Role of neural sonic hedgehog in the development of the mouse hypothalamus and thalamus [Elektronische Ressource] / von Nora Szabó

Nora Szabo

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Role of Neural Sonic hedgehog
in the Development of the
Mouse Hypothalamus and Thalamus


Von der Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades einer


DOKTORIN DER NATURWISSENSCHAFTEN
Dr. rer. nat.




genehmigte Dissertation
von

Dipl.-Biol. Nora Szabó
geboren am 26.08.1978 in Miercurea Ciuc


2009






















Referent: Prof. Dr. Herbert Hildebrandt,
Medizinische Hochschule Hannover

Korreferentin: Prof. Dr. Marta Szamel,
Medizinische Hochschule Hannover

Tag der Promotion: 15.12.2008
Table of Contents

TABLE OF CONTENTS

ZUSAMMENFASSUNG............................................................................... 6
ABSTRACT.................................................................................................... 7
1 GENERAL INTRODUCTION .............................................................. 8
1.1 Patterning of the embryonic forebrain................................................................. 8
1.2 Sonic hedgehog (Shh) ............................................................................................. 9
1.2.1 Shh in the forebrain.................................................................................................... 12
1.2.2 The Shh knockout mouse ........................................................................................... 13
1.3 The hypothalamus ................................................................................................ 15
1.3.1 The mammillary body ................................................................................................ 17
1.4 The thalamus (dorsal thalamus).......................................................................... 18
1.5 The transcription factor Foxb1 ........................................................................... 20
1.6 The Cre-loxP recombination system................................................................... 21
1.6.1 The ROSA26 reporter line ......................................................................................... 22
1.6.2 The Foxb1-Cre mouse line......................................................................................... 23
1.6.3 The floxed Shh mouse line......................................................................................... 24
1.7 Aim of the work .................................................................................................... 25
2 MATERIALS AND METHODS..........................................................26
2.1 Animal work.......................................................................................................... 26
2.2 Genotyping ............................................................................................................ 26
2.2.1 Primers for genotyping............................................................................................... 27
2.2.2 Genotyping of the mouse lines Foxb1-Cre, CMV-Cre, and Z/AP............................. 27
2.2.3 Genotyping of the Gbx2 mouse line........................................................................... 28
2.2.4 Genotyping of the Shh mouse line ............................................................................. 28
2.3 In situ hybridization ............................................................................................. 29
2.3.1 Probe preparation ....................................................................................................... 29
2.3.1.1 RNA isolation ........................................................................................................ 29
2.3.1.2 Reverse transcription ............................................................................................. 29
2.3.1.3 Primer design ......................................................................................................... 30
2.3.1.4 PCR I ..................................................................................................................... 31
2.3.1.5 PCR II (large scale PCR)....................................................................................... 32
2.3.1.6 RNA transcription.................................................................................................. 33
2.3.2 Whole mount in situ hybridization............................................................................. 34
2.3.3 In situ hybridization on sections................................................................................. 36
2.4 LacZ/ X-Gal staining............................................................................................ 38
3 Table of Contents
2.5 AP-staining............................................................................................................ 39
2.6 Nissl staining ......................................................................................................... 39
2.7 Explant experiments............................................................................................. 40
2.8 Electroporation of embryos ................................................................................. 40
2.9 Molecular biological methods for cloning experiments .................................... 41
2.9.1 Small-scale isolation of plasmid DNA....................................................................... 41
2.9.2 EndoFree plasmid Maxi protocol............................................................................... 42
2.9.3 Gel electrophoresis..................................................................................................... 42
2.9.4 Isolation of DNA fragments from agarose gels.......................................................... 43
2.9.5 Determination of nucleic acid concentrations............................................................ 43
2.9.6 Restriction digestion of DNA..................................................................................... 44
2.9.7 Ligation of DNA fragments ....................................................................................... 44
2.9.8 Transformation of bacteria......................................................................................... 44
2.9.9 The In-Fusion™ PCR Cloning Method ..................................................................... 45
2.9.9.1 Cloning of the exon 2-deleted Shh construct............................................................ 45
3 RESULTS ...............................................................................................47
3.1 First Manuscript ................................................................................................... 47
Neuroepithelial Sonic hedgehog is essential to specify the hypothalamic subregions
and to stabilize diencephalic against telencephalic fate............................................ 47
3.1.1 Introduction................................................................................................................ 48
3.1.2 Methods...................................................................................................................... 50
3.1.3 Results........................................................................................................................ 53
3.1.3.1 Foxb1-driven Cre abolishes Shh full length expression in the anterior ventral
neural plate and forebrain ...................................................................................... 53
3.1.3.2 Activation of Shh expression in the lateral hypothalamus depends on neural Shh 55
3.1.3.3 Early abolition of the Shh pathway in the Shh-c forebrain.................................... 56
3.1.3.4 Before and after ZLI and LH domain formation: early alteration of the
hypothalamus and prethalamus in the Shh-c embryo ............................................ 57
3.1.3.5 Division of the hypothalamic neuroepithelium into AD and PV........................... 59
3.1.3.6 Hypothalamic specification is altered in the Shh-c embryo .................................. 61
3.1.3.7 PV and prethalamus form one abnormal domain in the Shh-c embryo ................. 61
3.1.3.8 Cortical ectopia in the Shh-c embryo..................................................................... 61
3.1.3.9 Neural Shh vs Gli3 in diencephalic dorso-ventral patterning................................ 62
3.1.3.10 The Shh-c hypothalamus is transversally bisected by a dorsalized structure ........ 64
3.1.3.11 Neural Shh is required for the development of the lateral hypothalamus.............. 66
3.1.3.12 Differential alterations of AD and PV in the Shh-c hypothalamus........................ 68
3.1.3.13 Neural Shh is required to maintain expression of the PV survival factor Foxb1... 70
3.1.4 Discussion .................................................................................................................. 72
3.1.5 Authors' contributions ................................................................................................ 77
3.2 Second Manuscript............................................................................................... 78
The role of Shh of n