CRANFIELD UNIVERSITY C. A. FERREIRA DE SOUSA VERTEBRATE SOMITE DEVELOPMENT AND NEURAL PATTERNING SCHOOL OF HEALTH PhD THESIS 2013 Supervisor: D. Tannahill; C. Toro CRANFIELD UNIVERSITY SCHOOL OF HEALTH PhD THESIS C A FERREIRA DE SOUSA Vertebrate somite development and neural patterning Supervisor: D Tannahill; C Toro September 2013 This thesis is submitted for the degree of Doctor of Philosophy © Cranfield University 2013. Thesis is restricted for two years. No part of this publication may be reproduced without the written permission of the copyright owner. ABSTRACT The segmentation of the axial skeleton and peripheral nervous system involves a complex integration of multiple patterning molecules. For the latter, axon-repelling molecules in the posterior half-sclerotome are particularly important. This study built on a previously performed mouse microarray screen for novel candidate genes in the posterior half-sclerotome. Multiple candidates were selected for whole-mount in situ hybridization in chick. Two were expressed in the posterior half-sclerotome: thrombin receptor (F2R) and fibronectin leucine rich transmembrane protein-2 (Flrt2). Flrt2 was selected for siRNA-mediated knockdown and a new in ovo transfection technique for somites successfully developed. Scrambled siRNA-transfection did not affect morphogenesis, somite patterning or axon guidance. However, Flrt2 siRNA- transfection resulted in defects in notochord, dermomyotome and neural tube morphogenesis, and in the de-fasciculation and mis-targeting of spinal axons into the posterior half-sclerotome and dermomyotome. Hence, Flrt2 may be a chemorepellent for spinal axons. An unidentified peanut agglutinin (PNA)-binding glycoprotein in the posterior half-sclerotome was previously shown to repel spinal axons. In this project, the expression of a family of mucin-type O-glycosylation enzymes (which could glycosylate the PNA-binding protein) was investigated by whole-mount in situ hybridization in chick, but none was differentially expressed in the posterior half- sclerotome. One candidate for the PNA-binding glycoprotein, Presenilin1, was investigated because of previously published loss of spinal nerve segmentation in Presenilin1 mutants. However, analysis of Presenilin1-hypomorphic mutant mouse embryos showed this was not the PNA-binding molecule. Live-immunostaining for a second candidate, prolyl 4-hydroxylase, beta polypeptide (P4HB), showed its expression coincided with PNA-binding at the surface of posterior half-sclerotome cells. P4HB siRNA-transfection into somites reduced PNA binding and disrupted spinal axon segmentation and expression of a posterior sclerotome marker, Uncx4.1. Overall, these results suggest that P4HB is a strong candidate to be the key PNA-binding glycoprotein in the posterior half-sclerotome that repels spinal axons. For those that inspired me; TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABREVIATIONS CHAPTER 1- INTRODUCTION ................................................................................. 2 1.1- Vertebrate segmentation ..................................................................................... 1 1.1.1- Segmentation of the adult vertebrate .............................................................. 1 1.1.2- Embryonic segmentation ................................................................................ 4 1.2- Formation of the pre-somitic mesoderm during gastrulation ......................... 4 1.3- Somitogenesis ....................................................................................................... 8 1.4- Anteroposterior Polarization of the Presomitic Mesoderm ........................... 13 1.4.1- Molecular oscillation clock within somitogenesis ........................................ 18 1.4.2- Other signalling pathways important for somite formation .......................... 20 1.5- Somite fate .......................................................................................................... 21 1.5.1- Formation of the dermomyotome and myotome .......................................... 23 1.5.2- Sclerotome fate ............................................................................................. 24 1.6- Sclerotome and Peripheral Nervous System development ............................ 27 1.7- Neural crest formation and migration ............................................................. 37 1.7.1- Identification of a repulsive cue for DRG axons in the posterior half- sclerotome ............................................................................................................... 44 1.8- The chicken as a model system ......................................................................... 45 1.9- Overall Aims ...................................................................................................... 46 CHAPTER 2- MATERIALS AND METHODS ........................................................ 49 2.1- Incubation and staging of chicken embryos .................................................... 51 2.2- Synthesis of antisense RNA probes from chicken cDNA using PCR ............ 51 2.2.1- Total RNA isolation for cDNA synthesis ..................................................... 51 2.2.2- cDNA synthesis ............................................................................................ 52 2.2.3- Primer design ................................................................................................ 52 2.2.4- Polymerase chain reaction to prepare template for riboprobe synthesis ....... 53 2.2.5- Riboprobe synthesis ...................................................................................... 53 2.3- In situ hybridization .......................................................................................... 54 2.3.1- Embryo fixation and dehydration ................................................................. 54 2.3.2- Whole-mount in situ hybridization ............................................................... 54 2.3.4- Clearing and photography of embryos after in situ hybridization ................ 56 2.4- Transfection of embryonic tissue or cultured cells ........................................ 56 2.4.1- Preparation of siRNA or plasmid DNA with the transfection agent. ........... 56 2.4.2- Preparation of chick embryos for in ovo transfection ................................... 57 2.4.3- Somite targeting in ovo ................................................................................. 57 2.4.4- Neural tube targeting using in ovo electroporation ....................................... 58 2.4.5- Evaluation of siRNA impact after in ovo transfection .................................. 59 2.4.6 Culture of in ovo-transfected somite strips .................................................... 59 2.4.6.1- Preparation of in ovo-transfected somite strips for culture .................... 59 2.4.6.2- Culture of intact in ovo-transfected somite strips .................................. 60 2.4.6.3- Culture of dissociated in ovo-transfected somite strips .......................... 60 2.4.7- Transfection of retinal cells and astrocytes in culture ................................ 60 2.5- Immunohistochemistry ..................................................................................... 61 2.5.1- Whole-mount immunohistochemistry .......................................................... 61 2.5.2- Live immunostaining of cultured tissue/cells ............................................... 62 2.5.3- Controls for anti-PDI immunohistochemistry .............................................. 62 2.6- Vibratome sectioning ......................................................................................... 64 2.7- Alcian blue staining ........................................................................................... 64 CHAPTER 3- GENE EXPRESSION ATLAS ........................................................... 65 3.1- Introduction ....................................................................................................... 67 3.2- Positive controls: Tbx 18 and Uncx4.1 ............................................................. 68 3.3- Glypican-6 (Gpc6) ............................................................................................. 70 3.4- Hyaluronan synthase 2 (Has2) ......................................................................... 72 3.5- Phosphatase and tensin homolog (Pten) .......................................................... 75 3.6- Tissue Inhibitor of Metalloproteinase-3 (Timp3) ........................................... 77 3.7- Cystatin C (Cst3) ............................................................................................... 80 3.8- Coagulation factor II (thrombin) receptor (F2r) ............................................ 82 3.9- Ets2 ...................................................................................................................... 84 3.10- Fibroblast Activation Protein (Fap) ............................................................... 86 3.11- Insulin-like growth factor-binding protein 5 (Igfbp5) ................................. 88 3.12- Roundabout homolog 1 (Drosophila): Robo1 ................................................ 90 3.13- Transforming growth factor beta receptor II (Tgfbr2) ............................... 92 3.14- Fibronectin leucine rich transmembrane protein 2 (Flrt2) ......................... 94 CHAPTER 4- DEVELOPMENT OF A NEW IN OVO TRANSFECTION TECHNIQUE FOR TARGETING SOMITES