Transcription factor Zbtb20 controls regional specification of mammalian archicortex Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Eva Helga Rosenthal aus Kassel 1. Referent: Prof. Dr. Hans-Henning Arnold 2. Referent: Prof. Dr. Martin Korte eingereicht am: 24.02.2010 mündliche Prüfung (Disputation) am: 02.06.2010 Druckjahr 2010 Diese Arbeit wurde am Max-Planck-Institut für biophysikalische Chemie, -Karl-Friedrich-Bonhoeffer-Institut- in Göttingen in der Abteilung für Molekulare Zellbiologie (Direktor Prof. Dr. Peter Gruss) Arbeitsgruppe Molekulare Neuroentwicklungsbiologie durchgeführt. 1 Table of contents I. INTRODUCTION......................................................................................................6 1.1. Organization of mammalian forebrain with a focus on the cerebral cortex ................................... 6 1.1.1. Regionalization of telencephalon............................................................................................... 8 1.1.2. Cortical regionalization: inductive centers............................................................................... 9 1.1.3. Neurogenesis in developing cortex: germinative layers, asymmetric/symmetric divisions 10 1.1.4. Cortical layering ....................................................................................................................... 11 1.1.5. Functional arealization of mammalian cortex ....................................................................... 12 1.2. Hippocampal formation and retrosplenial cortex ........................................................................... 15 1.2.1. Hippocampus proper................................................................................................................ 16 1.2.2. Dentate gyrus ............................................................................................................................ 18 1.2.3. Subiculum.................................................................................................................................. 18 1.2.4. Retrosplenial cortex.................................................................................................................. 19 1.2.5. Principal cell of hippocampus proper and dentate and their generation ............................ 19 1.3. Transcription factor Zbtb20.............................................................................................................. 20 1.3.1. Conserved function of Zbtb20 ................................................................................................. 20 1.3.2. Molecular properties of the Zbtb20 protein........................................................................... 20 1.3.3. Zbtb20 expression in the mammalian brain........................................................................... 21 1.3.4. Zbtb20 in the regional patterning of the forebrain................................................................. 21 1.3.5. Zbtb20 expression and function outside central nervous system .......................................... 21 1.4. Aims of this study ............................................................................................................................... 22 II. RESULTS..............................................................................................................23 2.1. Screen for identification of genes expressed in Emx2-dependent manner in developing mouse cortex .......................................................................................................................................................... 23 2.2. Analysis of the function of Zbtb20 ..................................................................................................... 31 2.2.1. Expression pattern of Zbtb20 in the developing embryo ....................................................... 31 2.2.2. Targeted disruption of Zbtb20 ................................................................................................. 35 2.2.2.1. Generation of knockout construct........................................................................................ 36 2.2.2.2. Generation of homologous recombinant ES-cells ............................................................... 38 2 2.2.2.3. Generation of mouse chimaeras and screening for germline transmissions..................... 40 2.2.2.4. Confirmation of homologous recombination into the Zbtb20 gene locus.......................... 40 2.2.2.4.1. PCR...................................................................................................................................... 40 2.2.2.4.2. RNA-ISH ............................................................................................................................. 41 2.2.3. Expression analysis of Zbtb20 in developing embryo based on LacZ reporter activity of Zbtb20+/- mice..................................................................................................................................... 42 2.2.3.3. Expression in postnatal brain ............................................................................................... 46 2.2.3.4. Expression in different organs in adulthood ....................................................................... 48 2.2.4. Gross abnormalities in the Zbtb20 KO mice........................................................................... 48 2.2.4.1. Prenatal viability.................................................................................................................... 48 2.2.4.2. Growth retardation, premature death and other defects in postnatal Zbtb20-/- mice..... 49 2.2.5. Analysis of the brain phenotype in Zbtb20 KO mice ............................................................. 52 2.2.5.1. Morphological defects of the postnatal brain...................................................................... 52 2.2.5.2. Analysis of the molecular patterning of cortex in Zbtb20 KO embryos at stage E12.5 ... 55 2.2.5.3. Defect of the regional organization of the medial pallium at stage E16.5-18.5................. 58 2.2.5.3.1. Medial displacement of the border retrosplenial cortex/subiculum, Rsp/S................... 58 2.2.5.3.2. Medial displacement of the border S/CA1 of hippocampus............................................ 62 2.2.5.3.3. Medial displacement of the border CA1/CA2-3............................................................... 63 2.2.5.3.4. Mispatterning of the CA2/CA3 area of hippocampus ..................................................... 65 2.2.5.3.5. Patterning defects in the Zbtb20-/- medial pallium – a summary................................... 70 2.2.5.3.6. Abnormality of dentate gyrus in the Zbtb20-/- mutant brain ......................................... 71 2.2.5.3.7. Failure of neuronal differentiation in Zbtb20-/- ............................................................... 72 2.2.6. Defects of skeleton of Zbtb20 KO mice.................................................................................... 74 2.2.6.1. Defect in the composition of vertebral elements in absence of Zbtb20 .............................. 74 2.2.6.2. Malformations of elements in the axial skeleton................................................................. 75 2.2.6.3. Delayed ossification of the skeleton...................................................................................... 77 III. DISCUSSION .......................................................................................................82 3.1. Candidate genes acting in Emx2-dependent manner during corticogenesis ................................. 82 3.2. Function of TF Zbtb20 during development..................................................................................... 84 3.2.1. Zbtb20 controls body growth ................................................................................................... 84 3 3.2.2. Role of Zbtb20 in developing mammalian cortex ................................................................... 85 3.2.2.1. Zbtb20 and morphogenesis of hippocampus........................................................................ 86 3.2.2.2. Zbtb20 and patterning of medial pallium............................................................................. 87 3.2.2.3. Zbtb20 controls acquisition of archicortical areal identity................................................. 89 3.2.2.4. Zbtb20 in growth and differentiation of DG........................................................................ 95 3.2.2.6. Possible mechanisms of Zbtb20 dependent areal patterning of MP .................................. 96 3.2.3. Role of Zbtb20 for the normal antero-posterior axis specification of the skeleton.............. 97 3.2.4. Zbtb20 is necessary for the integrity of vertebrae and for normal ossification ................... 99 IV. SUMMARY ........................................................................................................102 V. MATERIALS AND METHODS ...........................................................................104 5.1. Organisms ......................................................................................................................................... 104 5.1.1. Mice.........................................................................................................................................