........................................................................... 35
3.1- Analysis of GDF15 expression in the embryonic and adult GE/SVZ
and hippocampus. ...........................................................................................35
3.2- GDF15 is not a mitogen and its addition at different concentrations to
neurosphere cultures does not affect NPC proliferation. ...........................38
3.3- Effect of GDF15 on proliferation and differentiation of NPC derived
from the embryonic GE..................................................................................39
-/-3.3.1- NPC derived from GDF15 embryonic GE in vitro give rise to less progeny
than their WT counterpart. ..................................................................................... 39
3.3.2- Absence of GDF15 leads to a decrease in EGFR expression in GE NPC. .. 41
3.3.3- Decrease on EGFR expression is not mediated by a change in FGF-2
signalling. ............................................................................................................... 48
3.3.4- GDF15 controls cell cycle exit of NPC differentiating in vitro. .................. 49
3.3.5- Role of GDF15 in the regulation of GE NPC in vivo.................................. 53
3.3.6- Absence of GDF15 leads to an increase in Mash1 expression. ................... 58
3.4- Effect of GDF15 on NPC of the hippocampus......................................60
-/-3.4.1- NPC derived from GDF15 embryonic hippocampus give rise to less
progeny in vitro than their WT counterpart............................................................ 60
3.4.2- Absence of GDF15 leads to a decrease in EGFR expression in hippocampal
NPC. ....................................................................................................................... 61
3.4.3- Decrease on EGFR expression is not mediated by a change in FGF-2
signaling. ................................................................................................................ 66
3.4.4- Analysis of EGFR expression in vivo.......................................................... 68
3.4.5- Role of GDF15 in the regulation of hippocampal NPC in vivo................... 70
3.4.7- Comparison of EGFR and PHH3 expression in vivo................................... 74
V Index
Chapter 4: Discussion....................................................................... 76
4.1- GDF15 is expressed by NPC from embryonic and adult GE/SVZ and
hippocampus....................................................................................................76
4.2- GDF15 does not directly affect NPCs proliferation in vitro................77
4.3- GDF15 directly regulates EGFR expression in NPC and not by
modulation of FGF-2 signalling.....................................................................79
4.4- GDF15 promotes cell cycle exit of GE derived progenitors in vitro...80
4.5- GDF15 provides a feed forward signal regulating the cell cycle of
proliferating progenitors in the developing GE...........................................80
-/-
4.6- Impaired EGFR expression in GDF15 hippocampal NPC leads to a
decrease in proliferation in the hippocampal subependyma in vivo..........83
References.......................................................................................... 87
Abbreviations. ................................................................................... 93
Acknowledgements. .......................................................................... 95

VI Summary
Summary.
TGF-b superfamily members play important roles in the regulation of multiple aspects
of neural stem cell behaviour. Growth/differentiation factor 15 (GDF15), a new member
of this superfamily recently cloned and characterized by our lab and others, has been
shown to be expressed at low levels in the rodent brain (Böttner(a) et al., 1999) and to
be particularly localised in neurogenic areas as the sub-ventricular zone (SVZ) of the
lateral ventricles (Schober et al., 2001). As a follow up of this observation, in this study
I investigated the possibility that GDF15 may play a role in the regulation of neural
precursor behaviour during brain devel